Changes of cell membrane fluidity for mesenchymal stem cell spheroids on biomaterial surfaces.

BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs) in the form of three-dimensional spheroids has been extensively demonstrated. The underlying mechanisms for the altered cellular behavior of spheroids have also been investigated. Cell membrane fluidity is a critically important physical property for the regulation of cell behavior, but it has not been studied for the spheroid-forming cells to date. AIM: To explore the association between cell membrane fluidity and the morphological changes of MSC spheroids on the surface of biomaterials to elucidate the role of membrane fluidity during the spheroid-forming process of MSCs. METHODS: We generated three-dimensional (3D) MSC spheroids on the surface of various culture substrates including chitosan (CS), CS-hyaluronan (CS-HA), and polyvinyl alcohol (PVA) substrates. The cell membrane fluidity and cell morphological change were examined by a time-lapse recording system as well as a high-resolution 3D cellular image explorer. MSCs and normal/cancer cells were pre-stained with fluorescent dyes and co-cultured on the biomaterials to investigate the exchange of cell membrane during the formation of heterogeneous cellular spheroids. RESULTS: We discovered that vesicle-like bubbles randomly appeared on the outer layer of MSC spheroids cultured on different biomaterial surfaces. The average diameter of the vesicle-like bubbles of MSC spheroids on CS-HA at 37 °C was approximately 10 µm, smaller than that on PVA substrates (approximately 27 µm). Based on time-lapse images, these unique bubbles originated from the dynamic movement of the cell membrane during spheroid formation, which indicated an increment of membrane fluidity for MSCs cultured on these substrates. Moreover, the membrane interaction in two different types of cells with similar membrane fluidity may further induce a higher level of membrane translocation during the formation of heterogeneous spheroids. CONCLUSION: Changes in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.

Functionalized Cellulose Nanofibers as Crosslinkers to Produce Chitosan Self-Healing Hydrogel and Shape Memory Cryogel.

Cellulose nanofibers functionalized with multiple aldehyde group were synthesized as the crosslinker to produce composite self-healing hydrogel and shape memory cryogel from chitosan. The hydrogel possessed effective self-healing (∼100% efficiency) and shear-thinning properties. The cryogel had macroporous structure, large water absorption (>4300%), and high compressibility. Both hydrogel and cryogel were injectable. In particular, the cryogel (nanocellulose/chitosan 1:6) revealed thermally induced shape memory, the mechanism of which was elucidated by in situ small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) as changes in orientation of the induced crystalline structure during the shape memory program. The shape memory cryogel with a large size (15 mm × 10 mm × 1.1 mm) injected through a 16 G syringe needle was recoverable in 37 °C water. Moreover, the cryogel was cytocompatible and promoted cell growth. The nanocellulose-chitosan composite hydrogel and cryogel are injectable and degradable biomaterials with adjustable mechanical properties for potential medical applications.

Effects of hydrophilic fullerene nanoarchitectured structures on the behaviour of neural stem cells.

The interaction between nanoarchitectonic fullerenes and cells is essential for their applications in the biological field. Herein we reported the preparation and investigation of the function of different types of water-dispersible self-assembled fullerenes. The hydrophobic self-assembled fullerenes were either surface-modified or chemically etched to become water dispersible. Different types of fullerenes were then examined for their effects on the behavior of neural stem cells (NSCs). Our results indicated that only the hydrophilic fullerene nanotubes (FNTs, diameter ∼480 nm) created by chemically etching were endocytosed by NSCs, which showed a spindle-like morphology after the uptake. Meanwhile, the FNTs did not increase the reactive oxygen species (ROS) production of the cells. The expression levels of neural-related genes (CNPase and ß-tubulin) were upregulated 1.5-fold in the presence of FNTs. The differentiation of NSCs depended on the size, shape, and surface functional group of various fullerenes. Besides, the addition of FNTs in a chitosan self-healing hydrogel did not influence the integrity, injectability, and self-healing properties of the composite hydrogel. These results revealed that FNTs induced the neural differentiation of NSCs in the composite hydrogel. The addition of FNTs at a low concentration (50 µg mL-1) was enough to create such effects in the composite hydrogel. The expression levels of the oligodendrocytic marker gene CNPase and the neuronal marker gene ß-tubulin were increased remarkably by ∼14.5- and ∼8.4-fold, respectively, by the composite self-healing hydrogel containing 50 µg mL-1 FNTs. The fullerene nanoarchitectured structures may have potential for use as nanovehicles and in neural tissue engineering in the future.

