Response Of Mesenchymal Stem Cells Research Articles

3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

The response of human mesenchymal stem cells to internal exposure to tritium β-rays.

There is no doubt that estimating the exposure risk of external and internal low-dose radiation is an imperative issue in radiobiological study. Human mesenchymal stem cells (hMSCs) are multipotent and self-renewing, supporting the regeneration of damaged tissue, including tissue damaged by radiation. However, the responses of hMSCs to internal exposure to radionuclides are still insufficiently understood. In order to evaluate the adverse effects produced by internal exposure to tritiated water (HTO) at a low dose, hMSCs were exposed to 2 × 107 Bq/ml HTO, and the biological effects after the exposure were examined. Apoptosis and DNA double-strand breaks (DSBs) were assayed to analyze the cellular response to the damage induced by HTO. Slight enhancement of apoptosis was found after treatment, except at the dose of 9 mGy. The number of DSBs at 24 h post-irradiation showed that the DNA damage was able to be efficiently repaired by the hMSCs. Moreover, the increasing proportion of the cell population in S phase proved that the persistence of residual γH2AX foci at lower concentrations of HTO was attributable to the secondary production of DSBs in DNA replication. Our work adds to the available data, helping us understand the risk of stem cell transformation due to internal exposure and its correlation with low-dose radiation–induced carcinogenesis.

Formation of γH2AX and pATM Foci in Human Mesenchymal Stem Cells Exposed to Low Dose-Rate Gamma-Radiation.

DNA double-strand breaks (DSB) are among the most harmful DNA lesions induced by ionizing radiation (IR). Although the induction and repair of radiation-induced DSB is well studied for acute irradiation, responses to DSB produced by chronic IR exposures are poorly understood, especially in human stem cells. The aim of this study was to examine the formation of DSB markers (γH2AX and phosphorylated kinase ATM, pATM, foci) in human mesenchymal stem cells (MSCs) exposed to chronic gamma-radiation (0.1 mGy/min) in comparison with acute irradiation (30 mGy/min) at cumulative doses of 30, 100, 160, 240 and 300 mGy. A linear dose-dependent increase in the number of both γH2AX and pATM foci, as well as co-localized γH2AX/pATM foci (“true” DSB), were observed after an acute radiation exposure. In contrast, the response of MSCs to a chronic low dose-rate IR exposure deviated from linearity towards a threshold model, for γH2AX, pATM foci and γH2AX/pATM foci, with an indication of a “plateau”. The state of equilibrium between newly formed DSB at a low rate during the protracted exposure time and the elimination of a fraction of DSB is proposed as a mechanistic explanation of the non-linear DSB responses following a low dose-rate irradiation. This notion is supported by the observation of the elimination of a substantial fraction of DSB 6 h after the cessation of the exposures. Our results demonstrate non-linear dose responses for γH2AX and pATM foci in human MSCs exposed to low dose-rate IR and showed the existence of a threshold, which may have implications for radiation protection in humans.

TiO2 nanotubes promote osteogenic differentiation of mesenchymal stem cells via regulation of lncRNA CCL3-AS

Titanium (Ti) is widely used as orthopedic implant materials. TiO2 nanotubes (TNTs) further improve the bioactivity of Ti, which enhance the osteogenic differentiation of mesenchymal stem cells (MSCs). However, the underlying mechanism is still unclear. In this study, we verified the response of MSCs on Ti and TNT substrates and explored the regulatory mechanism of long non-coding RNAs (lncRNAs). LncRNA and mRNA expression profiles were analyzed via RNA sequencing. Differential lncRNA and mRNA expression and predicted target genes of lncRNAs were performed by bioinformatics analysis. 1075 up-regulated and 1301 down-regulated genes, 26 up-regulated and 35 down-regulated lncRNAs were obtained according to the RNA-Seq. Expression of 8 lncRNAs were verified by qPCR, which was consistent with the sequencing data. To explore the function and target gene of lncRNA, lncRNA CCL3-AS and gene CCL3 were selected for further investigation. The fluorescence staining, alkaline phosphatase (ALP) activity and CCK-8 assay were performed. Besides, expressions of runt-related transcription factor 2 (Runx2), collagen type I (Col I), osteopontin (OPN) were detected by qPCR and western blot. These results indicate that lncRNA CCL3-AS could inhibit the osteogenic differentiation and enhance cell viability of MSCs on the TNT substrates, which was dependent on the regulation of CCL3. This study supplied a comprehensive understanding for further study using lncRNA modulators to surface design of titanium for enhancing osseointegration.

Biological responses of human bone mesenchymal stem cells to Ti and TiZr implant materials.

