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.

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.

Personalized tissue-engineered veins - long term safety, functionality and cellular transcriptome analysis in large animals.

Tissue engineering is a promising methodology to produce advanced therapy medicinal products (ATMPs). We have developed personalized tissue engineered veins (P-TEV) as an alternative to autologous or synthetic vascular grafts utilized in reconstructive vein surgery. Our hypothesis is that individualization through reconditioning of a decellularized allogenic graft with autologous blood will prime the tissue for efficient recellularization, protect the graft from thrombosis, and decrease the risk of rejection. In this study, P-TEVs were transplanted to vena cava in pig, and the analysis of three veins after six months, six veins after 12 months and one vein after 14 months showed that all P-TEVs were fully patent, and the tissue was well recellularized and revascularized. To confirm that the ATMP product had the expected characteristics one year after transplantation, gene expression profiling of cells from P-TEV and native vena cava were analyzed and compared by qPCR and sequencing. The qPCR and bioinformatics analysis confirmed that the cells from the P-TEV were highly similar to the native cells, and we therefore conclude that P-TEV is functional and safe in large animals and have high potential for use as a clinical transplant graft.

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.

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.

The Effect of Hypoxic and Normoxic Culturing Conditions in Different Breast Cancer 3D Model Systems.

The field of 3D cell cultures is currently emerging, and material development is essential in striving toward mimicking the microenvironment of a native tissue. By using the response of reporter cells to a 3D environment, a comparison between materials can be assessed, allowing optimization of material composition and microenvironment. Of particular interest, the response can be different in a normoxic and hypoxic culturing conditions, which in turn may alter the conclusion regarding a successful recreation of the microenvironment. This study aimed at determining the role of such environments to the conclusion of a better resembling cell culture model to native tissue. Here, the breast cancer cell line MCF7 was cultured in normoxic and hypoxic conditions on patient-derived scaffolds and compared at mRNA and protein levels to cells cultured on 3D printed scaffolds, Matrigel, and conventional 2D plastics. Specifically, a wide range of mRNA targets (40), identified as being regulated upon hypoxia and traditional markers for cell traits (cancer stem cells, epithelial-mesenchymal transition, pluripotency, proliferation, and differentiation), were used together with a selection of corresponding protein targets. 3D cultured cells were vastly different to 2D cultured cells in gene expression and protein levels on the majority of the selected targets in both normoxic and hypoxic culturing conditions. By comparing Matrigel and 3DPS-cultured cells to cells cultured on patient-derived scffolds, differences were also noted along all categories of mRNA targets while specifically for the GLUT3 protein. Overall, cells cultured on patient-derived scaffolds closely resembled cells cultured on 3D printed scaffolds, contrasting 2D and Matrigel-cultured cells, regardless of a normoxic or hypoxic culturing condition. Thus, these data support the use of either a normoxic or hypoxic culturing condition in assays using native tissues as a blueprint to optimize material composition.

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.

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.

Characterization of cell-free breast cancer patient-derived scaffolds using liquid chromatography-mass spectrometry/mass spectrometry data and RNA sequencing data.

Patient-derived scaffolds (PDSs) generated from primary breast cancer tumors can be used to model the tumor microenvironment in vitro. Patient-derived scaffolds are generated by repeated detergent washing, removing all cells. Here, we analyzed the protein composition of 15 decellularized PDSs using liquid chromatography-mass spectrometry/mass spectrometry. One hundred forty-three proteins were detected and their relative abundance was calculated using a reference sample generated from all PDSs. We performed heatmap analysis of all the detected proteins to display their expression patterns across different PDSs together with pathway enrichment analysis to reveal which processes that were connected to PDS protein composition. This protein dataset together with clinical information is useful to investigators studying the microenvironment of breast cancers. Further, after repopulating PDSs with either MCF7 or MDA-MB-231 cells, we quantified their gene expression profiles using RNA sequencing. These data were also compared to cells cultured in conventional 2D conditions, as well as to cells cultured as xenografts in immune-deficient mice. We investigated the overlap of genes regulated between these different culture conditions and performed pathway enrichment analysis of genes regulated by both PDS and xenograft cultures compared to 2D in both cell lines to describe common processes associated with both culture conditions. Apart from our described analyses of these systems, these data are useful when comparing different experimental model systems. Downstream data analyses and interpretations can be found in the research article "Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment" [1].

Structural Analysis of Experimentally Induced Disc Herniation-Like Changes in the Rat.

INTRODUCTION: A disc herniation has traditionally been considered as disc tissue that has slipped out from an intervertebral disc. However, it was recently suggested that the disc herniation mass is a product of bioactive substances from the disc and that the disc hernia would more likely be scar tissue than herniated disc material. In this study, we aimed to analyze the structural components of experimentally induced disc herniations and compare with scar tissue and nucleus pulposus, in the rat. METHODS: Twenty-eight rats had their L4-5 discs punctured. After three weeks, the nodule that had been formed over the puncture site, scar tissue from the spine musculature, and normal nucleus pulposus were harvested and processed for further analysis. RESULTS: Proteomics analysis demonstrated that the formed nodule was more similar to scar tissue than to nucleus pulposus. Gene expression analysis showed that there was no resemblance between any tissues when looking at inflammatory genes but that, there was a clear resemblance between the nodule and scar tissue when analyzing extracellular matrix-related genes. Analysis of the GAG and polysaccharide size distribution revealed that only the nodule and scar tissue contained the shorter versions, potentially short chain hyaluronic acid that is known to induce inflammatory responses. The hematoxylin and eosin stained sections of the nodule, disc tissue, and scar tissue indicated that the morphology of the nodule and scar tissue was very similar. CONCLUSIONS: The nodule formed after experimental disc puncture, and that resembles a disc hernia, has a more structural resemblance to scar tissue than disc tissue. The nodule is, therefore, more likely to be induced by disc-derived bioactive substances than being formed by herniated disc material.

Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment.

Tumor cells interact with the microenvironment that specifically supports and promotes tumor development. Key components in the tumor environment have been linked to various aggressive cancer features and can further influence the presence of subpopulations of cancer cells with specific functions, including cancer stem cells and migratory cells. To model and further understand the influence of specific microenvironments we have developed an experimental platform using cell-free patient-derived scaffolds (PDSs) from primary breast cancers infiltrated with standardized breast cancer cell lines. This PDS culture system induced a series of orchestrated changes in differentiation, epithelial-mesenchymal transition, stemness and proliferation of the cancer cell population, where an increased cancer stem cell pool was confirmed using functional assays. Furthermore, global gene expression profiling showed that PDS cultures were similar to xenograft cultures. Mass spectrometry analyses of cell-free PDSs identified subgroups based on their protein composition that were linked to clinical properties, including tumor grade. Finally, we observed that an induction of epithelial-mesenchymal transition-related genes in cancer cells growing on the PDSs were significantly associated with clinical disease recurrences in breast cancer patients. Patient-derived scaffolds thus mimics in vivo-like growth conditions and uncovers unique information about the malignancy-inducing properties of tumor microenvironment.