Cellulase-assisted platelet-rich plasma release from nanofibrillated cellulose hydrogel enhances wound healing.

Platelet-rich plasma (PRP) is a source of growth factors, which are implicated in active tissue regeneration. However, after transplantation the efficacy of these bioactive compounds is often diminished due to rapid degradation and untargeted localization. For this reason, we evaluated the potential of nanofibrillated cellulose (NFC) hydrogel as a PRP carrier. NFC hydrogel is an animal-free biomaterial that, when doped with cellulase, can assist the release of PRP in a wound site. In this study, we examined the effects of 0.5% (m/v) NFC hydrogel formulations, including PRP and cellulase, on the migration and proliferation of skin cells via an in vitro scratch wound model. The suitability of the 0.8% NFC hydrogel formulations for accelerated wound healing and PRP carrying was studied in vitro in diffusion studies and in vivo in a full-thickness excisional wound model in SKH1 mice. None of the NFC hydrogel formulations with or without PRP and cellulase disturbed the normal cell behavior in vitro, and cellulase was successfully used to degrade NFC. NFC hydrogel slowed fibroblast migration rate in vitro. In vivo, NFC hydrogel treatment showed significantly enhanced re-epithelialization compared to control and supported collagen deposition. In addition, angiogenesis was significantly induced via PRP release after degrading NFC hydrogel with cellulase without abnormal host reaction. This study demonstrates the potential of NFC hydrogel with cellulase as a carrier for PRP with controlled release in future skin tissue engineering applications.

Stiffness-Controlled Hydrogels for 3D Cell Culture Models.

Nanofibrillated cellulose (NFC) hydrogel is a versatile biomaterial suitable, for example, for three-dimensional (3D) cell spheroid culturing, drug delivery, and wound treatment. By freeze-drying NFC hydrogel, highly porous NFC structures can be manufactured. We freeze-dried NFC hydrogel and subsequently reconstituted the samples into a variety of concentrations of NFC fibers, which resulted in different stiffness of the material, i.e., different mechanical cues. After the successful freeze-drying and reconstitution, we showed that freeze-dried NFC hydrogel can be used for one-step 3D cell spheroid culturing of primary mesenchymal stem/stromal cells, prostate cancer cells (PC3), and hepatocellular carcinoma cells (HepG2). No difference was observed in the viability or morphology between the 3D cell spheroids cultured in the freeze-dried and reconstituted NFC hydrogel and fresh NFC hydrogel. Furthermore, the 3D cultured spheroids showed stable metabolic activity and nearly 100% viability. Finally, we applied a convolutional neural network (CNN)-based automatic nuclei segmentation approach to automatically segment individual cells of 3D cultured PC3 and HepG2 spheroids. These results provide an application to culture 3D cell spheroids more readily with the NFC hydrogel and a step towards automatization of 3D cell culturing and analysis.

Angiogenic Potential of Human Adipose-Derived Mesenchymal Stromal Cells in Nanofibrillated Cellulose Hydrogel.

Adipose-derived mesenchymal stromal cells (ASCs) hold great potential for cellular therapies by having immunomodulatory behavior and tissue regenerative properties. Due to the capability of ASCs to differentiate into endothelial cells (ECs) and other angiogenic cell types, such as pericytes, ASCs are a highly valuable source for stimulating angiogenesis. However, cellular therapies in tissue engineering have faced challenges in poor survival of the cells after transplantation, which is why a protective biomaterial scaffold is required. In this work, we studied the potential of nanofibrillated cellulose (NFC) hydrogel to be utilized as a suitable matrix for three-dimensional (3D) cell culturing of human-derived ASCs (hASCs) and studied their angiogenic properties and differentiation potential in ECs and pericytes. In addition, we tested the effect of hASC-conditioned medium and stimulation with angiopoietin-1 (Ang-1) on human umbilical vein endothelial cells (HUVECs) to induce blood vessel-type tube formation in NFC hydrogel. The hASCs were successfully 3D cell cultured in NFC hydrogel as they formed spheroids and had high cell viability with angiogenic features. Most importantly, they showed angiogenic potential by having pericyte-like characteristics when differentiated in EC medium, and their conditioned medium improved HUVEC viability and tube formation, which recalls the active paracrine properties. This study recommends NFC hydrogel for future use as an animal-free biomaterial scaffold for hASCs in therapeutic angiogenesis and other cell therapy purposes.

