Characterization and In Vitro Cytotoxicity Safety Screening of Fractionated Organosolv Lignin on Diverse Primary Human Cell Types Commonly Used in Tissue Engineering.

There is limited data assessing the cytotoxic effects of organosolv lignin with cells commonly used in tissue engineering. Structural and physico-chemical characterization of fractionated organosolv lignin showed that a decrease of the molecular weight (MW) is accompanied by a less branched conformation of the phenolic biopolymer (higher S/G ratio) and an increased number of aliphatic hydroxyl functionalities. Enabling stronger polymer-solvent interactions, as proven by the Hansen solubility parameter analysis, low MW organosolv lignin (2543 g/mol) is considered to be compatible with common biomaterials. Using low MW lignin, high cell viability (70-100%) was achieved after 2 h, 24 h and 7 days using the following lignin concentrations: MSCs and osteoblasts (0.02 mg/mL), gingival fibroblasts and keratinocytes (0.02 to 0.04 mg/mL), periodontal ligament fibroblasts and chondrocytes (0.02 to 0.08 mg/mL). Cell viability was reduced at higher concentrations, indicating that high concentrations are cytotoxic. Higher cell viability was attained using 30/70 (w/v) NaOH vs. 40/60 (w/v) EtOH as the initial lignin solvent. Hydrogels containing low MW lignin (0.02 to 0.3 mg/mL) in agarose dose-dependently increased chondrocyte attachment (cell viability 84-100%) and hydrogel viscosity and stiffness to 3-11 kPa, similar to the pericellular matrix of chondrocytes. This suggests that low MW organosolv lignin may be used in many tissue engineering fields.

Grafting Diels-Alder moieties on cellulose nanocrystals through carbamation.

The Diels-Alder reaction is a promising click chemistry for the design of advanced materials from cellulose nanocrystals (CNCs). Transferring such chemistry to cellulose nanocrystals requires the precise grafting of reactive Diels-Alder moeities under heterogeneous conditions without compromising the nanocrystals morphology. In this study toluene diisocyanate is used as a spacer to graft Diels-Alder moieties viz the furyl and protected maleimido moieties onto cellulose nanocrystals. A factorial experimental design reveals that reaction time and reactant molar ratio positively affect the grafting efficiency, as evidenced by FTIR and CHNS elemental analysis. The surface degree of substitution was analyzed via CHNS elemental analysis and XPS and found to range between 0.05 to 0.30, with a good agreement between the two techniques. 13C CP/MAS NMR confirmed that the grafted moieties and CNCs are intact after reaction. Side reactions were also observed and their impact on performing controllable click chemistry between cellulose nanocrystals is discussed.

Lignin-Assisted Stabilization of an Oriented Liquid Crystalline Cellulosic Mesophase, Part B: Toward the Molecular Origin and Mechanism.

Lignin valorization has been scarcely considered in the form of liquid crystalline polymer blends. Recently, a stabilizing effect of organosolv lignin (OSL) on the oriented mesophase of hydroxypropyl cellulose (HPC) was observed and related to drastic improvements in tensile properties of the blends. With a view to elucidating this relaxation phenomenon, different molecular weight fractions and derivatives of organosolv lignin are synthesized, blended in solution with the liquid crystalline cellulosic polymer and analyzed in regard to their effect on the microstructural evolution of shear-aligned HPC chains. The rheological and rheo-optical investigations suggest a crucial contribution of the lower molecular weight oligomers and the phenolic hydroxy functionalities to the stabilization of the oriented cellulosic mesophase. The results provide an indication of the molecular origin and mechanism and might be of special interest for the production of anisotropic materials from liquid crystalline cellulosic polymers.

Thermodynamic and Kinetic Study of Diels-Alder Reaction between Furfuryl Alcohol and N-Hydroxymaleimides-An Assessment for Materials Application.

