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Surface chemistry of nanocellulose fibers directs monocyte/macrophage response Kai Hua, Eva Ålander, Tom Lindstrom, Albert Mihranyan, Maria Stromme, and Natalia Ferraz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00727 • Publication Date (Web): 06 Aug 2015 Downloaded from http://pubs.acs.org on August 11, 2015

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Surface chemistry of nanocellulose fibers directs monocyte/macrophage response *

§

§

*

*

Kai Hua , Eva Ålander , Tom Lindström , Albert Mihranyan , Maria Strømme , Natalia *,‡

Ferraz *

Nanotechnology and Functional Materials, Department of Engineering Sciences, Uppsala

University, Box 534, 75121 Uppsala, Sweden. §

Innventia AB, Drottning Kristinas väg 55, 11486, Stockholm, Sweden.

Keywords: nanocellulose, nanofibrillated cellulose, monocytes/macrophages, biocompatibility, inflammation

Abstract

The

effect

of

surface

functionalization

of

nanofibrillated

cellulose

(NFC)

on

monocyte/macrophage (MM) behavior is investigated in order to increase the understanding about how the physicochemical properties of nanocelluloses influence the interactions of such materials with biological systems. Films of anionic (a-), cationic (c-) and unmodified (u-) NFC were synthesized and characterized in terms of surface charge. THP-1 monocytes were cultured on the surface of the films for 24h in the presence and absence of lipopolysaccharide and the cell response was evaluated in terms of cell adhesion, morphology and secretion of TNF-α, IL-10 and IL-1ra. The results show that MMs cultured on carboxymethylated-NFC films (a-NFC) are activated towards a pro-inflammatory phenotype, while u-NFC promotes a mild activation of the studied cells. The presence of hydroxypropyltrimethylammonium groups on c-NFC, however, does not promote the activation of MMs, indicating that c-NFC 1 ACS Paragon Plus Environment

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closely behaves as an “inert” material in terms of MM activation. None of the materials is able to directly activate the MMs towards an anti-inflammatory response. These results may constitute the fundamentals for the design of future NFC-based materials with the ability of controlling MM activation and expand the use of NFC in biomedical applications.

1. Introduction Cellulose, the most abundant natural polymer on earth, consists on repeated units of β (1-4) bound D-glucopyranose and is synthesized by higher plants, certain types of bacteria, algae, fungi and tunicates.1 Properties such as hydrophilicity, mechanical strength, broad possibility of chemical modifications and biocompatibility have largely contributed to making cellulose one of the most interesting polymers for industrial applications in biotechnology and biomedicine.2 In the era of nanotechnology, cellulose has regained attention in the form of nanocellulose. The increasing concern for sustainability of resources has contributed to the growing interest of cellulose-based products, where nanocellulose emerges as a highly interesting version of the wide-spread polymer for novel applications. Nanocellulose consists of cellulose fibrils or crystallites with at least one dimension in the nanoscale and combines the properties of cellulose described above with specific nanomaterial characteristics like high specific surface area and high aspect ratio together with tailorable mechanical, rheological and optical properties.2-4 Nanocellulose materials are generally classified in three main groups: nanofibrillated cellulose (NFC, also called microfibrillated cellulose or cellulose nanofibrils), cellulose nanocrystals (CNC, also referred as nanocrystalline cellulose or cellulose nanowhiskers) and bacterial cellulose (BC, also called microbial cellulose).5, 6 The classification relies on the 2 ACS Paragon Plus Environment