Three-dimensional printing of chitosan cryogel as injectable and shape recoverable scaffolds.

Cryogel has macroporous structure and advantages of mechanical stability and injectability for biomedical applications. Three-dimensional (3D) printing is a customized manufacturing technology. However, there is little research on 3D printing of cryogel. In this work, we developed a 3D-printable chitosan cryogel using difunctional polyurethane nanoparticles as the crosslinker that reacted with chitosan at 4 °C for 4 h to form a stable feeding hydrogel (pre-cryogel) for 3D printing. The printed pre-cryogel was frozen at -20 °C to form 3D-printed chitosan cryogel. The 3D-printed cryogel had properties similar to those of bulk cryogel such as high compressibility, elastic recovery, and water absorption (≈3200%). Results from cell experiments indicated that the 3D-printed chitosan cryogel scaffolds provided good mechanical integrity for proliferation and chondrogenic differentiation of human adipose-derived adult stem cells. The 3D-printed chitosan cryogel scaffolds with injectability and shape recovery property are potential biomaterials for customized tissue engineering and minimally invasive surgery.

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration.

Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G0/G1 cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 µg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.

Revealing the Phagosomal pH Regulation and Inflammation of Macrophages after Endocytosing Polyurethane Nanoparticles by A Ratiometric pH Nanosensor.

The effect of the intracellular pH of macrophages after taking up biodegradable polymer nanoparticles (NPs) on immunomodulating functions has not been explored so far. Previous studies have demonstrated that biodegradable polyurethane (PU) NPs exhibit immunosuppressive activity. Yet, the intracellular mechanism is not clearly understood. In this study, a uniquely designed pH nanosensor is employed for tracking the intracellular pH value of macrophages to reveal the intracellular journey of PU NPs and to clarify the intracellular pH effect on the corresponding inflammatory response. First, fluorescent mesoporous silica nanoparticles (FRMSNs) is used to detect the pH change in macrophages after endo/phagocytosis of PU NPs. Second, PU is coated on the external surface of FRMSNs to examine the intracellular trafficking process of PU in the macrophages. The results show that the majority of PU-coated FRMSNs remain to stay at the cytosol-early endosome/phagosome regions. The intracellular pH value and other supporting results show that the immune response of PU NPs may be correlated to their internalization journey. The retardation in the degradation process of the PU NPs may intervene with the lysosome activity and repress the immunostimulatory effect, which contributes to the low immune response of PU NPs.

Identification of potential descriptors of water-soluble fullerene derivatives responsible for antitumor effects on lung cancer cells via QSAR analysis.

Water-soluble fullerene derivatives are actively investigated as potential drugs for cancer treatment due to their favorable membranotropic properties. Herein, cytotoxic effects of twenty fullerene derivatives with different solubilizing addends were evaluated in three different types of non-small-cell lung carcinoma (NSCLC). The potential structural descriptors of the solubilizing addends related to the inhibitory activities on each type of lung cancer cell were investigated by the quantitative structure-activity relationship (QSAR) approach. The determination coefficient r2 for the recommended QSAR model were 0.9325, 0.8404, and 0.9011 for A549, H460, and H1299 cell lines, respectively. The results revealed that the chemical features of the fullerene-based compounds including aromatic bonds, sulfur-containing aromatic rings, and oxygen atoms are favored properties and promote the inhibitory effects on H460 and H1299 cells. Particularly, thiophene moiety is the key functional group, which was positively correlated with strong inhibitory effects on the three types of lung cancer cells. The useful information obtained from our regression models may lead to the design of more efficient inhibitors of the three types of NSCLC.

