Nanocellulose, and particularly nanofibrillated cellulose (NFC), has been proposed for a diversity of applications in industry and in the biomedical field. Its unique physicochemical and structural features distinguish nanocellulose from traditional materials and enable its use as an advance nanomaterial. However, its nanoscale features may induce unknown biological responses. Limited studies with NFC are available and the biological impacts of its use have not been thoroughly explored. This study assesses the in vitro biological responses elicited by wood-derived NFC gels, when human dermal fibroblasts, lung MRC-5 cells and THP-1 macrophage cells are exposed to the nanomaterial. Furthermore, whether the presence of surface charged groups (i.e. carboxymethyl and hydroxypropyltrimethylammonium groups) on NFC can induce distinct biological responses is investigated. The introduction of surface charged groups resulted in individual nanofibrils, while fibril aggregates predominated in the unmodified NFC gel suspensions as observed by transmission electron microscopy. In the presence of proteins, the surface modified NFCs formed compact agglomerates while the agglomeration pattern of the unmodified NFC was similar in the presence of proteins and in physiological buffer. Unmodified and modified NFC gels did not induce cytotoxicity in human dermal fibroblasts, lung and macrophage cells. No significant ROS production by THP-1 macrophages was found and no cellular uptake was observed. However, an inflammatory response was detected when THP-1 macrophages were treated with unmodified NFC as assessed by an increase in TNF-α and IL1-β levels, an effect that was absent when surface charged groups were introduced into NFC. Taken together, the data presented here show the absence of cytotoxic effects associated with the exposure to unmodified, carboxymethylated and hydroxypropyltrimethylammonium-modified NFCs. Unmodified NFC presented a pro-inflammatory effect which can be further moderated by introducing surface modifications such as carboxymethyl and hydroxypropyltrimethylammonium groups into the nanofibrils. The present findings suggest that the inflammatory response to NFC might be driven by the material surface chemistry, and thus open up for the possibility of designing safe nanocellulose materials.

The influence of nanostructure on the cytocompatibility of cellulose films is analyzed providing insight into how physicochemical properties of surface modified microfibrillated cellulose (MFC) and Cladophora nanocellulose (CC) affect the materials cytocompatibility. CC is modified through TEMPO-mediated oxidation and glycidyltrimethylammonium chloride (EPTMAC) condensation to obtain anionic and cationic nanocellulose samples respectively, while anionic and cationic MFC samples are obtained by carboxymethylation and EPTMAC condensation respectively. Films of unmodified, anionic and cationic MFC and CC are prepared by vacuum filtration and characterized in terms of specific surface area, pore size distribution, degree of crystallinity, surface charge and water content. Human dermal fibroblasts are exposed to culture medium extracts of the films in an indirect contact cytotoxicity test. Moreover, cell adhesion and viability are evaluated in a direct contact assay and the effects of the physicochemical properties on cell behavior are discussed. In the indirect cytotoxicity test no toxic leachables are detected, evidencing that the CC and MFC materials are non-cytotoxic, independently of the chemical treatment that they have been subjected to. The direct contact tests show that carboxymethylated-MFC presents a more cytocompatible profile than unmodified and trimethylammonium-MFC. TEMPO–CC promotes fibroblast adhesion and presents cell viability comparable to the results obtained with the tissue culture material Thermanox. We hypothesize that the distinct aligned nanofiber structure present in the TEMPO–CC films is responsible for the improved cell adhesion. Thus, by controlling the surface properties of cellulose nanofibers, such as chemistry, charge, and orientation, cell adhesion properties can be promoted.