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source and dimensions of the nanocellulose material, the latter being mainly a consequence of the processing method used to obtain the material.6 BC is synthesized from low molecular weight sugar (e.g. glucose) or other carbon sources by bacteria such as Gluconacetobacter xylinus (bottom-up method), while top-down methods are used to obtain NFC and CNC from wood, cotton, wheat straw, hemp, etc.1, 6 By acid hydrolysis cellulose crystals are extracted from native cellulose, resulting in rodlike nanocrystals with typical dimensions of 5-30 nm in diameter and 100-500 nm in length (from plant cellulose) or longer crystals (up to several micrometers) when the source is tunicates or algae.5 When cellulose fibers are delaminated by mechanical treatment individual microfibrils are obtained. The mechanical treatment consists of high-pressure homogenization and/or grinding and is generally preceded by chemical or enzymatic pre-treatments.6 The fibril dimensions and the degree of crystallinity depend on the defibrillation process, the pretreatment method and the cellulose source.7 Wood is the most important source of NFC and the individual fibrils are typically 3-5 nm in diameter and several micrometers in length, forming 20-50 nm thick aggregates.3 NFC forms gels at very low concentrations in water and the rheology of such gels is a key property for applications of NFC as food structuring and dispersion stabilizing.1 NFC gel suspensions can be used to prepare films by simply filtrating the suspension and letting it dry or by dispersion casting.3 NFC films present properties such as high tensile strength, optical transparency, low thermal expansion and oxygen-barrier characteristics that make them viable candidates for, e.g., food and pharmaceutical packaging, electronic devices, and printing applications.7-10 Extensive work is currently carried out by numerous research groups in order to better understand the influence of NFC characteristics in films, nanocomposites and paper coating.6, 7, 11

However, the influence of NFC’s physicochemical properties on the interactions with

biological systems has not yet been fully investigated. This is most probably because NFC is a 3 ACS Paragon Plus Environment

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novel candidate for biomedical applications, in contrast to other members of the NC family, such as BC2, 5, 12 and CNC,13-16 that have been extensively studied for such applications. Some investigations of NFC-based biomedical materials have, however, been reported including applications for tissue engineering,17-20 drug delivery,21-25 protein immobilization,2631

antibacterial agents,32-35 and medical devices.36-38 The effect of NFC on biological systems has been investigated by means of toxicological

evaluations with the aim of gathering information about the human and environmental hazard of the nanomaterial.39-41 When considering specific biomedical applications, some studies have addressed the biocompatibility of NFC.19, 20, 42-46 Kollar et al. investigated the inflammatory response of different types of cellulose including NFC, considering the potential application of the materials in wound healing therapy.42 Lou et al. and Bhattaxharya et al. explored the use of NFC hydrogels as 3D scaffolds for culturing human pluripotent stems cells and liver cells, respectively.20,

43

Chang and Wang cultured fibroblasts cells on NFC-calcium peroxide

composites that allow the release of H2O2 or O2 useful for, e.g., wound healing dressings and tissue regeneration.19 Hua et al. investigated the biocompatibility of unmodified, cationic and anionic NFC films used as culture substrates for human dermal fibroblasts cells.44 Alexandrescu et al. evaluated the cytocompatibility of NFC air-dried films and aerogels using mouse fibroblasts and suggested the use of such materials for regenerative medicine and wound healing.46 The aim of the present work is to bring forward new knowledge about how the surface chemistry of NFC impact cell-material interactions in order to allow for new applications of this type of material in a number of biomedicine applications including wound healing dressings, coatings of implant materials and membranes for blood purification. The fact that NFC is obtained from a sustainable resource and that it can be produced at a relatively low-

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cost make it a more advantageous material compared to other types of biopolymer materials presently used in biomedical applications.47 Films of NFC with different surface functionalization are investigated. Cationic and anionic groups are introduced during carboxymethylation and epoxypropyltrimethylammonium chloride (EPTMAC) pre-treatments of wood pulp, respectively. Enzymatic pre-treatment of the wood pulp results in NFC with low content of carboxyl groups and is herein referred as unmodified NFC. The response of monocytes/macrophages to the anionic, cationic and unmodified NFC films is studied due to the central role of these cells in the host reaction to biomaterials, including inflammatory response, wound healing and tissue repair. To the authors’ knowledge this is the first work that investigates the effect of NFC surface chemistry on the behavior of monocytes/macrophages in vitro.