Angiogenic Effects and Crosstalk of Adipose-Derived Mesenchymal Stem/Stromal Cells and Their Extracellular Vesicles with Endothelial Cells.

Adipose-derived mesenchymal stem/stromal cells (ASCs) are an adult stem cell population able to self-renew and differentiate into numerous cell lineages. ASCs provide a promising future for therapeutic angiogenesis due to their ability to promote blood vessel formation. Specifically, their ability to differentiate into endothelial cells (ECs) and pericyte-like cells and to secrete angiogenesis-promoting growth factors and extracellular vesicles (EVs) makes them an ideal option in cell therapy and in regenerative medicine in conditions including tissue ischemia. In recent angiogenesis research, ASCs have often been co-cultured with an endothelial cell (EC) type in order to form mature vessel-like networks in specific culture conditions. In this review, we introduce co-culture systems and co-transplantation studies between ASCs and ECs. In co-cultures, the cells communicate via direct cell-cell contact or via paracrine signaling. Most often, ASCs are found in the perivascular niche lining the vessels, where they stabilize the vascular structures and express common pericyte surface proteins. In co-cultures, ASCs modulate endothelial cells and induce angiogenesis by promoting tube formation, partly via secretion of EVs. In vivo co-transplantation of ASCs and ECs showed improved formation of functional vessels over a single cell type transplantation. Adipose tissue as a cell source for both mesenchymal stem cells and ECs for co-transplantation serves as a prominent option for therapeutic angiogenesis and blood perfusion in vivo.

Clinical Study of Nanofibrillar Cellulose Hydrogel Dressing for Skin Graft Donor Site Treatment.

Objective: Skin graft donor site management is a concern particularly for elderly patients and patients with poor wound healing competence, and also because donor sites are a source of pain and discomfort. Although different types of dressings exist, there is no consensus regarding optimal dressing type on donor site care to promote healing, reduce pain, and improve patients' comfort. Approach: This prospective, single-center clinical trial evaluated the performance of nanofibrillar cellulose (NFC) wound dressing (FibDex® by UPM-Kymmene Corporation) for treatment of donor sites compared with a polylactide-based copolymer dressing. The study enrolled 24 patients requiring skin grafting with mean age of 49 ± 18. The primary outcome measure was wound healing time. Secondary outcomes, the epithelialization, subjective pain, the scar appearance assessed using the Patient and Observer Scar Assessment Scale (POSAS), and skin elasticity and transepidermal water loss (TEWL), were evaluated at 1 and 6 months postoperatively. Results: No statistically significant differences were observed between NFC and copolymer dressings regarding wound healing time, epithelialization, experience of pain, or TEWL. Significant differences were observed in the POSAS results for thickness and vascularity in the Observer score, in the favor of NFC over copolymer dressing. Moreover, skin elasticity was significantly improved with NFC dressing in terms of viscoelasticity and elastic modulus at 1 month postoperatively. Innovation: NFC dressing is a new, green sustainable product for wound treatment without animal or human-origin components. Conclusion: NFC dressing provides efficient wound healing at skin graft donor sites and is comparable or even preferable compared with the copolymer dressing.

Nanofibrillar cellulose wound dressing supports the growth and characteristics of human mesenchymal stem/stromal cells without cell adhesion coatings.

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.

Effects of nanofibrillated cellulose hydrogels on adipose tissue extract and hepatocellular carcinoma cell spheroids in freeze-drying.

