Kristin Syverud

3D Bioprinting of Carboxymethylated-Periodate Oxidized Nanocellulose Constructs for Wound Dressing Applications

Nanocellulose has a variety of advantages, which make the material most suitable for use in biomedical devices such as wound dressings. The material is strong, allows for production of transparent films, provides a moist wound healing environment, and can form elastic gels with bioresponsive characteristics. In this study, we explore the application of nanocellulose as a bioink for modifying film surfaces by a bioprinting process. Two different nanocelluloses were used, prepared with TEMPO mediated oxidation and a combination of carboxymethylation and periodate oxidation. The combination of carboxymethylation and periodate oxidation produced a homogeneous material with short nanofibrils, having widths <20 nm and lengths <200 nm. The small dimensions of the nanofibrils reduced the viscosity of the nanocellulose, thus yielding a material with good rheological properties for use as a bioink. The nanocellulose bioink was thus used for printing 3D porous structures, which is exemplified in this study. We also demonstrated that both nanocelluloses did not support bacterial growth, which is an interesting property of these novel materials.

Cytotoxicity tests of cellulose nanofibril-based structures

Cellulose nanofibrils based on wood pulp fibres are most promising for biomedical applications. Bacterial cellulose has been suggested for some medical applications and is presently used as wound dressing. However, cost-efficient processes for mass production of bacterial cellulose are lacking. Hence, fibrillation of cellulose wood fibres is most interesting, as the cellulose nanofibrils can efficiently be produced in large quantities. However, the utilization of cellulose nanofibrils from wood requires a thorough verification of its biocompatibility, especially with fibroblast cells which are important in regenerative tissue and particularly in wound healing. The cellulose nanofibril structures used in this study were based on Eucalyptus and Pinus radiata pulp fibres. The nanofibrillated materials were manufactured using a homogenizer without pre-treatment and with 2,2,6,6-tetramethylpiperidine-1-oxy radical as pre-treatment, thus yielding nanofibrils low and high level of anionic charge, respectively. From these materials, two types of nanofibril-based structures were formed; (1) thin and dense structures and (2) open and porous structures. Cytotoxicity tests were applied on the samples, which demonstrated that the nanofibrils do not exert acute toxic phenomena on the tested fibroblast cells (3T3 cells). The cell membrane, cell mitochondrial activity and the DNA proliferation remained unchanged during the tests, which involved direct and indirect contact between the nano-structured materials and the 3T3 cells. Some samples were modified using the crosslinking agent polyethyleneimine (PEI) or the surfactant cetyl trimethylammonium bromide (CTAB). The sample modified with CTAB showed a clear toxic behaviour, having negative effects on cell survival, viability and proliferation. CTAB is an antimicrobial component, and thus this result was as expected. The sample crosslinked with PEI also had a significant reduction in cell viability indicating a reduction in DNA proliferation. We conclude that the neat cellulose nanostructured materials tested in this study are not toxic against fibroblasts cells. This is most important as nano-structured materials based on nanofibrils from wood pulp fibres are promising as substrate for regenerative medicine and wound healing.

Emulsions Stabilized by Microfibrillated Cellulose: The Effect of Hydrophobization, Concentration and O/W Ratio

The properties of emulsions stabilized by nanosized cellulose particles (denominated also microfibrillated cellulose; MFC) were investigated. The wettability (hydrophobicity) of the MFC was changed through surface modification with chlorodimethyl isopropylsilane. The modified MFC was characterized with Fourier transform infrared spectroscopy (FTIR), and x-ray photoelectron spectroscopy (XPS). Using MFC of intermediate hydrophobicity resulted in water-in-oil emulsions within a range of oil/water ratios. The emulsifying capacity of the MFC was found to be proportional to both the relative amount of MFC used and its wettability (degree of surface substitution; DSS). The emulsions separated rapidly from the excess oil phase; the separated emulsion layers were highly stable towards gravity-induced sedimentation and, once a stable MFC network was formed, were also stable towards coalescence. The influence of the oil/water ratio, the concentration and DSS of the MFC on the stability of the emulsions toward coalescence, and the inversion to oil-in-water emulsions was also investigated. The results verify that MFC can be efficiently used as a stabilizer of oil/water emulsions within a range of oil/water ratios and particle concentrations.

