Characterization of carboxylated cellulose nanofibrils and oligosaccharides from Kraft pulp fibers and their potential elicitor effect on the gene expression of Capsicum annuum.

Biomass-derived oligo- and polysaccharides may act as elicitors, i.e., bioactive molecules that trigger plant immune responses. This is particularly important to increase the resistance of plants to abiotic and biotic stresses. In this study, cellulose nanofibrils (CNF) gels were obtained by TEMPO-mediated oxidation of unbleached and bleached kraft pulps. The molecular structures were characterized with ESI and MALDI MS. Analysis of the fine sequences was achieved by MS and MS/MS of the water-soluble oligosaccharides obtained by acid hydrolysis of the CNF gels. The analysis revealed the presence of two families: one corresponding to homoglucuronic acid sequences and the other composed by alternating glucose and glucuronic acid units. The CNF gels, alone or with the addition of the water-soluble oligosaccharides, were tested on Chili pepper (Capsicum annuum). Based on the characterization of the gene expression with Next Generation Sequencing (NGS) of the C. annuum's total messenger RNA, the differences in growth of the C. annuum seeds correlated well with the downregulation of the pathways regulating photosynthesis. A downregulation of the response to abiotic factors was detected, suggesting that these gels would improve the resistance of the C. annuum plants to abiotic stress due to, e.g., water deprivation and cold temperatures.

Sustainable Materials from Organosolv Fibers and Lignin, Kraft Fibers, and Their Blends.

The aim of this study was to investigate new materials from organosolv fibers, organosolv lignin, kraft fibers, and their blends. The organosolv fibers showed reprecipitated lignin on the surface, a comparably low fiber length of 0.565 mm on average, and a high fines content of 82.3%. Handsheets were formed and thermopressed at 175 °C and 50 MPa, yielding dense materials (1050-1100 kg/m3) with properties different to that of regular paper products. The thermopressing of organosolv fibers alone produced materials with similar or better tensile strength (σb = 18.6 MPa) and stiffness (E* = 2.8 GPa) to the softwood Kraft reference pulp (σb = 14.8 MPa, E* = 1.8 GPa). The surface morphology was also smoother with fewer cavities. As a result, the thermopressed organosolv fibers exhibited higher hydrophobicity (contact angle > 95°) and had the lowest overall water uptake. Combinations of Kraft fibers with organosolv fibers or organosolv lignin showed reduced wetting and a higher density than the Kraft fibers alone. Furthermore, the addition of organosolv lignin to Kraft fibers greatly improved tensile stiffness and strength (σb = 23.8 MPa, E* = 10.5 GPa), likely due to the lignin acting as a binder to the fiber network. In conclusion, new thermopressed materials were developed and tested, which show promising potential for sustainable fiber materials with improved water resistance.

The Effect of Cellulose Nanofibres on Dewatering during Wet-Forming and the Mechanical Properties of Thermoformed Specimens Made of Thermomechanical and Kraft Pulps.

Due to environmental concerns regarding single-use plastic materials, major efforts are being made to develop new material concepts based on biodegradable and renewable resources, e.g., wood pulp. In this study, we assessed two types of wood pulp fibres, i.e., thermomechanical pulp (TMP) and Kraft pulp fibres, and tested the performance of the fibres in wet-moulding and thermopressing trials. Kraft pulp fibres appeared to retain more water than TMP, increasing the dewatering time during wet-moulding and apparently increasing the compression resistance of the pulp during thermoforming. Additionally, cellulose nanofibres (CNF) were added to the pulps, which improved the mechanical properties of the final thermopressed specimens. However, the addition of CNF to the pulps (from 2 to 6%) had a further decrease in the dewatering efficiency in the wet-moulding process, and this effect was more pronounced in the Kraft pulp specimens. The mechanical performance of the thermoformed specimens was in the same range as the plastic materials that are conventionally used in food packaging, i.e., modulus 0.6-1.2 GPa, strength 49 MPa and elongation 6-9%. Finally, this study demonstrates the potential of wood pulps to form three-dimensional thermoformed products.