Thermoresponsive and Conductive Chitosan-Polyurethane Biocompatible Thin Films with Potential Coating Application.

Conductive thin films have great potential for application in the biomedical field. Herein, we designed thermoresponsive and conductive thin films with hydrophilicity, strain sensing, and biocompatibility. The crosslinked dense thin films were synthesized and prepared through a Schiff base reaction and ionic interaction from dialdehyde polyurethane, N-carboxyethyl chitosan, and double-bonded chitosan grafted polypyrrole. The thin films were air-dried under room temperature. These thin films showed hydrophilicity and conductivity (above 2.50 mS/cm) as well as responsiveness to the deformation. The tensile break strength (9.72 MPa to 15.07 MPa) and tensile elongation (5.76% to 12.77%) of conductive thin films were enhanced by heating them from 25 °C to 50 °C. In addition, neural stem cells cultured on the conductive thin films showed cell clustering, proliferation, and differentiation. The application of the materials as a conductive surface coating was verified by different coating strategies. The conductive thin films are potential candidates for surface modification and biocompatible polymer coating.

An injectable, self-healing phenol-functionalized chitosan hydrogel with fast gelling property and visible light-crosslinking capability for 3D printing.

Self-healing hydrogels attract broad attention as cell/drug carriers for direct injection into damaged tissues or as bioinks for three-dimensional (3D) printing of tissue-like constructs. For application in 3D printing, the self-healing hydrogels should maintain the steady rheological properties during printing process, and be further stabilized by secondary post-printing crosslinking. Here, a chitosan self-healing hydrogel is developed for injectable hydrogel and printable ink using phenol-functionalized chitosan and dibenzaldehyde-terminated telechelic poly(ethylene glycol). Phenol functionalization of chitosan can introduce unique interaction that allows the hydrogel to possess fast gelling rate, good self-healing ability, and long-range critical gel behavior, as well as secondary visible light-crosslinking capability. The hydrogel is easily pre-formed in a syringe and extruded through a 26-gauge needle to produce a continuous and stackable filament. The cell-laden hydrogel is successfully printed into a 3D construct. Moreover, the hydrogel is developed for modular 3D printing, where hydrogel modules (LEGO-like building blocks) are individually printed and assembled into an integrated construct followed by secondary visible light-crosslinking. The versatile phenol-functionalized chitosan self-healing hydrogel will open up numerous potential applications, particularly in 3D bioprinting and modular 3D bioprinting.

Toward Understanding the Antitumor Effects of Water-Soluble Fullerene Derivatives on Lung Cancer Cells: Apoptosis or Autophagy Pathways?

Here we report the synthesis and investigation of anticancer effects of a series of water-soluble fullerene derivatives bearing amino acid (F1-F7) and thioacid (F8-F10) residues. Compounds F4 and F10 efficiently inhibited proliferation of lung cancer cells in vitro while being nontoxic to endothelial cells. It was revealed that the cancer cell death was caused by either autophagy (F4) or apoptosis (F10). Both fullerene derivatives strongly inhibited the tumor growth in the zebrafish xenograft model. In contrast to the vast majority of known cytostatics, fullerene derivatives do not show any significant acute toxicity effects in mice. Importantly, functional groups attached to the carbon cage affect interaction of the compounds with cancer cells, thus enabling realization of two different cell death mechanisms. The obtained results pave a way to the development of a new generation of selective antitumor drugs suppressing efficiently the proliferation of cancer cells while being nontoxic to normal cells.

Biomaterial substrate-derived compact cellular spheroids mimicking the behavior of pancreatic cancer and microenvironment.