2. Experimental section

2.1 Chemicals and reagents RPMI 1640 culture medium, Dulbecco’s phosphate buffered saline (PBS), lactate dehydrogenase (LDH) in vitro toxicology assay kit, dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), polymyxin B (PMB), ethanol, hexamethyldisilazane (HMDS) and glutaraldehyde were purchased from Sigma Aldrich. Alamar blue cell viability reagent, TNFα IL-1ra and IL-10 Human ELISA kits were purchased from Invitrogen. Unmodified-NFC, carboxymethylated-NFC and hydroxypropyltrimethylammonium-NFC were provided by Innventia AB (Sweden).

2.2 Preparation of materials NFC was produced from commercial, never dried, bleached sulfite softwood dissolving pulp (Domsjö fabriker AB, Sweden). Unmodified-NFC (u-NFC) was prepared by enzymatic 5 ACS Paragon Plus Environment

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pretreatment of the bleached sulfite softwood pulp as described by Pääkkö et al.48 Carboxymethylated-NFC and hydroxypropyltrimethylammonium-NFC were prepared by carboxymehylation and EPTMAC quaternization pretreatments of the softwood dissolving pulp as previously described by Hua et al.44 For simplicity carboxymethylated-NFC is referred as anionic-NFC (a-NFC) and hydroxypropyltrimethylammonium-NFC as cationic NFC (c-NFC). NFC films were prepared by dispersing 300 mg of NFC (u-NFC, a-NFC and c-NFC) in 50 ml of deionized water using high energy ultrasonic treatment (Vibtacell 600 W, 20 kHz, USA), followed by vacuum filtration with a nylon filter membrane (0.1 µm mesh size). After air-drying, 0.2-0.3 mm thin NFC films were obtained. Finally, the NFC films were cut into 13 mm diameter discs and sterilized by autoclaving at 1.5 MPa for 15 min, excepting the c-NFC samples which were sterilized by UV radiation.

2.3 Characterization: surface functional group quantification The number of carboxyl groups presents on a-NFC and u-NFC samples was determined by conductometric titration of the pulp. Prior to titration, the pulp was washed to different counter-ion forms as follows. First the pulp was set to its hydrogen counter-ion form. A sample containing 2 g of dry pulp was dispersed in 1000 ml of deionized water and then 0.01 M HCl was added, adjusting the pH to 2. The excess of HCl was washed away after 30 minutes with deionized water on a Büchner funnel until the conductivity was below 5 µS cm1

. The pulp was then set to its sodium counter-ion form. The pulp was dispersed in deionized

water and then 0.001 M NaHCO3 was added, after which the pH was set to 9 using NaOH. After 30 more minutes, the excess NaOH was washed away with deionized water on a Büchner funnel until the conductivity was below 5 µS cm-1. After this procedure, the sample was once more set to its hydrogen counter ion form and washed to a conductivity below 5 µS

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cm-1. Finally the total charge density of the pulp was determined using the conductometric titration procedure described by Katz et al.49 Since unmodified NFC does not contain nitrogen, the amount of cationic charges [hydroxypropyltrimethylammonium (HPTMA)] in the c-NFC was determined by elemental analysis of total nitrogen in the pulp. The instrument used was an Antek MultiTek Nitrogen, Sulfur & Halides Analyze (ANTEK instruments Inc., USA). The method used was pyrochemiluminescence with urea for calibration.

2.4 Cell culture Human THP-1 monocytes were cultured in RPMI 1640 culture medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 IU ml-1 penicillin and 100 µg ml-1 streptomycin in an incubator at 37oC, 5% CO2 in a humidified atmosphere. Cell viability prior to THP-1 seeding on the NFC films was assessed using trypan blue staining (95-99% viable cells).