Effects of bagasse microfibrillated cellulose and cationic polyacrylamide on key properties of bagasse paper.

This study explores the benefits of using bagasse microfibrillated cellulose (MFC) in bagasse paper. Two different types of MFC were produced from DED bleached soda bagasse pulp. The MFC was added to soda bagasse pulp furnishes in different amounts. Cationic polyacrylamide (C-PAM) was selected as retention aid. The results show that addition of MFC increased the strength of paper as expected. Interestingly, 1% MFC in combination with 0.1% C-PAM yielded similar drainage time as the reference pulp, which did not contain MFC. In addition, the samples containing 1% MFC and 0.1% C-PAM yielded (i) a significant increment of the tensile index, (ii) a minor decrease of opacity and (iii) preserved Gurley porosity. Hence, this study proves that small fractions of MFC in combination with adequate retention aids can have positive effects with respect to paper properties, which is most interesting from an industrial point of view.

On the structure and oxygen transmission rate of biodegradable cellulose nanobarriers

Cellulose nanofibrils have been proposed for novel barrier concepts, based on their capability to form smooth, strong and transparent films, with high oxygen barrier properties. A series of cellulose-based films were manufactured and tested with respect to their oxygen transmission rate (OTR) capabilities. The obtained OTR levels were considerably better than the levels recommended for packaging applications. Part of the nanofibrillated material applied in this study was produced with 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) mediated oxidation as pretreatment. Films made of TEMPO-pretreated samples yielded lower OTR values. The minimum obtained OTR value was 3.0 mL m-2 day-1 atm-1 with a corresponding oxygen permeability of 0.04 mL mm m-2 day-1 atm-1, tested at 50% relative humidity. The good barrier properties are due to the compact and dense structure of the films, as revealed by field-emission scanning electron microscopy. A relationship between OTR and the structure of the corresponding nanofibril-based films was confirmed.

Pretreatment-dependent surface chemistry of wood nanocellulose for pH-sensitive hydrogels:

Nanocellulose from wood is a promising material with potential in various technological areas. Within biomedical applications, nanocellulose has been proposed as a suitable nano-material for wound dressings. This is based on the capability of the material to self-assemble into 3D micro-porous structures, which among others have an excellent capacity of maintaining a moist environment. In addition, the surface chemistry of nanocellulose is suitable for various applications. First, OH-groups are abundant in nanocellulose materials, making the material strongly hydrophilic. Second, the surface chemistry can be modified, introducing aldehyde and carboxyl groups, which have major potential for surface functionalization. In this study, we demonstrate the production of nanocellulose with tailor-made surface chemistry, by pre-treating the raw cellulose fibres with carboxymethylation and periodate oxidation. The pre-treatments yielded a highly nanofibrillated material, with significant amounts of aldehyde and carboxyl groups. Importantly, the poly-anionic surface of the oxidized nanocellulose opens up for novel applications, i.e. micro-porous materials with pH-responsive characteristics. This is due to the swelling capacity of the 3D micro-porous structures, which have ionisable functional groups. In this study, we demonstrated that nanocellulose gels have a significantly higher swelling degree in neutral and alkaline conditions, compared to an acid environment (pH 3). Such a capability can potentially be applied in chronic wounds for controlled and intelligent release of antibacterial components into biofilms.