Biocomposites containing poly(lactic acid) and chitosan for 3D printing - Assessment of mechanical, antibacterial and in vitro biodegradability properties.

New bone repair materials are needed for treatment of trauma- and disease-related skeletal defects as they still represent a major challenge in clinical practice. Additionally, new strategies are required to combat orthopedic device-related infections (ODRI), given the rising incidence of total joint replacement and fracture fixation surgeries in increasingly elderly populations. Recently, the convergence of additive manufacturing (AM) and bone tissue engineering (BTE) has facilitated the development of bone healthcare to achieve personalized three-dimensional (3D) scaffolds. This study focused on the development of a 3D printable bone repair material, based on the biopolymers poly(lactic acid) (PLA) and chitosan. Two different types of PLA and chitosan differing in their molecular weight (MW) were explored. The novel feature of this research was the successful 3D printing using biocomposite filaments composed of PLA and 10 wt% chitosan, with clear chitosan entrapment within the PLA matrix confirmed by Scanning Electron Microscopy (SEM) images. Tensile testing of injection molded samples indicated an increase in stiffness, compared to pure PLA scaffolds, suggesting potential for improved load-bearing characteristics in bone scaffolds. However, the potential benefit of chitosan on the biocomposite stiffness could not be reproduced in compression testing of 3D printed cylinders. The antibacterial assays confirmed antibacterial activity of chitosan when dissolved in acetic acid. The study also verified the biodegradability of the scaffolds, with a process producing an acidic environment that could potentially be neutralized by chitosan. In conclusion, the study indicated the feasibility of the proposed PLA/chitosan biocomposite for 3D printing, demonstrating adequate mechanical strength, antibacterial properties and biodegradability, which could serve as a new material for bone repair.

Polysaccharides and Structural Proteins as Components in Three-Dimensional Scaffolds for Breast Cancer Tissue Models: A Review.

Breast cancer is the most common cancer among women, and even though treatments are available, efficiency varies with the patients. In vitro 2D models are commonly used to develop new treatments. However, 2D models overestimate drug efficiency, which increases the failure rate in later phase III clinical trials. New model systems that allow extensive and efficient drug screening are thus required. Three-dimensional printed hydrogels containing active components for cancer cell growth are interesting candidates for the preparation of next generation cancer cell models. Macromolecules, obtained from marine- and land-based resources, can form biopolymers (polysaccharides such as alginate, chitosan, hyaluronic acid, and cellulose) and bioactive components (structural proteins such as collagen, gelatin, and silk fibroin) in hydrogels with adequate physical properties in terms of porosity, rheology, and mechanical strength. Hence, in this study attention is given to biofabrication methods and to the modification with biological macromolecules to become bioactive and, thus, optimize 3D printed structures that better mimic the cancer cell microenvironment. Ink formulations combining polysaccharides for tuning the mechanical properties and bioactive polymers for controlling cell adhesion is key to optimizing the growth of the cancer cells.

Carboxylated nanocellulose for wound healing applications - Increase of washing efficiency after chemical pre-treatment and stability of homogenized gels over 10 months.

To commercialize a biomedical product as a medical device, reproducibility of production and time-stability are important parameters. Studies of reproducibility are lacking in the literature. Additionally, chemical pre-treatments of wood fibres to produce highly fibrillated cellulose nanofibrils (CNF) seem to be demanding in terms of production efficiency, being a bottleneck for industrial upscaling. In this study, we evaluated the effect of pH on the dewatering time and washing steps of 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO)-mediated oxidized wood fibres when applying 3.8 mmol NaClO/g cellulose. The results indicate that the method does not affect the carboxylation of the nanocelluloses, and levels of approximately 1390 µmol/g were obtained with good reproducibility. The washing time of a Low-pH sample was reduced to 1/5 of the time required for washing a Control sample. Additionally, the stability of the CNF samples was assessed over 10 months and changes were quantified, the most pronounced were the increase of potential residual fibre aggregates, reduction of viscosity and increase of carboxylic acid content. The cytotoxicity and skin irritation potential were not affected by the detected differences between the Control and Low-pH samples. Importantly, the antibacterial effect of the carboxylated CNFs against S. aureus and P. aeruginosa was confirmed.