2.4.1 Indirect cytotoxicity test The tests were carried out according to the ISO-10993-5 procedure.50 NFC films were extracted for 24±2 h in cell culture medium at 37oC, 5% CO2, and the surface/volume ratio was 6 cm2 ml-1. THP-1 monocytes were suspended in extract medium at a density of 300 000 cells ml-1, and 150 000 cells per well were added to a 24-well tissue culture plate. Cells were cultured for 24±2 h at 37oC, 5% CO2 in a humidified atmosphere. The negative control was the medium extract of tissue culture plate (TCP) and the positive control was 5% DMSO in culture medium. Samples were run in triplicate. The viability of THP-1 monocytes cultured in extract medium was determined by the alamar blue assay. The cells were collected in Eppendorf tubes and centrifuged at 1000 rpm for 5 min. The supernatants were removed from the tubes and cells were re-suspended in 500 7 ACS Paragon Plus Environment

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µl of alamar blue stock solution diluted 1: 10 in cell culture medium, transferred to a 24-well tissue culture plate and incubated at 37oC, 5% CO2 in a humidified atmosphere for 90 min. Aliquots of 100 µl were transferred from each well to a 96 well plate, and the fluorescence intensity was measured at 560 nm excitation wavelength and 590 nm emission wavelength by employing a spectrofluorometer (Tecan infinite® M200). The results were presented as percentage of cell viability of the negative control. The presented data were given as the mean±the standard error of the mean for n = 5.

2.4.2 THP-1 monocytes culture on NFC films NFC films were placed on 24-well plates and presoaked with culture medium. THP-1 cells were seeded onto the different NFC films in the presence and absence of LPS (1 ng ml-1) at a cell density of 150 000 cells per well and cultured for 24±2 h at 37oC, 5% CO2 in a humidified atmosphere. Cells seeded on thermanox (TMX) discs with and without LPS served as controls. The same experiments were performed in the presence of PMB at a final concentration of 25 µg ml-1 in order to inhibit the potential effects of any endotoxin present in the NFC samples.51 NFC samples under each of the described experimental conditions were run in triplicate and the experiments repeated three times. LDH assay. The number of adherent cells on NFC films was determined by a LDH in vitro toxicology assay kit (Sigma Aldrich). After 24±2 h culture, NFC films were transferred to a new 24-well plate, incubated with lysis solution and thereafter the LDH activity was measured following the manufacturer’s instructions. Results are expressed as arbitrary units and reported as mean value ± standard error of the mean for n=3.

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A control was done to determine if the culture medium that had been in contact with the NFC films could have interfered with the LDH enzymatic activity. No changes in LDH activity due to contact with the NFC films were observed. Cytokine measurements. Cytokine production was quantified by enzyme immunoassays after 24±2 h cell culture. The levels of TNF-α, IL-10 and IL-1ra in the culture supernatants were analyzed by ELISA kits (Human TNF-α human, human IL-10 and human IL-1ra ELISA kits, Invitrogen), following the manufacturer’s instructions. The cytokines levels were obtained as pg ml-1 using a standard curve constructed using the recombinant cytokines standards provided by the kits and are reported as mean value ± standard error of the mean for n=3. Scanning electron microscopy (SEM). After 24±2 h cell culture, the NFC films were removed from the wells, carefully washed with PBS and dehydrated through a series of ethanol concentrations (25, 50, 70, 80, 90 and 100% (v/v)), followed by incubations with HMDS solutions (HMDS : ethanol 1:2, HMDS : ethanol 2:1 and 100% HMDS). After dehydration, samples were air-dried and cell number and morphology were evaluated using a SEM instrument (Leo 1550, Zeiss, Germany).

2.5 Statistical analysis The data were analyzed by one way ANOVA with LSD and Tamhane post hoc test using IBM SPSS statistics v.19. Normal distribution was evaluated by Shapiro-Wilk test and equal variances were evaluated by Levenes’s homogeneity of variance test. p value < 0.05 was considered statistically significant. Data are presented as the mean ± the standard error of the mean for n = 3.

3. Results and discussion

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3.1 Characterization During NFC production different functional groups can be introduced on the fiber surface through the pretreatment processes. In the present study, carboxymethylation and EPTMAC pretreatments

were

applied

in

order

to

obtain

carboxymethylated-NFC

and

hydroxypropyltrimethylammonium-NFC, respectively. The quantification of the surface groups on the pulp fibers confirmed the incorporation of carboxymethyl and HPTMA groups on a-NFC and c-NFC, respectively, and showed higher surface group density for c-NFC than for a-NFC (Table 1). NFC obtained after mild enzymatic hydrolysis pretreatment is herein referred as unmodified NFC (u-NFC), reflecting that no specific surface groups were introduced during the production process. As expected, u-NFC presented low carboxyl group content, most probably due to the presence of residual hemicellulose.