The study of Diels-Alder reactions in materials science is of increasing interest. The main reason for that is the potential thermoreversibility of the reaction. Aiming to predict the behavior of a material modified with maleimido and furyl moieties, 1H NMR and UV-Vis solution studies of the Diels-Alder reaction between furfuryl alcohol and two N-hydroxymaleimides are explored in the present study. Rate constants, activation energy, entropy, and enthalpy of formation were determined from each technique for both reacting systems. Endo and exo isomers were distinguished in 1H NMR, and the transition from a kinetic, controlled Diels-Alder reaction to a thermodynamic one could be observed in the temperature range studied. A discussion on the effect of that on the application in a material was performed. The approach selected considers a simplified equilibrium of the Diels-Alder reaction as the kinetic model, allowing materials scientists to evaluate the suitability of using the reacting molecules for the creation of thermoresponsive materials. The proposed approach determines the kinetic constants without the direct influence of the equilibrium constant value, thereby allowing a more objective data analysis. The effects of the selection of kinetic model, analytical method, and data treatment are discussed.

Lignin-Assisted Stabilization of an Oriented Liquid Crystalline Cellulosic Mesophase, Part A: Observation of Microstructural and Mechanical Behavior.

Liquid crystalline polymer blends containing lignin have been scarcely studied in the literature, albeit demonstrating potential for the design of high-performance lignin-based materials. In this study, organosolv lignin is blended in solution with hydroxypropyl cellulose (HPC), a lyotropic cellulose derivative, and its impact on the dynamics of the cellulosic liquid crystalline mesophase is investigated. Rheological measurements and rheo-optical investigations under crossed polarizers reveal that lignin enhances the persistence of the shear-induced orientation of the cellulosic macromolecules. In shear-cast films, the retention of the microstructural organization or band texture entails a drastic increase of the mechanical anisotropy and properties with lignin content. For the origin of the textural stabilization, we propose a specific "jacketing"-like effect of lignin on HPC. This study indicates the possibility of a beneficial impact of lignin on the relaxation behavior of liquid crystalline cellulosic polymers.

Lignin in Bio-Based Liquid Crystalline Network Material with Potential for Direct Ink Writing.

The flow-induced supramolecular arrangement, or band texture, present in water-soluble anisotropic films prepared from blend solutions of hydroxypropyl cellulose and organosolv lignin is locked via esterification with bio-based polycarboxylic acids. Subsequent to shear casting of the blend solutions, the chemical cross-linking with citric acid-based cross-linkers and a dimerized fatty acid yields water-insoluble, anisotropic films prone to swelling in water. The liquid crystalline networks are analyzed by means of polarized optical microscopy, tensile testing, Fourier transform infrared, and swelling experiments. Depending on the cross-linker, the dry "banded" films reach up to 3.5 GPa in tensile modulus, 80 MPa in tensile strength along the shear direction, and 5 MJ/m3 toughness across the shear direction. Films are softened upon water uptake causing a reversible extinguishment of the banded texture without interfering with the specimens' anisotropy. Rheological studies point to the applicability of highly concentrated blend solutions to direct ink writing. The implementation of the findings to the additive manufacturing of cross-linked 3D structures demonstrates the potential of a resource-friendly processing of fully bio-based materials.

Direct Ink Writing of Fully Bio-Based Liquid Crystalline Lignin/Hydroxypropyl Cellulose Aqueous Inks: Optimization of Formulations and Printing Parameters.