Functional surfaces, films, and coatings with lignin - a critical review.

Lignin is the most abundant polyaromatic biopolymer. Due to its rich and versatile chemistry, many applications have been proposed, which include the formulation of functional coatings and films. In addition to replacing fossil-based polymers, the lignin biopolymer can be part of new material solutions. Functionalities may be added, such as UV-blocking, oxygen scavenging, antimicrobial, and barrier properties, which draw on lignin's intrinsic and unique features. As a result, various applications have been proposed, including polymer coatings, adsorbents, paper-sizing additives, wood veneers, food packaging, biomaterials, fertilizers, corrosion inhibitors, and antifouling membranes. Today, technical lignin is produced in large volumes in the pulp and paper industry, whereas even more diverse products are prospected to be available from future biorefineries. Developing new applications for lignin is hence paramount - both from a technological and economic point of view. This review article is therefore summarizing and discussing the current research-state of functional surfaces, films, and coatings with lignin, where emphasis is put on the formulation and application of such solutions.

Gene-Expression Analysis of Human Fibroblasts Affected by 3D-Printed Carboxylated Nanocellulose Constructs.

Three-dimensional (3D) printing has emerged as a highly valuable tool to manufacture porous constructs. This has major advantages in, for example, tissue engineering, in which 3D scaffolds provide a microenvironment with adequate porosity for cell growth and migration as a simulation of tissue regeneration. In this study, we assessed the suitability of three cellulose nanofibrils (CNF) that were obtained through 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO)-mediated oxidation. The CNFs were obtained by applying three levels of carboxylation, i.e., 2.5, 3.8, and 6.0 mmol sodium hypochlorite (NaClO) per gram of cellulose. The CNFs exhibited different nanofibrillation levels, affecting the corresponding viscosity and 3D printability of the CNF gels (0.6 wt%). The scaffolds were manufactured by micro-extrusion and the nanomechanical properties were assessed with nanoindentation. Importantly, fibroblasts were grown on the scaffolds and the expression levels of the marker genes, which are relevant for wound healing and proliferation, were assessed in order to reveal the effect of the 3D-scaffold microenvironment of the cells.

Influence of Compounding Parameters on the Tensile Properties and Fibre Dispersion of Injection-Moulded Polylactic Acid and Thermomechanical Pulp Fibre Biocomposites.

Thermomechanical pulp (TMP) fibres can serve as renewable, cost-efficient and lightweight reinforcement for thermoplastic polymers such as poly(lactic acid) (PLA). The reinforcing ability of TMP fibres can be reduced due to various factors, e.g., insufficient dispersion of the fibres in the matrix material, fibre shortening under processing and poor surface interaction between fibres and matrix. A two-level factorial design was created and PLA together with TMP fibres and an industrial and recyclable side stream were processed in a twin-screw microcompounder accordingly. From the obtained biocomposites, dogbone specimens were injection-moulded. These specimens were tensile tested, and the compounding parameters statistically evaluated. Additionally, the analysis included the melt flow index (MFI), a dynamic mechanical analysis (DMA), scanning electron microscopy (SEM) and three-dimensional X-ray micro tomography (X-µCT). The assessment provided insight into the microstructure that could affect the mechanical performance of the biocomposites. The temperature turned out to be the major influence factor on tensile strength and elongation, while no significant difference was quantified for the tensile modulus. A temperature of 180 °C, screw speed of 50 rpm and compounding time of 1 min turned out to be the optimal settings.

Nanocelluloses - Nanotoxicology, Safety Aspects and 3D Bioprinting.