The aim of this study was to evaluate the effects of two nanofibrillated cellulose (NFC) hydrogels on two human derivatives during freeze-drying. Native NFC hydrogel is a suitable platform to culture 3D cell spheroids and a hydrogel processed further, called anionic NFC (ANFC) hydrogel, is an excellent platform for controlled release of proteins. Moreover, it has been shown to be compatible with freeze-drying when correct lyoprotectants are implemented. Freeze-drying is a method, where substance is first frozen, and then vacuum dried trough sublimation of water in order to achieve dry matter without the loss of the original three-dimensional structures. The first chosen human derivative was adipose tissue extract (ATE) which is a cell-free growth factor-rich preparation capable of promoting growth of regenerative cells. The release of growth factors from the freeze-dried mixture of ATE and ANFC was compared to that of non-freeze-dried control mixtures. The release profiles remained at the same level after freeze-drying. The second derivative was hepatocellular carcinoma (HepG2) cell spheroids which were evaluated before and after freeze-drying. The 3D structure of the HepG2 cell spheroids was preserved and the spheroids retained 18% of their metabolic activity after rehydration. However, the freeze-dried and rehydrated HepG2 cell spheroids did not proliferate and the cell membrane was damaged by fusion and formation of crystals.

Nanofibrillar cellulose hydrogels and reconstructed hydrogels as matrices for controlled drug release.

Concentrated 3% and 6.5% anionic nanofibrillar cellulose (ANFC) hydrogels were introduced as matrix reservoirs for controlled delivery applications of small molecules and proteins. A further aim was to study how the freeze-drying and subsequent rehydration of ANFC hydrogel affects the rheological properties and drug release of selected model compounds from the reconstructed hydrogels. It was demonstrated that the 3% and 6.5% ANFC hydrogels can be freeze-dried with suitable excipients into highly porous aerogel structures and redispersed back into the hydrogel form without significant change in the rheological properties. Freeze-drying did not affect the drug release properties from redispersed ANFC hydrogels, indicating that these systems could be stored in the dry form and only redispersed when needed. For large molecules, the diffusion coefficients were significantly smaller when higher ANFC fiber content was used, indicating that the amount of ANFC fibers in the hydrogel can be used to control the release rate. The release of small molecules was controlled with the ANFC fiber content only to a moderate extent. The results indicate that ANFC hydrogel can be used for controlled delivery of several types of molecules and that the hydrogel can be successfully freeze-dried and redispersed.

MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line.

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

Hepatocyte growth factor activator inhibitor-1 is induced by bone morphogenetic proteins and regulates proliferation and cell fate of neural progenitor cells.

BACKGROUND: Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner. CONCLUSIONS: This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of a potential value in stem cell therapies in various brain diseases.

Interferon-gamma produced by microglia and the neuropeptide PACAP have opposite effects on the viability of neural progenitor cells.

Inflammation is part of many neurological disorders and immune reactions may influence neuronal progenitor cells (NPCs) contributing to the disease process. Our knowledge about the interplay between different cell types in brain inflammation are not fully understood. It is important to know the mechanisms and factors involved in order to enhance regeneration and brain repair. We show here that NPCs express receptors for interferon-gamma (IFNgamma), and IFNgamma activates the signal transducer and activator of transcription (STAT) protein-1. IFNgamma reduced cell proliferation in NPCs by upregulation of the cell cycle protein p21 as well as induced cell death of NPCs by activating caspase-3. Studies of putative factors for rescue showed that the neuropeptide, Pituitary adenylate cyclase-activating polypeptide (PACAP) increased cell viability, the levels of p-Bad and reduced caspase-3 activation in the NPCs. Medium from cultured microglia contained IFNgamma and decreased the viability of NPCs, whilst blocking with anti-IFNgamma antibodies counteracted this effect. The results show that NPCs are negatively influenced by IFNgamma whereas PACAP is able to modulate its action. The interplay between IFNgamma released from immune cells and PACAP is of importance in brain inflammation and may affect the regeneration and recruitment of NPCs in immune diseases. The observed effects of IFNgamma on NPCs deserve to be taken into account in human anti-viral therapies particularly in children with higher rates of brain stem cell proliferation.