Titanium-zirconium alloy (TiZr1317) is a new material used for biological implants. There are several studies on the effects of TiZr implants on the biological characteristics of human bone mesenchymal stem cells (hBMSCs). The purpose of this study was to investigate the biological responses of hBMSCs to implant holes affected by the physicochemical properties of oral implants (TiSLA , TiSLActive , TiZrSLA , and TiZrSLActive ). Grade 4 Ti and TiZr (13-17% Zr) substrates were modified by sand-blasted large-grit acid-etched (SLA) or hydrophilic sand-blasted large-grit acid-etched (SLActive), resulting in four types of surface with complex microstructures corresponding to the commercially-available implants SLA, RoxolidSLA, SLActive, and RoxolidSLActive (Institute Straumann AG, Basel, Switzerland). Physicochemical properties were detected and the biological responses of hBMSCs were observed. Surface morphology characterization by scanning electron microscopy and atomic force microscopy revealed differences between the four groups. SLActive had higher surface energy/wettability than SLA, indicating that increased surface energy/wettability can promote the absorption of osteogenic proteins and enhance osseointegration. hBMSCs seeded on SLActive substrates exhibited better performance in terms of cell attachment, proliferation and osteoblastic differentiation than cells seeded on SLA. Because of their more suitable physicochemical properties, TiSLActive and TiZrSLActive materials demonstrated more pronounced effects on the biological responses of hBMSCs compared with TiSLA and TiZrSLA .

Absence of cytotoxic and inflammatory effects following in vitro exposure of chondrogenically-differentiated human mesenchymal stem cells to adenosine, lidocaine and Mg2+ solution

BackgroundALM solution, a combination of adenosine, lidocaine and Mg2+, is an emerging small volume therapy that has been shown to prevent and correct coagulopathy and surgery-related inflammation in preclinical models, though its application in orthopaedic surgery is yet to be demonstrated. The effect of ALM solution on chondrocytes is unknown. The aim of this preliminary study was to investigate the effect of ALM solution on viability and inflammatory responses of chondrogenically-differentiated human bone marrow-derived mesenchymal stem cells (chondro-MSC), in vitro.MethodsChondro-MSC were exposed to media only, saline (0.9% NaCl or 1.3% NaCl) only, or saline containing ALM (1 mM adenosine, 3 mM lidocaine, 2.5 mM Mg2+) or tranexamic acid (TXA, 100 mg/ml) for 1 or 4 h. Responses to ALM solutions containing higher lidocaine concentrations were also compared. Chondrocyte viability was determined using WST-8 colorimetric assays and inflammatory cytokine (TNF-α, IL-1β, IL-8) and matrix metalloproteinases (MMP-3, MMP-12, MMP-13) concentrations using multiplex bead arrays.ResultsThe viability of chondro-MSC was significantly greater after 1 h treatment with ALM compared to saline (96.2 ± 7.9 versus 75.6 ± 7.3%). Extension of exposure times to 4 h had no significant adverse effect on cell viability after treatment with ALM (1 h, 85.4 ± 5.6 v 4 h, 74.0 ± 15.2%). Cytotoxicity was evident following exposure to solutions containing lidocaine concentrations greater than 30 mM. There were no significant differences in viability (80 ± 5.4 v 57.3 ± 16.2%) or secretion of IL-8 (60 ± 20 v 160 ± 50 pg/ml), MMP-3 (0.95 ± 0.6 v 3.4 ± 1.6 ng/ml), and MMP-13 (4.2 ± 2.4 v 9.2 ± 4.3 ng/ml) in chondro-MSC exposed to saline, ALM or TXA.ConclusionsShort-term, in vitro exposure to clinically-relevant concentrations of ALM solution had no adverse inflammatory or chondrotoxic effects on human chondro-MSC, with responses comparable to saline and TXA. These findings provide support for continued evaluation of ALM solution as a possible therapeutic to improve outcomes following orthopaedic procedures.

Mesenchymal Stem Cells Alleviate Acute Lung Injury and Inflammatory Responses Induced by Paraquat Poisoning.