Following the recent demonstration of the potential to direct ink write lyotropic blends of organosolv lignin (OSL) and hydroxypropyl cellulose (HPC), this study aims to optimize the formulations and direct ink writing parameters for fully bio-based lignin/HPC inks. A prescreening identifies the theoretical window of printability for different compositions for formulations based on OSL solutions of 45, 47.5, and 50% solid contents and OSL/HPC wt %/wt % ratios of 30/70, 40/60, and 50/50. Measurements of shear-viscosity and recovery behavior evidence the shear-thinning contribution of HPC and the viscosity recovery contribution of lignin. Shape fidelity, morphology, and mechanical properties of printed monolayers from the prescreened formulations reveal band texture morphology in printed parts and the best performance for the inks with a solid content of 49% and an OSL/HPC ratio of 50/50 (Young's modulus of 995 MPa, ultimate tensile strength of 18.5 MPa, and toughness of 2.8 MJ m-3). The ink composition has a more significant impact on final properties than the printing parameters, albeit a decreased printing speed of 5 mm s-1 and increased pressure of 3.5 bar contribute favorably to properties. Finally, multiple layer objects were successfully printed for compositions having solid contents of 49 and 50.5% and OSL/HPC ratios of 40/60 and 50/50.

Acetyl Groups in Typha capensis: Fate of Acetates during Organosolv and Ionosolv Pulping.

During biomass fractionation, any native acetylation of lignin and heteropolysaccharide may affect the process and the resulting lignin structure. In this study, Typha capensis (TC) and its lignin isolated by milling (MWL), ionosolv (ILL) and organosolv (EOL) methods were investigated for acetyl group content using FT-Raman, ¹H NMR, 2D-NMR, back-titration, and Zemplén transesterification analytical methods. The study revealed that TC is a highly acetylated grass; extractive free TC (TCextr) and TC MWL exhibited similar values of acetyl content: 6 wt % and 8 wt % by Zemplén transesterification, respectively, and 11 wt % by back-titration. In contrast, lignin extracted from organosolv and [EMIm][OAc] pulping lost 80% of the original acetyl groups. With a high acetyl content in the natural state, TC could be an interesting raw material in biorefinery in which acetic acid could become an important by-product.

Preparation and Chemical/Microstructural Characterization of Azacrown Ether-Crosslinked Chitosan Films.

Chemically stable porous azacrown ether-crosslinked chitosan films were prepared by reacting varying molar amounts of N,N-diallyl-7,16-diaza-1,4,10,13-tetraoxa-dibenzo-18-crown-6 (molar equivalents ranging from 0, 0.125, 0.167, 0.25 and 0.5) with chitosan. Their chemical and structural properties were characterized by solid state-nuclear magnetic resonance (NMR), elemental, Fourier transform infrared (FTIR), microscopy, and X-ray analyses, as well as gel content. NMR and FTIR analyses of the reaction products suggested that new -CH2- crosslink bridges were produced between the amine groups of chitosan (Ch) and the allyl groups of the azacrown (DAC). The crosslinking chemistry between allyl and amine groups of the reactants was further evidenced with solution NMR studies on model compound of glucosamine with the azacrown. X-ray diffraction analysis of the Ch/azacrown films using wide angle X-ray scattering (WAXS), including synchrotron-WAXS, revealed that the crystalline arrangement of chitosan (Ch) was partially destroyed with increasing grafting of azacrown ether proportion on the Ch polymer chain. Solubility and gel content determination confirmed network formation with a gel content as high as 84-95 wt %. Microstructural analysis revealed microporous morphology with high surface area. The morphology and structure of the azacrown ether-crosslinked chitosan films could be tailored by stoichiometry of the reacting species.

Imidazole, a New Tunable Reagent for Producing Nanocellulose, Part I: Xylan-Coated CNCs and CNFs.

Imidazole is reported to be an effective reactant for the production of nanocellulose from hardwood pulp. The morphologies and surface properties of the nanocellulose can be simply tailored according to the water content in the imidazole system: with pure imidazole, cellulose nanofibrils (CNFs) in a yield of 10 wt % can be produced. With 25 wt % of water in imidazole, cellulose nanocrystals (CNCs) are obtained in 20 wt % yield. Both nanocelluloses exhibit crystallinity indices in the order of 70%. Interestingly, they retain the original xylan from the pulp with ca. 9⁻10 wt % of residual xylan content.

Swelling and hydrolysis kinetics of Kraft pulp fibers in aqueous 1-butyl-3-methylimidazolium hydrogen sulfate solutions.