Table 1: Surface charge groups content of the different NFC types under study

Surface charged groups (mmol g-1) a b

u-NFC

a-NFC

c-NFC

0.03a

0.53a

1.60b

carboxyl groups hydroxypropyltrimethylammonium groups

We have previously reported a detailed characterization of the types of NFC films under the present study and showed that they present similar degrees of crystallinity. The introduction of charges onto the NFC fibers resulted in non-porous films with densely packed fibers and lower specific surface areas compared to the unmodified films.44

3.2 Indirect cytotoxicity test

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The potential release of toxic products from the NFC films was investigated by indirect cytotoxicity tests. Figure 1 shows the viability of THP-1 monocytes cultured in extract medium of the different NFC films for 24 h. Cell viability for u-NFC, a-NFC and c-NFC extracts were (108±2)%, (105±2)% and (99±3)% of the negative control, respectively. Since these values are well-above the toxicity limit of 70% defined by the ISO standard50 with 95% confidence interval, the NFC films can be regarded as non-cytotoxic to the THP-1 cell line, independently of the pretreatment employed during NFC production.

Figure 1. Cell viability of THP-1 monocytes cultured with extracts of NFC samples. The data are expressed as percentage of the negative control (TCP extract medium). Culture medium containing 5% DMSO served as positive control. Data represent the mean ± SE for n=3. Cell viability values greater than 70% of the negative control indicate a non-cytotoxic effect. 3.3 THP-1 monocytes response to NFC films We analyzed the response of THP-1 cells to u-NFC, a-NFC and c-NFC in the presence and absence of LPS stimulus by evaluating cell adhesion, morphology and cytokine secretion.

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THP-1 cells are non-adherent cells unless they are exposed to the right stimulus, e.g. when differentiated into macrophages or interacting with artificial surfaces. As expected, THP-1 monocytes cultured on TMX (a standard cell culture material) presented low cell adhesion. However, when THP-1 monocytes were cultured in the presence of the NFC films, cells notably adhered to the NFC material surfaces and no significant difference was found between cell adhesion on u-NFC, a-NFC and c-NFC (Figure 2, (-)LPS). We refer to THP-1 monocytes cultured for 24 h on the materials surfaces as monocytes/macrophages (MMs), reflecting that most probably they represent a mixture of monocytes and differentiated macrophages.

Figure 2. Adherent THP-1 cells on NFC films and TMX. Data represent the mean ± SE for n=3. The notations (-)LPS and (+)LPS indicate absence and presence, respectively, of LPS in the culture medium. * denotes statistically significant difference (p < 0.05).

SEM micrographs depicted in Figure 3 (left panels) show THP-1 cell adhesion patterns on the different NFC films and TMX, confirming the higher cell adhesion on u-NFC, a-NFC and 12 ACS Paragon Plus Environment

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c-NFC as compared to on TMX. When studying the morphology of the adherent cells, mainly rounded single cells were observed on c-NFC and TMX, while cells on a-NFC tended to form clusters and presented many, short filipodia (Figure 4, left panels). Cells cultured on u-NFC presented both single cells and small clusters, with few and short filipodia (Figure 4, left panels).