BackgroundMesenchymal stem cells (MSCs) show anti-oxidative and anti-inflammatory effects that have prompted further research into their potential applications in treating paraquat (PQ) poisoning cases in emergency rooms. We assessed the protective effects, underlying mechanisms, and secondary inflammatory responses of MSCs on PQ-induced acute lung injury.Material/MethodsSprague-Dawley rats were injected intraperitoneally with PQ (20 μg per gram of body weight). MSCs were injected through the caudal vein 1 h after PQ treatment. The severity of lung injury and oxidative stress and levels of inflammatory mediators were examined with and without MSC grafting. Expression levels of TLR4, NF-κB, p65, Nrf2, HO-1, and activated caspase-3 protein were determined by Western blotting.ResultsAdministration of MSCs significantly decreased the levels of TNF-α, IL-1β, and IL-6 and polymorphonuclear neutrophil (PMN) count in the bronchoalveolar lavage fluid (BALF) of rats with PQ-induced ALI. In addition, MSC also effectively reduced the wet-to-dry lung weight ratio, lung injury score, and the levels of MDA and 8-OHdG. Conversely, MSC increased SOD and GSH-PX activity in the lung tissue. Moreover, MSC significantly upregulated HO-1, Nrf-2 protein expression in the lung tissue. In contrast, the levels of TLR4, NF-κB p65 and activated caspase-3 protein were decreased in MSC-treated rats (P<0.05).ConclusionsTreatment with MSCs overexpressed Nrf2 gene and activated downstream antioxidant HO-1, leading to inhibit oxidative stress, cell apoptosis and inflammatory response in lung tissue, thereby significantly improving PQ-induced acute lung injury in rats.

Polysaccharide-Based Hybrid Self-Healing Hydrogel Supports the Paracrine Response of Mesenchymal Stem Cells

The aim of stem cell therapy is to repair damaged tissues. Some of the challenges facing its success include cell retention and survival at the wound site. While the retention of cells has been addressed by employing scaffolds, the survival of transplanted cells in the repair tissue is however low. It is hypothesized that the observed regeneration is more a result of migration of tissue repairing cells from adjoining tissues in response to paracrine factors secreted by implanted cells than by the implanted cells per se. In this study, we report the synthesis of a self-healing hybrid hydrogel that is injectable. The hybrid hydrogel was developed using the dynamic equilibrium of Schiff base linkage between the aldehyde groups on carboxymethyl cellulose dialdehyde (CMC-D) and amino groups on carboxymethyl chitosan (CMCh). The hydrogel stiffness and kinetics of gelation were observed to be modulated with different molecular weights of chitosan. In vitro studies demonstrated the cytocompatibility, hemocompatibility, and biodegradability of the hydrogel. The chemotactic, proliferative, and wound-healing response of cells to the paracrine factors secreted from the mesenchymal stem cell (MSC)-hydrogel composite confirmed the ability of the hydrogel to support the paracrine response of stem cells. Our results suggest that the synthesized hydrogel-MSC composite could serve as a potential scaffold for studying the in vitro response of cells to the paracrine factors released by the encapsulated cells as well as a cell delivery vehicle for in vivo applications.

Survival/Adaptation of Bone Marrow-Derived Mesenchymal Stem Cells After Long-Term Starvation Through Selective Processes.

After in vivo transplantation, mesenchymal stem cells (MSC) face an ischemic microenvironment, characterized by nutrient deprivation and reduced oxygen tension, which reduces their viability and thus their therapeutic potential. Therefore, MSC response to models of in vitro ischemia is of relevance for improving their survival and therapeutic efficacy. The aim of this study was to understand the survival/adaptive response mechanism that MSC use to respond to extreme culture conditions. Specifically, the effect of a long-term starvation on human bone marrow (hBM)-derived MSC cultured in a chemically defined medium (fetal bovine serum-free [SF] and human SF), either in hypoxic or normoxic conditions. We observed that hBM-MSC that were isolated and cultured in SF medium and subjected to a complete starvation for up to 75 days transiently changed their behavior and phenotype. However, at the end of that period, hBM-MSC retained their characteristics as determined by their morphology, DNA damage resistance, proliferation kinetic, and differentiation potential. This survival mode involved a quiescent state, confirmed by increased expression of cell cycle regulators p16, p27, and p57 and decreased expression of proliferating cell nuclear antigen (PCNA), Ki-67, mTOR, and Nanog. In addition, Jak/STAT (STAT6) antiapoptotic activity selected which cells conserved stemness and that supported metabolic, bioenergetic, and scavenging requirements. We also demonstrated that hBM-MSC exploited an autophagic process which induced lipid β-oxidation as an alternative energy source. Priming MSC by concomitant starvation and culture in hypoxic conditions to induce their quiescence would be of benefit to increase MSC survival when transplanted in vivo. Stem Cells 2019;37:813-827.

