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The diversity and safety of nanofibrillated cellulose (NFC) hydrogels have gained a vast amount of interest at the pharmaceutical site in recent years. Moreover, this biomaterial has a high potential to be utilized as a protective matrix during the freeze-drying of heat-sensitive pharmaceuticals and biologics to increase their properties for long-term storing at room temperature and transportation. Since freeze-drying and subsequent reconstitution have not been optimized for this biomaterial, we must find a wider understanding of the process itself as well as the molecular level interactions between the NFC hydrogel and the most suitable lyoprotectants. Herein we optimized the reconstitution of the freeze-dried NFC hydrogel by considering critical quality attributes required to ensure the success of the process and gained insights of the obtained experimental data by simulating the effects of the used lyoprotectants on water and NFC. We discovered the correlation between the measured characteristics and molecular dynamics simulations and obtained successful freeze-drying and subsequent reconstitution of NFC hydrogel with the presence of 300 mM of sucrose. These findings demonstrated the possibility of using the simulations together with the experimental measurements to obtain a more comprehensive way to design a successful freeze-drying process, which could be utilized in future pharmaceutical applications.
Background: In the field of regenerative medicine, delivery of human adipose-derived mesenchymal stem/stromal cells (hASCs) has shown great promise to promote wound healing. However, a hostile environment of the injured tissue has shown considerably to limit the survival rate of the transplanted cells, and thus, to improve the cell survival and retention towards successful cell transplantation, an optimal cell scaffold is required. The objective of this study was to evaluate the potential use of wood-derived nanofibrillar cellulose (NFC) wound dressing as a cell scaffold material for hASCs in order to develop a cell transplantation method free from animal-derived components for wound treatment. Methods: Patient-derived hASCs were cultured on NFC wound dressing without cell adhesion coatings. Cell characteristics, including cell viability, morphology, cytoskeletal structure, proliferation potency, and mesenchymal cell and differentiation marker expression, were analyzed using cell viability assays, electron microscopy, immunocytochemistry, and quantitative or reverse transcriptase PCR. Student's t test and one-way ANOVA followed by a Tukey honestly significant difference post hoc test were used to determine statistical significance.Results: hASCs were able to adhere to NFC dressing and maintained high cell survival without cell adhesion coatings with a cell density-dependent manner for the studied period of 2 weeks. In addition, NFC dressing did not induce any remarkable cytotoxicity towards hASCs or alter the morphology, proliferation potency, filamentous actin structure, the expression of mesenchymal vimentin and extracellular matrix (ECM) proteins collagen I and fibronectin, or the undifferentiated state of hASCs.Conclusions: As a result, NFC wound dressing offers a functional cell culture platform for hASCs to be used further for in vivo wound healing studies in the future.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Background The segmentation of 3D cell nuclei is essential in many tasks, such as targeted molecular radiotherapies (MRT) for metastatic tumours, toxicity screening, and the observation of proliferating cells. In recent years, one popular method for automatic segmentation of nuclei has been deep learning enhanced marker-controlled watershed transform. In this method, convolutional neural networks (CNNs) have been used to create nuclei masks and markers, and the watershed algorithm for the instance segmentation. We studied whether this method could be improved for the segmentation of densely cultivated 3D nuclei via developing multiple system configurations in which we studied the effect of edge emphasizing CNNs, and optimized H-minima transform for mask and marker generation, respectively. Results The dataset used for training and evaluation consisted of twelve in vitro cultivated densely packed 3D human carcinoma cell spheroids imaged using a confocal microscope. With this dataset, the evaluation was performed using a cross-validation scheme. In addition, four independent datasets were used for evaluation. The datasets were resampled near isotropic for our experiments. The baseline deep learning enhanced marker-controlled watershed obtained an average of 0.69 Panoptic Quality (PQ) and 0.66 Aggregated Jaccard Index (AJI) over the twelve spheroids. Using a system configuration, which was otherwise the same but used 3D-based edge emphasizing CNNs and optimized H-minima transform, the scores increased to 0.76 and 0.77, respectively. When using the independent datasets for evaluation, the best performing system configuration was shown to outperform or equal the baseline and a set of well-known cell segmentation approaches. Conclusions The use of edge emphasizing U-Nets and optimized H-minima transform can improve the marker-controlled watershed transform for segmentation of densely cultivated 3D cell nuclei. A novel dataset of twelve spheroids was introduced to the public.
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Nanofibrillar cellulose (NFC)-derived dressings such as films, hydrogels, and aerogels are one of the favorable materials for wound healing due to their proper mechanical properties and water holding ability. However, the therapeutic differences between native and anionic NFC materials are rarely studied. In this report, we compared the differences and addressed the regenerative potential of native and anionic wood-derived NFC hydrogels for wound treatment. In vitro characteristics of the hydrogels were detected using scanning electron microscopy, rheological measurements, and swelling and hemolytic activity assays. Skin regeneration at an early stage after hydrogel treatment was analyzed using an in vivo splinted excisional full-thickness skin wound model in C57BL/6 mice. Both native NFC and anionic NFC (ANFC) hydrogel with differing mechanical and surface properties were shown to be biocompatible. Surprisingly, wounds treated with NFC and ANFC hydrogel did not show any statistical difference compared with control wounds and progressed through normal wound closure, inflammatory response, re-epithelialization, vascularization, and tissue maturation with no signs of fibrosis. The data show here for the first time the therapeutic performance of native and anionic NFC hydrogel in a wound mimicking human wound healing mechanisms. The mechanical properties of native and anionic NFC hydrogels such as the capability to modify material stiffness may also prove to be valuable in the management of wounds in the future.
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