Structure of nanofibrillated cellulose layers at the o/w interface

The nature of layers formed by cellulose nanofibrils that had been surface modified (hydrophobized) at the oil/water (o/w) interface was investigated. The aim of the study was to clarify the mechanism underlying the excellent ability of these nanoparticles to stabilize emulsions. Layers of hydrophobized nanofibrillated cellulose spread at the o/w interface were deposited on glass slides by the Langmuir-Blodgett deposition technique. Overall evaluation of layer structures was performed by image analysis based on a Quadtree decomposition of images obtained from a flatbed scanner. A more detailed characterization of the layer structures was performed by Atomic Force Microscopy (AFM), and Field-Emission Scanning Electron Microscopy (FE-SEM). The results show that nanofibrils that were able to stabilize emulsions occur as single, dispersed fibrils or form large, network-like aggregates at the o/w interface. Fibrils that were insufficiently hydrophobized and therefore did not stabilize emulsions were only partially deposited and formed small, compact aggregates. We conclude that it is likely that the network formation is the main mechanism by which the fibrils prevent coalescence of emulsion droplets.

Controlling the elastic modulus of cellulose nanofibril hydrogels—scaffolds with potential in tissue engineering

AbstractCellulose nanofibrils (CNF) form hydrogels at low concentrations. These hydrogels are held together by transient interactions such as entanglement of fibrils, non-specific ionic interactions and hydrogen bonds; and are thus vulnerable for changes in the chemical environment or the influence of mechanical forces. By a covalent crosslinking of the fibrils, stable permanent gels can be formed. In this study we have produced CNF by using TEMPO mediated oxidation followed by fibrillation. During this procedure, carboxyl and aldehyde groups are introduced on the CNF surfaces. The aldehyde groups are suitable sites for crosslinking, as aldehydes readily form covalent bonds to primary amines through formation of Schiff bases. For this purpose the diamines ethylenediamine and hexamethylenediamine, differing with four carbon atoms in the chain, were used as crosslinker molecules. The results show that by varying the concentration and length of the crosslinker molecules, the elastic modulus of the gels could be controlled. The reversible gels were in this way transformed to irreversible gels by a simple water based reaction. Controlling gel strength is one important premise for the use of CNF in applications such as tissue engineering.

Cross-linking cellulose nanofibrils for potential elastic cryo-structured gels

Cellulose nanofibrils were produced from P. radiata kraft pulp fibers. The nanofibrillation was facilitated by applying 2,2,6,6-tetramethylpiperidinyl-1-oxyl-mediated oxidation as pretreatment. The oxidized nanofibrils were cross-linked with polyethyleneimine and poly N-isopropylacrylamide-co-allylamine-co-methylenebisacrylamide particles and were frozen to form cryo-structured gels. Samples of the gels were critical-point dried, and the corresponding structures were assessed with scanning electron microscopy. It appears that the aldehyde groups in the oxidized nanofibrils are suitable reaction sites for cross-linking. The cryo-structured materials were spongy, elastic, and thus capable of regaining their shape after a given pressure was released, indicating a successful cross-linking. These novel types of gels are considered potential candidates in biomedical and biotechnological applications.

Hemicellulose-reinforced nanocellulose hydrogels for wound healing application

Polysaccharides are finding an increasing number of applications in medical and pharmaceutical fields thanks to their biodegradability, biocompatibility, and in some cases bioactivity. Two approaches were applied to use hemicelluloses as crosslinkers to tune the structural and mechanical properties of nanofibrillated cellulose (NFC) hydrogel scaffolds, and thus to investigate the effect of these properties on the cellular behavior during wound healing application. Different types of hemicellulose (galactoglucomannan (GGM), xyloglucan (XG), and xylan) were introduced into the NFC network via pre-sorption (Method I) and in situ adsorption (Method II) to reinforce the NFC hydrogels. The charge density of the NFC, the incorporated hemicellulose type and amount, and the swelling time of the hydrogels were found to affect the pore structure, the mechanical strength, and thus the cells’ growth on the composite hydrogel scaffolds. The XG showed the highest adsorption capacity on the NFC, the highest reinforcement effect, and facilitated/promoted cell growth. The pre-sorbed XG in the low-charged NFC network with a lower weight ratio (NFC/XG-90:10) showed the highest efficacy in supporting the growth and proliferation of fibroblast cells (NIH 3T3). These all-polysaccharide composite hydrogels may work as promising scaffolds in wound healing applications to provide supporting networks and to promote cells adhesion, growth, and proliferation.