Nanocelluloses have good rheological properties that facilitate the extrusion of nanocellulose gels in micro-extrusion systems. It is considered a highly relevant characteristic that makes it possible to use nanocellulose as an ink component for 3D bioprinting purposes. The nanocelluloses assessed in this book chapter include wood nanocellulose (WNC), bacterial nanocellulose (BNC), and tunicate nanocellulose (TNC), which are often assumed to be non-toxic. Depending on various chemical and mechanical processes, both cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC) can be obtained from the three mentioned nanocelluloses (WNC, BNC, and TNC). Pre/post-treatment processes (chemical and mechanical) cause modifications regarding surface chemistry and nano-morphology. Hence, it is essential to understand whether physicochemical properties may affect the toxicological profile of nanocelluloses. In this book chapter, we provide an overview of nanotoxicology and safety aspects associated with nanocelluloses. Relevant regulatory requirements are considered. We also discuss hazard assessment strategies based on tiered approaches for safety testing, which can be applied in the early stages of the innovation process. Ensuring the safe development of nanocellulose-based 3D bioprinting products will enable full market use of these sustainable resources throughout their life cycle.

3D Printed Nanocellulose Scaffolds as a Cancer Cell Culture Model System.

Current conventional cancer drug screening models based on two-dimensional (2D) cell culture have several flaws and there is a large need of more in vivo mimicking preclinical drug screening platforms. The microenvironment is crucial for the cells to adapt relevant in vivo characteristics and here we introduce a new cell culture system based on three-dimensional (3D) printed scaffolds using cellulose nanofibrils (CNF) pre-treated with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) as the structural material component. Breast cancer cell lines, MCF7 and MDA-MB-231, were cultured in 3D TEMPO-CNF scaffolds and were shown by scanning electron microscopy (SEM) and histochemistry to grow in multiple layers as a heterogenous cell population with different morphologies, contrasting 2D cultured mono-layered cells with a morphologically homogenous cell population. Gene expression analysis demonstrated that 3D TEMPO-CNF scaffolds induced elevation of the stemness marker CD44 and the migration markers VIM and SNAI1 in MCF7 cells relative to 2D control. T47D cells confirmed the increased level of the stemness marker CD44 and migration marker VIM which was further supported by increased capacity of holoclone formation for 3D cultured cells. Therefore, TEMPO-CNF was shown to represent a promising material for 3D cell culture model systems for cancer cell applications such as drug screening.

Biocomposite Inks for 3D Printing.

Three-dimensional (3D) printing has evolved massively during the last years and is demonstrating its potential in tissue engineering, wound dressings, cell culture models for drug testing, and prosthesis, to name a few [...].

Characterization and Antibacterial Properties of Autoclaved Carboxylated Wood Nanocellulose.

Cellulose nanofibrils (CNFs) were obtained by applying a chemical pretreatment consisting of autoclaving the pulp fibers in sodium hydroxide, combined with 2,2,6,6-tetramethylpiperidinyl-1-oxyl-mediated oxidation. Three levels of sodium hypochlorite were applied (2.5, 3.8, and 6.0 mmol/g) to obtain CNF qualities (CNF_2.5, CNF_3.8, and CNF_6.0) with varying content of carboxyl groups, that is, 1036, 1285, and 1593 µmol/g cellulose. The cytotoxicity and skin irritation potential (indirect tests) of the CNFs were determined according to standardized in vitro testing for medical devices. We here demonstrate that autoclaving (121 °C, 20 min), which was used to sterilize the gels, caused a modification of the CNF characteristics. This was confirmed by a reduction in the viscosity of the gels, a morphological change of the nanofibrils, by an increase of the ultraviolet-visible absorbance maxima at 250 nm, reduction of the absolute zeta potential, and by an increase in aldehyde content and reducing sugars after autoclaving. Fourier-transform infrared spectroscopy and wide-angle X-ray scattering complemented an extensive characterization of the CNF gels, before and after autoclaving. The antibacterial properties of autoclaved carboxylated CNFs were demonstrated in vitro (bacterial survival and swimming assays) on Pseudomonas aeruginosa and Staphylococcus aureus. Importantly, a mouse in vivo surgical-site infection model on S. aureus revealed that CNF_3.8 showed pronounced antibacterial effect and performed as good as the antiseptic Prontosan wound gel.

Optimized alginate-based 3D printed scaffolds as a model of patient derived breast cancer microenvironments in drug discovery.