Pancreatic stromal cells especially pancreatic stellate cells (PSCs) play a critical role in the progression of human pancreatic ductal adenocarcinoma (PDAC). However, the exact interaction between cancer cells and PSCs remains to be elucidated in order to develop more effective therapeutic approaches to treat PDAC. The microenvironment of PDAC shows higher hyaluronan (HA) levels, which is associated with poor prognosis of PDAC patients. In the current study, an efficient three-dimensional tumor spheroid model for PDAC was established. The pancreatic cancer cells and PSCs were co-cultured on hyaluronan grafted chitosan (CS-HA) coated plates to generate 3D tumor-like co-spheroids. The pancreatic cancer cells and PSCs (1:9 ratio) co-cultured on CS-HA coated plates were assembled into tumor-like co-spheroids with 3D core-shell structure in 48 h. These spheroids displayed potent in vitro tumorigenicity such as up-regulated expression of stemness and migration markers. The migration rate of cancer cells in spheroids (from 1:9 cell ratio) was much faster (3.2-fold) than that of cancer cells alone. Meanwhile, this unique co-spheroidal cancer cell structure with the outer wrap of PSCs contributed to the chemo-resistance of pancreatic cancer cells to gemcitabine as well as sensitivity to the combined gemcitabine and Abraxane treatment in vitro. The metastatic nature of the spheroids was confirmed by the zebrafish xenograft model in vivo. The compact and dynamic pancreatic cancer-PSC co-spheroids generated by the unique 3D co-culture platform on CS-HA biomaterials can mimic the PSC-constituting microenvironment of PDAC and demonstrate the chemo-resistant, invasive, and metastatic phenotypes. They have potential applications in personalized and high-throughput drug screening.

Polyurethane Nanoparticle-Loaded Fenofibrate Exerts Inhibitory Effects on Nonalcoholic Fatty Liver Disease in Mice.

Polyurethane (PU) nanoparticles are potential drug carriers. We aimed to study the in vitro and in vivo efficacy of biodegradable PU nanoparticles loaded with fenofibrate (FNB-PU) on nonalcoholic fatty liver disease (NAFLD). FNB-PU was prepared by a green process, and its preventive effects on NAFLD were investigated on HepG2 cells and mice. FNB-PU showed sustained in vitro FNB release profile. Compared to FNB crude drug, FNB-PU significantly decreased triglyceride content in HepG2 cells incubated with oleic acid and in livers of mice with NAFLD induced by a methionine choline deficient diet, and increased plasma FNB concentration of the mice. FNB-PU increased absorption of FNB and therefore enhanced the inhibitory effects of FNB on NAFLD.

A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting.

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering.

Biomaterial Substrate-Mediated Multicellular Spheroid Formation and Their Applications in Tissue Engineering.

Three-dimentional (3D) multicellular aggregates (spheroids), compared to the traditional 2D monolayer cultured cells, are physiologically more similar to the cells in vivo. So far there are various techniques to generate 3D spheroids. Spheroids obtained from different methods have already been applied to regenerative medicine or cancer research. Among the cell spheroids created by different methods, the substrate-derived spheroids and their forming mechanism are unique. This review focuses on the formation of biomaterial substrate-mediated multicellular spheroids and their applications in tissue engineering and tumor models. First, the authors will describe the special chitosan substrate-derived mesenchymal stem cell (MSC) spheroids and their greater regenerative capacities in various tissues. Second, the authors will describe tumor spheroids derived on chitosan and hyaluronan substrates, which serve as a simple in vitro platform to study 3D tumor models or to perform cancer drug screening. Finally, the authors will mention the self-assembly process for substrate-derived multiple cell spheroids (co-spheroids), which may recapitulate the heterotypic cell-cell interaction for co-cultured cells or crosstalk between different types of cells. These unique multicellular mono-spheroids or co-spheroids represent a category of 3D cell culture with advantages of biomimetic cell-cell interaction, better functionalities, and imaging possibilities.

Krüppel-like transcription factor 4 contributes to maintenance of telomerase activity in stem cells.