1Butyl-3-methylimidazolium hydrogen sulfate ([Bmim]HSO4) is efficient at extracting cellulose nanocrystals from pulp fibers. To shed some light on the respective contributions of swelling and hydrolysis of pulp fibers by [Bmim]HSO4, the physical, structural and morphological characteristics of hardwood Kraft pulp fibers were monitored under various conditions of temperature, water content and time. Swelling was largely compounded by hydrolysis at the highest temperatures (120°C) as evidenced by mass loss and reduced degree of polymerization (DPn) at this temperature. At 120°C only, water content appeared to play a significant role on the extent of hydrolysis. At this temperature, a heterogeneous kinetic model involving weak links and amorphous regions best described the experimental data. Hydrolysis rates were maximum at 25% water content in the aqueous ionic liquid.

Synthesis and Characterization of Macrocyclic Polyether N,N'-Diallyl-7,16-diaza-1,4,10,13-tetraoxa-dibenzo-18-crown-6.

In this study an efficient and direct production procedure for a macrocyclic polyether N,N'-diallyl-7,16-diaza-1,4,10,13-tetraoxa-dibenzo-18-crown-6 from the reaction of catechol and N,N-bis(2-chloroethyl)prop-2-en-1-amine in n-butanol in the presence of a strong base is reported. The synthesis involves a two-step addition of sodium hydroxide to enhance the cyclization process, and at the end of the reaction, the reaction mixture is neutralized and the solvent replaced with water in-situ through distillation to afford a relatively pure precipitate that is easily recrystallized from acetone. The yield of the macrocycle was 36%-45% and could be scaled-up to one-mole quantities. The structure and purity of this compound was verified on the basis of elemental analysis, IR, UV-Vis, ¹H-, (13)C-NMR, 2D-NMR, mass spectroscopy, and thermal analysis. The white crystalline compound has a sharp melting point of 124 °C and a crystallization temperature of 81.4 °C determined by differential scanning calorimetry. Our motivation behind the synthesis of the bibracchial lariat azacrown polyether ligand was to examine its possible applications in ion-selective polymer-supported materials.

Ionic liquid-mediated technology to produce cellulose nanocrystals directly from wood.

We report for the first time the direct extraction of cellulose nanocrystals (CNCs) from wood by means of a 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]) treatment. A native cellulosic product could be recovered in 44% yield with respect to wood cellulose content. The product was analyzed for morphological (TEM, AFM, XRD), chemical (FTIR, (13)C CP/MAS NMR), thermal (DSC, TGA) and surface properties (Zeta potential, contact angle). These analyses evidenced the presence of partially acetylated (surface DS=0.28) nanocrystals of native cellulose I microstructure, with a crystallinity index of about 75% and aspect ratio of 65. Direct production of CNCs from wood is ascribed to the simultaneous capability of [EMIM][OAc] to (1) dissolve lignin in situ while only swelling cellulose, (2) decrease intermolecular cohesion in wood via acetylation, and (3) to catalyze cellulose hydrolysis.

Role of Cellulose Nanocrystals on the Microstructure of Maleic Anhydride Plasma Polymer Thin Films.

Recently, it was shown that the microstructure of a maleic anhydride plasma polymer (MAPP) could be tailored ab initio by adjusting the plasma process parameters. In this work, we aim to investigate the ability of cellulose nanocrystals (CNCs) to induce topographical structuration. Thus, a new approach was designed based on the deposition of MAPP on CNCs model surfaces. The nanocellulosic surfaces were produced by spin-coating the CNC suspension on a silicon wafer substrate and on a hydrophobic silicon wafer substrate patterned with circular hydrophilic microsized domains (diameter of 86.9 ± 4.9 µm), resulting in different degrees of CNC aggregation. By depositing the MAPP over these surfaces, it was possible to observe that the surface fraction of nanostructures increased from 20% to 35%. This observation suggests that CNCs can act as nucleation points resulting in more structures, although a critical density of the CNCs is required.