Fibre Laser Treatment of Beta TNZT Titanium Alloys for Load-Bearing Implant Applications: Effects of Surface Physical and Chemical Features on Mesenchymal Stem Cell Response and Staphylococcus aureus Bacterial Attachment

A mismatch in bone and implant elastic modulus can lead to aseptic loosening and ultimately implant failure. Selective elemental composition of titanium (Ti) alloys coupled with surface treatment can be used to improve osseointegration and reduce bacterial adhesion. The biocompatibility and antibacterial properties of Ti-35Nb-7Zr-6Ta (TNZT) using fibre laser surface treatment were assessed in this work, due to its excellent material properties (low Young’s modulus and non-toxicity) and the promising attributes of fibre laser treatment (very fast, non-contact, clean and only causes changes in surface without altering the bulk composition/microstructure). The TNZT surfaces in this study were treated in a high speed regime, specifically 100 and 200 mm/s, (or 6 and 12 m/min). Surface roughness and topography (WLI and SEM), chemical composition (SEM-EDX), microstructure (XRD) and chemistry (XPS) were investigated. The biocompatibility of the laser treated surfaces was evaluated using mesenchymal stem cells (MSCs) cultured in vitro at various time points to assess cell attachment (6, 24 and 48 h), proliferation (3, 7 and 14 days) and differentiation (7, 14 and 21 days). Antibacterial performance was also evaluated using Staphylococcus aureus (S. aureus) and Live/Dead staining. Sample groups included untreated base metal (BM), laser treated at 100 mm/s (LT100) and 200 mm/s (LT200). The results demonstrated that laser surface treatment creates a rougher (Ra value of BM is 199 nm, LT100 is 256 nm and LT200 is 232 nm), spiky surface (Rsk &gt; 0 and Rku &gt; 3) with homogenous elemental distribution and decreasing peak-to-peak distance between ripples (0.63 to 0.315 µm) as the scanning speed increases (p &lt; 0.05), generating a surface with distinct micron and nano scale features. The improvement in cell spreading, formation of bone-like nodules (only seen on the laser treated samples) and subsequent four-fold reduction in bacterial attachment (p &lt; 0.001) can be attributed to the features created through fibre laser treatment, making it an excellent choice for load bearing implant applications. Last but not least, the presence of TiN in the outermost surface oxide might also account for the improved biocompatibility and antibacterial performances of TNZT.

Microtopography based on inverse opal structures regulates the behavior of bone marrow‐derived mesenchymal stem cells

Physical cues from the extracellular microenvironment play an important role in regulating cell behavior, such as adhesion, migration, and differentiation. Many studies have shown that different physical parameters (eg, stiffness and topography) could modulate the in vitro differentiation of mesenchymal stem cells (MSCs), which had multilineage differentiation potential and could be easily isolated from various tissues such as bone marrow, adipose tissue, and the umbilical cord. However, the underlying mechanism of the topographical influence on MSCs and the detailed cell‐substrate interaction remain unclear. Here, we present oriented elliptical inverse opal structures for regulating the morphology and alignment of bone marrow‐derived MSCs. The inverse opal structures were made through a convenient bottom‐up approach of self‐assembly, which is facile and cost effective. MSCs cultured on the oriented structures were highly aligned and extended highly oriented thick lamellipodia. Moreover, the oriented substrates cracked along the lateral boundary of the cells, suggesting that a strong cell‐substrate interaction was induced by the response of MSCs to the oriented topography. These features of the oriented elliptical topography indicated their promising value in stem cell research and tissue engineering.

Adhesion and Proliferation of Human Adipose-Derived Stem Cells on Titania Nanotube Surfaces

When a biomaterial is implanted, the body reacts similar to an injury and stem cells are recruited to the implant site. Since, stem cells play an important role in tissue repair in the body, their interaction with biomaterials is critical for long-term success of medical devices. Surfaces with nanostructured features have been shown to alter cellular functionality in vitro and even improve fixation of the implant to the surrounding bone tissues in vivo. In this study, the effect of titania nanotube (NT) size and cell density on the adhesion and proliferation of human adipose-derived stem cells (ASCs) was evaluated. Although many studies have evaluated mesenchymal stem cells response on nanostructured surfaces, very few studies have explored the response of adipose-derived stem cells on titanium nanotube surfaces and none have explored the effect of cell density concurrent with nanotube size on ASC proliferation. Proliferation behavior of ASCs on NT surfaces and titanium control were investigated for a week using three different cell densities. The optimal cell density was 2500 cells/well and the smaller diameter NT exhibited higher ASC proliferation. This study confirms that NT surfaces promote ASC adhesion and proliferation. By more fully understanding the effect of nanostructure size on adhesion and proliferation of stem cells, implants could be specifically designed to achieve the optimal stem cell response from the tissue in which they are implanted. Additionally, the favorable response of ASC to these NT surfaces and determination of optimal cell density suggests a potential use in orthopedic tissue regeneration. When a biomaterial is implanted, the body reacts similar to an injury and stem cells are recruited to the implant site. Since, stem cells play an important role in tissue repair in the body, their interaction with biomaterials is critical for long-term success of medical devices. This study confirms that nanotube surfaces promote stem cell adhesion and proliferation.