The cancer microenvironment influences tumor progression and metastasis and is pivotal to consider when designingin vivo-like cancer models. Current preclinical testing platforms for cancer drug development are mainly limited to 2D cell culture systems that poorly mimic physiological environments and traditional, low throughput animal models. The aim of this work was to produce a tunable testing platform based on 3D printed scaffolds (3DPS) with a simple geometry that, by extracellular components and response of breast cancer reporter cells, mimics patient-derived scaffolds (PDS) of breast cancer. Here, the biocompatible polysaccharide alginate was used as base material to generate scaffolds consisting of a 3D grid containing periostin and hydroxyapatite. Breast cancer cell lines (MCF7 and MDA-MB-231) produced similar phenotypes and gene expression levels of cancer stem cell, epithelial-mesenchymal transition, differentiation and proliferation markers when cultured on 3DPS and PDS, contrasting conventional 2D cultures. Importantly, cells cultured on 3DPS and PDS showed scaffold-specific responses to cytotoxic drugs (doxorubicin and 5-fluorouracil) that were different from 2D cultured cells. In conclusion, the data presented support the use of a tunable alginate-based 3DPS as a tumor model in breast cancer drug discovery.

Photopolymerization of Bio-Based Polymers in a Biomedical Engineering Perspective.

Photopolymerization is an effective method to covalently cross-link polymer chains that can be shaped into several biomedical products and devices. Additionally, polymerization reaction may induce a fluid-solid phase transformation under physiological conditions and is ideal for in vivo cross-linking of injectable polymers. The photoinitiator is a key ingredient able to absorb the energy at a specific light wavelength and create radicals that convert the liquid monomer solution into polymers. The combination of photopolymerizable polymers, containing appropriate photoinitiators, and effective curing based on dedicated light sources offers the possibility to implement photopolymerization technology in 3D bioprinting systems. Hence, cell-laden structures with high cell viability and proliferation, high accuracy in production, and good control of scaffold geometry can be biofabricated. In this review, we provide an overview of photopolymerization technology, focusing our efforts on natural polymers, the chemistry involved, and their combination with appropriate photoinitiators to be used within 3D bioprinting and manufacturing of biomedical devices. The reviewed articles showed the impact of different factors that influence the success of the photopolymerization process and the final properties of the cross-linked materials.

Oxygenated Nanocellulose-A Material Platform for Antibacterial Wound Dressing Devices.

Both carboxylated cellulose nanofibrils (CNF) and dissolved oxygen (DO) have been reported to possess antibacterial properties. However, the combination for use as wound dressings against biofilm infections in chronic wounds is less known. The present study reports the development of oxygenated CNF dispersions that exhibit strong antibacterial effect. Carboxylated CNF dispersions with different oxidation levels were oxygenated by the OXY BIO System and tested for antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. The results reveal that the higher oxidation level of the CNFs, the better antibacterial effect. Scanning electron microscopy of bacterial biofilms revealed that a potential mechanism of action of the CNFs is the formation of a network surrounding and entrapping the bacteria. This effect is further potentiated by the oxygenation process. A CNF sample (concentration 0.6 wt %) that was oxygenated to a DO level of 46.4 mg/L demonstrated a strong antibacterial effect against S. aureusin vivo using a mouse model of surgical site infection. The oxygenated CNF dispersion reduced the bacterial survival by 71%, after 24 h treatment. The potent antibacterial effect indicates that oxygenated nanocellulose is a promising material for antibacterial wound dressings.

Characterization of Porous Structures of Cellulose Nanofibrils Loaded with Salicylic Acid.

Bleached and unbleached pulp fibers were treated with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) mediated oxidation to obtain cellulose nanofibrils (CNFs). The resulting bleached and unbleached CNFs were mixed with salicylic acid (0, 5, 10, 20 wt%) before casting and freeze-drying or 3D-printing. A series of methods were tested and implemented to characterize the CNF materials and the porous structures loaded with salicylic acid. The CNFs were characterized with atomic force microscopy and laser profilometry, and release of salicylic acid was quantified with UV-visible absorbance spectroscopy, conductivity measurements, and inductive coupled plasma mass spectrometry (ICP-MS). Fourier-transform infrared spectroscopy (FTIR) complemented the analyses. Herein, we show that aerogels of bleached CNFs yield a greater release of salicylic acid, compared to CNF obtained from unbleached pulp. The results suggest that biodegradable constructs of CNFs can be loaded with a plant hormone that is released slowly over time, which may find uses in small scale agricultural applications and for the private home market.