Cellulose nanocrystals' production in near theoretical yields by 1-butyl-3-methylimidazolium hydrogen sulfate ([Bmim]HSO4)-mediated hydrolysis.

We report on near theoretical yield production of cellulose I nanocrystals (CNCs) using a two-step hydrolysis with the mildly acidic ionic liquid (IL) 1-butyl-3-methylimidazolium hydrogen sulfate ([Bmim]HSO4) in aqueous solution from common cellulosic sources. Two successive Taguchi experimental plans were performed to evaluate the impact of selected reaction parameters (T, t, H2O:IL ratio) and their interactions on the CNCs' yield from bleached softwood kraft pulp (SWP), bleached hardwood kraft pulp (HWP) and microcrystalline cellulose (MCC). With these experimental plans, the molar yield for extraction of nanocrystals was optimized to near theoretical levels, reaching 57.7±3.0%, 57.0±2.0%, and 75.6±3.0%, for SWP, HWP and MCC, respectively. The reaction yields corresponded to a relative crystalline region recovery of 84.1±5.3%, 71.7±1.3%, 76.0±2.0% from SWP, HWP and MCC, respectively. The collected nanocrystals exhibited high aspect ratios (36-43), negligible sulfur content (0.02-0.21%) and high solvent dispersibility in comparison to those obtained with the traditional sulfuric acid method. Additionally these near theoretical yields were achieved for mild reaction conditions with the combined severity factor of 2 and 3 for MCC and pulp, respectively. Overall this two-stage IL-mediated preparation of nanocrystals combines the advantages of achieving high product quality, high reaction yields and mild conditions.

Substitution pattern elucidation of hydroxypropyl Pinus pinaster (Ait.) bark polyflavonoid derivatives by ESI(-)-MS/MS.

The structure of condensed tannins (CTs) from Pinus pinaster bark extract and their hydroxypropylated derivatives with four degrees of substitution (DS 1, 2, 3 and 4) has been characterized for the first time using negative-ion mode electrospray ionization tandem mass spectrometry (ESI(-)-MS/MS). The results showed that P. pinaster bark CTs possess structural homogeneity in terms of monomeric units (C(15), catechin). The oligomer sizes were detected to be dimers to heptamers. The derivatives showed typical phenyl-propyl ether mass fragmentation by substituent elimination (58 amu) and inherent C(15) flavonoid fissions. The relative abundance of the product ions revealed a preferential triple, tetra-/penta- and octa- hydroxypropylation substitution pattern in the monomer, dimer and trimer derivatives, respectively. A defined order of -OH reactivity towards propylene oxide was established by means of multistage experiments (A-ring ≥ B-ring > C-ring). A high structural heterogeneity of the modified oligomers was detected.

Rheological properties and tunable thermoplasticity of phenolic rich fraction of pyrolysis bio-oil.

In this work we report on the preparation, characterization, and properties of a thermally treated lignin-derived, phenolic-rich fraction (PRF) of wood pyrolysis bio-oil obtained by ethyl acetate extraction. The PRF was characterized for viscoelastic and rheological behavior using dynamic mechanical analysis (DMA) and cone and plate rheology. A unique thermoplastic behavior was evidenced. Heat-treated PRFs acquire high modulus but show low temperatures of thermal flow which can be systematically manipulated through the thermal pretreatment. Loss of volatiles, changes in molecular weight, and glass transition temperature (Tg) were investigated using thermogravimetric analysis (TGA), mass spectrometry (MS), and differential scanning calorimetry (DSC), respectively. Underlying mechanisms for the thermal and rheological behavior are discussed with regard to interactions between pyrolytic lignin nanoparticles present in the system and the role of volatile materials on determining the properties of the material resembling in several aspects to colloidal suspension systems. Low thermal flow temperatures and reversible thermal effects can be attributed to association of pyrolytic lignin particles due to intermolecular interactions that are easily ruptured at higher temperatures. The thermoplastic behavior of PRF and its low Tg is of particular interest, as it gives opportunities for application of this fraction in several melt processing and adhesive technologies.