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Figure 3. Representative SEM micrographs at ~300X magnification of THP-1 monocytes cultured for 24 h on u-NFC, a-NFC, c-NFC and TMX, in the presence (right panels) and absence of LPS (left panels). Cells adhered in similar amount on the surface of u-NFC, a-NFC and c-NFC, independently of LPS treatment. Few cells were found on the surface of TMX. 14 ACS Paragon Plus Environment

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Figure 4. Representative SEM micrographs at ~1kX magnification of THP-1 monocytes cultured for 24 h on u-NFC, a-NFC, c-NFC and TMX in the presence (right panels) and absence of LPS (left panels). Mainly rounded single cells are found on c-NFC and TMX. Cells on a-NFC tended to form clusters and presented many, short filipodia while u-NFC presented both single cells and small cell clusters, with few and short filipodia. 15 ACS Paragon Plus Environment

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We evaluated whether the different NFC films could influence the secretion of cytokines by MMs by measuring the levels of the pro-inflammatory cytokine TNF-α and the antiinflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) in culture supernatant after 24 h of culture. First, the possibility that cytokine production could be a consequence of endotoxin contamination in the NFC samples was ruled out by measuring the cytokine levels in the presence of PMB. In such experiments THP-1 cells were cultured on the NFC films in the presence of 25 µg ml-1 PMB, a concentration that was shown to inhibit TNF-α secretion in LPS stimulated THP-1 monocytes (Figure 5). Results showed that there was no significant difference in TNF-α secretion by cells cultured on NFC films in the presence or absence of PMB (Figure 5), thus confirming that cytokine production is a sole consequence of cellmaterial interactions and not due to material endotoxin contamination.

Figure 5. TNF-α level secreted by THP-1 monocytes on NFC films in the presence and absence of PMB. Cells cultured on TMX with and without LPS served as positive and negative control, respectively. When PMB was added to the positive control, the secretion of TNF-α was reduced to a level comparable to the one found for the negative control. There 16 ACS Paragon Plus Environment

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was no significant difference in TNF-α secretion by cells cultured on NFC films in the presence or absence of PMB, showing that cytokine secretion is not a consequence of material LPS contamination. * denotes statistically significant difference (p < 0.05).

Figure 6a ((-)LPS) shows TNF-α levels secreted by THP-1 cells cultured on the different NFC films. Significantly higher levels were found for a-NFC and u-NFC when compared with the control TMX. Connected to this it should be noted that Schutte et al. established that tissue culture plate was suitable as a “blank” for inducing cytokine production from MMs.52 c-NFC presented TNF-α levels that were not significantly different from those found for TMX, while the corresponding levels for u-NFC were significantly higher than for c-NFC but lower than for a-NFC. Thus, in terms of TNF-α secretion, a-NFC was shown to be the most pro-inflammatory NC-based film, followed by u-NFC. c-NFC did not promote secretion of significant levels of the pro-inflammatory cytokine TNF-α. To evaluate the anti-inflammatory properties of the NFC films, we studied the secretion of IL-10 and IL-1ra. IL-10 levels were below the detection limit of the ELISA kit (11 pg ml-1) (results not shown). For IL-1ra, significantly higher levels were obtained for cells cultured on a-NFC compared with u-NFC, c-NFC and the control TMX (Figure 6b, (-)LPS). u-NFC induced higher levels than c-NFC, while cells cultured on c-NFC behaved similarly to those cultured on TMX in terms of secretion of the anti-inflammatory cytokine.

Figure 6: Cytokines secreted by THP-1 monocytes cultured on NFC films and TMX for 24h. 17 ACS Paragon Plus Environment

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a) TNF-α levels, b) IL-1ra levels. The notations (-)LPS and (+)LPS indicate absence and presence, respectively, of LPS in the culture medium. Data represent the mean ± SE for n=3. * denotes statistically significant difference (p < 0.05).