Biocomposites of Bio-Polyethylene Reinforced with a Hydrothermal-Alkaline Sugarcane Bagasse Pulp and Coupled with a Bio-Based Compatibilizer.

Bio-polyethylene (BioPE, derived from sugarcane), sugarcane bagasse pulp, and two compatibilizers (fossil and bio-based), were used to manufacture biocomposite filaments for 3D printing. Biocomposite filaments were manufactured and characterized in detail, including measurement of water absorption, mechanical properties, thermal stability and decomposition temperature (thermo-gravimetric analysis (TGA)). Differential scanning calorimetry (DSC) was performed to measure the glass transition temperature (Tg). Scanning electron microscopy (SEM) was applied to assess the fracture area of the filaments after mechanical testing. Increases of up to 10% in water absorption were measured for the samples with 40 wt% fibers and the fossil compatibilizer. The mechanical properties were improved by increasing the fraction of bagasse fibers from 0% to 20% and 40%. The suitability of the biocomposite filaments was tested for 3D printing, and some shapes were printed as demonstrators. Importantly, in a cradle-to-gate life cycle analysis of the biocomposites, we demonstrated that replacing fossil compatibilizer with a bio-based compatibilizer contributes to a reduction in CO2-eq emissions, and an increase in CO2 capture, achieving a CO2-eq storage of 2.12 kg CO2 eq/kg for the biocomposite containing 40% bagasse fibers and 6% bio-based compatibilizer.

Life cycle assessment of bagasse fiber reinforced biocomposites.

This study aims to evaluate the life cycle environmental implications of producing fiber-reinforced biocomposite pellets, compared with sugarcane- and petroleum-based polyethylene (PE) pellets. Life Cycle Assessment (LCA) methodology is used to evaluate the production of four types of pellets. LCA allows the evaluation of the benefits of improving the production of biobased materials by replacing part of the sugarcane bioPE with bagasse fibers. The functional unit selected was the production of 1 kg of plastic pellets. Primary data were collected from laboratory tests designed to obtain pulp fibers from bagasse and mix them with sugarcane bioPE. Two processes were studied to obtain fibers from bagasse: soda fractionation and hot water-soda fractionation. The results from the LCA show environmental improvements when reducing the amount of bioPE by replacing it with bagasse fibers in the categories of global warming, ozone formation, terrestrial acidification and fossil resource scarcity, when comparing to 100% sugarcane bioPE, and a reduction in global warming and fossil resource scarcity when compared to fossil-based PE. In contrast, results also indicate that there could be higher impacts in terms of ozone formation, freshwater eutrophication, and terrestrial acidification. Even though biocomposites result as a preferred option to bioPE, several challenges need to be overcome before a final recommendation is placed. The sensitivity analysis showed the importance of the energy source on the impacts of the processing of fibers. Thus, using clean energy to produce biobased materials may reduce the impacts related to the production stage. These results are intended to increase the attention of the revalorization of these residues and their application to generate more advanced materials. Further outlook should also consider a deeper evaluation of the impacts during the production of a plastic object and possible effects of the biobased materials during final disposal.

Relationship between rheological and morphological characteristics of cellulose nanofibrils in dilute dispersions.

The morphology of cellulose nanofibrils (CNFs), the rheological characteristics of their dispersions, and the corresponding relationships, are fundamental for understanding the properties of the material. This work aims at understanding how the morphological characteristics of the CNFs affect the rheology of the dispersions in the dilute region and to establish a relationship between both properties. A strong relationship was observed between the intrinsic viscosity of the CNF dispersions and their aspect ratio, which can be correlated through the expression ρ[η]=0.051p1.85. When comparing the model obtained in this work to the wormlike chain model, it was possible to verify that these models are independent of the flexibility of the CNFs. Regarding the fibrillation process, the dynamic viscosity only reflects part of the behavior of the morphological properties of the CNFs and does not provide reliable data that would allow these characteristics to be inferred, while the intrinsic viscosity does allow this relationship.