Cross-linked chitosan/chitin crystal nanocomposites with improved permeation selectivity and pH stability.

This study is aimed at developing and characterizing cross-linked bionanocomposites for membrane applications using chitosan as the matrix, chitin nanocrystals as the functional phase, and gluteraldehyde as the cross-linker. The nanocomposites' chemistry and morphology were examined by estimation of gel content, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and atomic force microscopy (AFM), whereby the occurrence of cross-linking and nanoscale dispersion of chitin in the matrix was confirmed. Besides, cross-linking and chitin whiskers content were both found to impact the water uptake mechanism. Cross-linking provided dimensional stability in acidic medium and significantly decreased the equilibrium water uptake. Incorporation of chitin nanocrystals provided increased permeation selectivity to chitosan in neutral and acidic medium.

Carbon-13 cross-polarization magic-angle-spinning nuclear magnetic resonance investigation of the interactions between maleic anhydride grafted polypropylene and wood polymers.

The chemical interactions between maleic anhydride grafted polypropylene (MAPP) and wood were studied with solid-state carbon-13 cross-polarization magic-angle-spinning nuclear magnetic resonance ((13)C CPMAS NMR) spectroscopy. MAPP was synthesized with 100% (13)C enrichment at the C(1) and C(4) carbons to allow detection of the [1,4-(13)C(2)]MAPP functional groups and was melt blended with cellulose, lignin, and maple wood. In the cellulose/MAPP blend, changes in (13)C CPMAS NMR corrected signal intensities for the anhydride and dicarboxylic maleic acid functionalities suggested that esterification may have occurred predominantly from the more numerous diacid carbons. A single proton longitudinal relaxation in the rotating frame, (H)T(1rho), for the MAPP and the cellulose carbons in the blend suggested that they were spin coupled, i.e., homogeneous on a 10-200 Angstrom scale. Esterification was also suggested in the lignin/MAPP blend. Furthermore, the more significant changes in the intensities of the carbonyl signals and (H)T(1rho) values suggested that lignin may be more reactive to MAPP than cellulose. Finally, when maple was melt blended with MAPP, the same trends in the (13)C CP-MAS NMR spectra and (H)T(1rho) behavior were observed as when MAPP was blended with cellulose or lignin. This study therefore clarifies that during melt compounding of wood with MAPP, esterification occurs with wood polymers, preferentially with lignin. Understanding the interactions of MAPP with wood is of significance for the development of natural-fiber-reinforced thermoplastic composites.

Bioengineering bacterial cellulose/poly(ethylene oxide) nanocomposites.

By adding poly(ethylene oxide) (PEO) to the growth medium of Acetobacter xylinum, finely dispersed bacterial cellulose (BC)/PEO nanocomposites were produced in a wide range of compositions and morphologies. As the BC/PEO w/w ratio increased from 15:85 to 59:41, the cellulose nanofibers aggregated in larger bundles, indicating that PEO mixed with the cellulose on the nanometer scale [corrected]. Fourier transform infrared spectroscopy suggested intermolecular hydrogen bonding and also preferred crystallization into cellulose Ibeta in the BC/PEO nanocomposites. The fine dispersion of cellulose nanofibers hindered the crystallization of PEO, lowering its melting point and crystallinity in the nanocomposites although remaining bacterial cell debris also contributed to the melting point depression. The decomposition temperature of PEO also increased by approximately 15 degrees C, and the tensile storage modulus of PEO improved significantly especially above 50 degrees C in the nanocomposites. It is argued that this integrated manufacturing approach to fiber-reinforced thermoplastic nanocomposites affords a good flexibility for tailoring morphology and properties. These results further pose the question of the necessity to remove bacterial cells to achieve desirable materials properties in biologically derived products.