It should be mentioned that LDH release was measured in culture supernatants after 24h culture to reassure that the measured levels of cytokines were not a consequence of passive release due to disruption of cell membrane. The results showed that the LDH activity was low for the NFC films culture supernatants and comparable to the values obtained with cells cultured on TMX (results not shown). Thus, the significantly higher levels of cytokines found for the u-NFC and a-NFC films as compared to the control are a consequence of cell activation and not due to material toxicity. These results also confirm the non-toxic profile of the NFC films when cells are directly exposed to the materials. We choose not to normalize the levels of cytokines by the number of adherent cells since THP-1 monocytes are not adherent cells and especially in the experiments involving TMX most of them will remain in suspension and might also be responsible for the observed cytokine levels.53 In the experiments with the NFC films cell-material transient contact may activate the cells. Moreover, TNF-α is released over time and some of the adherent cells might detach from the surfaces during the culture period 54, 55 but will still have contributed to the measured levels of cytokines. Therefore, the contribution of non-adherent cells to the secretion of cytokines cannot be dismissed. To summarize, a-NFC and u-NFC films promote the activation of MMs to a proinflammatory state, with the extent of activation connected with the latter being smaller, while cells cultured on c-NFC behave in a similar manner as cells cultured on the control TMX, i.e. showing no signs of inflammatory activation. Even though a-NFC and u-NFC presented significantly higher levels of IL-1ra compared to the control TMX, this is most probably an effect of a feedback loop taking place to limit the inflammatory response initiated by the materials, rather than a direct effect of the NFC films.56 Therefore, it can be concluded that 18 ACS Paragon Plus Environment

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none of the materials was able to directly activate the MM cells towards an anti-inflammatory response. We also studied the response of THP-1 monocytes to the NFC films in the presence of LPS, i.e. after inflammatory activation. The aim was to investigate if, under an inflammatory stimulus, the NFC films could influence the cell response and maybe redirect cell activation to an anti-inflammatory state. When THP-1 cells were stimulated with LPS, no significant difference in cell adhesion pattern was found compared with unstimulated cells (Figures 2 and 3). However, a significant increase in the levels of TNF-α was observed for all studied materials when comparing LPS treated cells with the unstimulated condition (Figure 6a). In the presence of LPS, a-NFC was the material that promoted secretion of the highest levels of TNF-α followed by u-NFC and TMX, while c-NFC was the material that induced the lowest levels of the pro-inflammatory cytokine (Figure 6a, (+)LPS). The levels of IL-1ra in the presence of LPS were also significantly higher for all studied materials compared to those obtained in the experiments in the absence of LPS (Figure 6b). LPS stimulates MMs to produce IL-1ra

57

so the higher levels of IL-1ra in TMX/LPS

conditions compared to the unstimulated control were expected. The high levels of IL-1ra found for a-NFC and u-NFC are most probably a response to the pro-inflammatory state produced by the material (Figure 6a, (+)LPS). No detectable levels of IL-10 were found for the studied materials in the presence of LPS. LPS stimulation is expected to induce IL-10 production in monocytes as a control mechanism (e.g. to suppress TNF-α production).58 However, to drive such mechanism, IL-10 needs to bind to its receptor, which may cause a decrease in the unbound IL-10 (i.e. the measurable IL10 in the culture supernatant).59 Cell behavior on c-NFC under LPS stimulus deserves special attention. In this case, cells seemed to have a mild response to the LPS stimulus, maintaining the same adhesion pattern in terms of number and morphology and secreting cytokines at levels below those found for cells 19 ACS Paragon Plus Environment

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cultured on the control TMX/LPS. The fact that the levels of cytokines found for c-NFC are low correlates very well with the cell morphology of adherent cells; they appeared rounded and did not seem to interact with the material surface. Therefore, the possibility that the low detected levels were due to the adsorption of the cytokines on the material surface can be dismissed. Moreover, the material surfaces are rapidly covered by a layer of serum proteins, limiting the available surface for cytokine adsorption. It can also be hypothesized that the cationic surface of the c-NFC films partially binds and neutralizes the anionic LPS (acting in a similar way that the cationic decapeptide PMB), thus moderating the secretion of TNF-α. The present work showed that NFC films with different surface chemistry promote distinct MM responses, reflected by morphological changes of adherent MMs and the secretion levels of cytokines. MMs cultured on a-NFC were activated towards a pro-inflammatory phenotype, while u-NFC promoted a mild activation of the studied cells. However, the presence of HPTMA groups on the NFC fibers (c-NFC) did not promote the activation of MMs, showing that c-NFC closely behaves as an “inert” material in terms of MM activation. The same tendency was found when MMs were stimulated with LPS. The effect of surface chemistry on MM interaction with biomaterials has been widely investigated; however the mechanisms by which chemical functional groups affect the adhesion and/or activation of MMs by artificial surfaces are not yet fully understood.55, 60, 61 Some authors claim that the immune reaction to biomaterials is independent of the surface chemistry.52, 62, 63 Schutte et al. stated that macrophages may respond to any non-specific array of protein in a similar fashion, even though material surface chemistry will influence the protein adsorption.52 On the other hand, it is believed that the materials surface chemistry may dictate patterns of MM behavior by an indirect effect, i.e. by determining the type, amount and conformation of the adsorbed proteins which in turn dictate the behavior of MMs,55, 60, 64 or by a direct effect of the material surface functional groups on the activation of MMs.65