The zinc finger Krüppel-like transcription factor 4 (KLF4) has been implicated in cancer formation and stem cell regulation. However, the function of KLF4 in tumorigenesis and stem cell regulation are poorly understood due to limited knowledge of its targets in these cells. In this study, we have revealed a surprising link between KLF4 and regulation of telomerase that offers important insight into how KLF4 contributes to cancer formation and stem cell regulation. KLF4 sufficiently activated expression of the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), in telomerase-low alternative lengthening of telomeres (ALT), and fibroblast cells, while downregulation of KLF4 reduced its expression in cancerous and stem cells, which normally exhibits high expression. Furthermore, KLF4-dependent induction of hTERT was mediated by a KLF4 binding site in the proximal promoter region of hTERT. In human embryonic stem cells, expression of hTERT replaced KLF4 function to maintain their self-renewal. Therefore, our findings demonstrate that hTERT is one of the major targets of KLF4 in cancer and stem cells to maintain long-term proliferation potential.

Recruitment of Rad51 and Rad52 to short telomeres triggers a Mec1-mediated hypersensitivity to double-stranded DNA breaks in senescent budding yeast.

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Here we demonstrated that the budding yeast Saccharomyces cerevisiae type I survivors derived from telomerase-deficient cells were hypersensitive to DNA damaging agents. Assays to track telomere lengths and drug sensitivity of telomerase-deficient cells from spore colonies to survivors suggested a correlation between telomere shortening and bleomycin sensitivity. Our genetic studies demonstrated that this sensitivity depends on Mec1, which signals checkpoint activation, leading to prolonged cell-cycle arrest in senescent budding yeasts. Moreover, we also observed that when cells equipped with short telomeres, recruitments of homologous recombination proteins, Rad51 and Rad52, were reduced at an HO-endonuclease-catalyzed double-strand break (DSB), while their associations were increased at chromosome ends. These results suggested that the sensitive phenotype may be attributed to the sequestration of repair proteins to compromised telomeres, thus limiting the repair capacity at bona fide DSB sites.

Importin KPNA2, NBS1, DNA repair and tumorigenesis.

During the past 20 years, the MRE11-RAD50-NBS1 complex has become an increasingly important focus in basic and clinical cancer research. One main conceptual step forward was made with the discovery of NBS1 and the understanding of its critical pathophysiological role in Nijmegen breakage syndrome. Major efforts were carried out to define the role in DNA repair of this complex. Recently, basic research has continuously extended our understanding of the complexity of the NBS1 complex. MRE11-RAD50-NBS1 complex can no longer be viewed as having a single role in DNA damage repair since it also serves as a sensor and a mediator in cell cycle checkpoint signaling. Meanwhile, studies have challenged the concept that NBS1 only functions as a tumor suppressor in preserving genome integrity in the nucleus. It may also provide an oncogenic role in the cytoplasm which is associated with the PI3-kinase/AKT-activation pathway. Consistent with this aspect, a growing body of clinical evidence suggests that NBS1 contains a deleterious character that depends on its subcellular localization. This review focuses on recent experimental evidences demonstrating how NBS1 is translocated into the nucleus by an importin KPNA2 which mediates NBS1 subcellular localization and the functions of the NBS1 complex in tumorigenesis.

Telomere configuration influences the choice of telomere maintenance pathways.

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In yeast cells that lack telomerase, telomeres are maintained by alternative type I and type II recombination mechanisms. Previous studies identified several proteins to control the choice between two types of recombinations. Here, we demonstrate that configuration of telomeres also plays a role to determine the fate of telomere replication in progeny. When diploid yeasts from mating equip with a specific type of telomeric structure in their genomes, they prefer to maintain this type of telomere replication in their descendants. While inherited telomere structure is easier to be utilized in progeny at the beginning stage, the telomeres in type I diploids can gradually switch to the type II cells in liquid culture. Importantly, the TLC1/tlc1 yeast cells develop type II survivors suggesting that haploid insufficiency of telomerase RNA component, which is similar to a type of dyskeratosis congenital in human. Altogether, our results suggest that both protein factors and substrate availability contribute to the choice among telomere replication pathways in yeast.