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We hypothesize that the different surface chemistry of the NFC films is the main reason behind the differences found in MM-material interactions. Previous material characterization data showed that the incorporation of cationic and anionic surface groups on the NFC fibers resulted in NFC films with decreased surface area and with nanofibers more densely packed compared to the unmodified material.44 However, no major differences were found between the two types of charged materials besides the presence of the ionic groups44 and the surface group density (Table 1). The presence of HPTMA groups on the NFC films seems to have a passivating effect on MM activation. The cell-material interactions did not promote the secretion of proinflammatory cytokines. Interestingly, the number of adherent cells on c-NFC was comparable with that on the u-NFC and a-NFC, showing that cell adhesion and activation did not correlate with each other. Although previous dogma has indicated that macrophage activation proportionally correlates with macrophage adhesion, several authors have presented evidence that challenged this.66-68 Young et al. proposed that adhesion and activation of monocytes on a biomaterial surface are mediated by different ligand-receptor interactions.68 We hypothesize that MMs found a protein layer on c-NFC that promoted interactions with the adhesion receptors of the THP-1 cells but did not favor the interaction with the activation receptors. On the other hand we can assume that the surface chemistry on a-NFC and u-NFC resulted in different composition and/or conformation of adsorbed proteins which promoted both adhesion and activation of the cells. In the presence of an activating stimulus (LPS) MMs reacted differently to the different NFC films, i.e. the material surface chemistry promoted distinct cytokine profiles in response to the pro-inflammatory stimulus, thus indicating a predominant effect of the material surface chemistry. Other authors have studied the effect of NFC on MMs and found no evidence of inflammatory effects when cells were exposed to different concentrations of NFC gels.41, 42, 69 21 ACS Paragon Plus Environment

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The materials investigated in such studies and in the present work differ in their structure (gels vs. films) and functionalization of the nanofibers. Thus, highlighting that not only the material surface chemistry (as it has been shown in the present work), but also the material structure could result in drastic changes in the way MMs interact with NFC materials.

4. Conclusions NFC films have the ability to direct MM activity depending on the surface chemical groups of the nanofibers. The introduction of carboxymethyl groups resulted in NFC films that promoted inflammation, while the presence of HPTMA groups passivated the surface of the NFC films in terms of inflammatory response. These results may constitute the fundamentals for the design of future NFC-based materials with the ability to direct MM activity depending on the surfaced chemical groups of the nanofibers.

AUTHOR INFORMATION Corresponding Author ‡

E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The project was funded by FORMAS, The Bo Rydin Foundation and The Olle Engkvist Byggmästare Foundation.

ACKNOWLEDGMENTS 22 ACS Paragon Plus Environment

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The cell studies were performed at the BioMat platform, Science for Life Laboratory, Uppsala University. K.H. thanks the China Scholarship Council (CSC) for financial support. Dr. Jonas Lindh at the Division of Nanotechnology and Functional Materials, Uppsala University is acknowledged for his help with imaging processing.

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For Table of Contents use only

Surface chemistry of nanocellulose fibers directs monocyte/macrophage response Kai Hua, Eva Ålander, Tom Lindström, Albert Mihranyan, Maria Strømme, Natalia Ferraz

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