Improved chondrogenic response of mesenchymal stem cells to a polyethersulfone/polyaniline blended nanofibrous scaffold.

Owing to the fact that the cartilage tissue is not able to repair itself, the treatment of the joint damages is very difficult by current methods. Induction of tissue repair requires suitable cell and extracellular matrix. Providing these two parts can only be done using tissue engineering. In the present study, polyethersulfone (PES) and polyaniline (PANI) blend was electrospined for nanofibrous scaffold fabrication. Mesenchymal stem cells were isolated from human adipose tissue (AT-MSCs), and after characterization cultured on the PES-PANI scaffold and culture plate. Electron microscopic and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assays were used for biocompatibility evaluation of the scaffold and the chondrogenic differentiation potential of AT-MSCs were investigated by staining of proteoglycans and gene and protein expression evaluation. Alcian blue staining, real-time reverse-transcriptase polymerase chain reaction and Western blot results showed that chondrogenic differentiation potential of AT-MSCs was significantly increased when grown on PES-PANI nanofibers and was compared to the one grown on a culture plate. According to the results, PES-PANI has a promising potential to be used as a biomedical implant in patients with joints lesion, such as arthritis and osteoarthritis.

Effects of titania nanotube surfaces on osteogenic differentiation of human adipose-derived stem cells

The surface of an implant is important for successful osseointegration and long-term stability as it can aid in cell migration and proliferation, cell differentiation and allow extracellular matrix production. Earlier studies have shown that nanostructuring the surface of titanium can enhance mesenchymal stem cell (MSC) migration, proliferation, and differentiation. Although many studies have evaluated MSC response on nanostructured surfaces, there are only a few studies that have explored the response of adipose-derived stem cells (ADSC) on titania nanotube surfaces. Because ADSC exhibit great potential in regenerative medicine and have already proven effective in developing new treatments, this study aims to further understand how ADSC interact with titania nanotube surfaces. The results of this study indicate that titania nanotube surfaces enhance ADSC proliferation and differentiation that is also dependent on the size of nanotubes. Additionally, the favorable response of ADSC on nanotube surfaces suggests a potential application in orthopedic tissue regeneration.

Effects of palmitate and astaxanthin on cell viability and proinflammatory characteristics of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have broad immunomodulatory activities. These cells are a stable source of cytokine production such as interleukin-6 (IL6), monocyte chemoattractant protein-1 (MCP-1/CCL2) and vascular endothelial growth factor (VEGF). Fatty acid elevation in chronic metabolic diseases alters the microenvironment of MSCs and thereby, might affect their survival and cytokine production. In the present study, we investigated the effects of palmitate, the most abundant saturated free fatty acid (FFA) in plasma, and astaxanthin, a potent antioxidant, on cell viability and apoptosis in human bone marrow-driven mesenchymal stem cells. We also elucidated how palmitate and astaxanthin influence the inflammation in MSCs. Human mesenchymal stem cells were collected from an aspirate of the femurs and tibias marrow compartment. The effect of palmitate on cell viability, caspase activity and pro-inflammatory cytokines expression and secretion were evaluated. In addition, activation of the MAP kinases and NF-kB signaling pathways were investigated. The results showed that astaxanthin protected MSCs from palmitate-induced cell death. We found that palmitate significantly enhanced IL-6, VEGF and MCP-1 expression, and secretion in MSC cells. Increased cytokine expression was parallel to the enhanced phosphorylation of P38, ERK and IKKα-IKKβ. In addition, pretreatment with JNK, ERK, P38, and NF-kB inhibitors could correspondingly attenuate palmitate-induced expression of VEGF, IL-6, and MCP-1. Our results demonstrated that fatty acid exposure causes inflammatory responses in MSCs that can be alleviated favorably by astaxanthin treatment.

Glancing-Angle Deposition of Nanostructures on an Implant Material Surface.

Cell-compatible and antibacterial surfaces are needed for implants, which frequently have complex and rough surfaces. Bio-inspired columnar nanostructures can be grown on flat substrates; however, the application of these nanostructures on clinically relevant, complex, and rough surfaces was pending. Therefore, a titanium plasma spray (TPS) implant surface was coated with titanium nano-spikes via glancing angle magnetron sputter deposition (GLAD) at room temperature. Using GLAD, it was possible to cover the three-dimensional, highly structured macroscopic surface (including cavities, niches, clefts, and curved areas) of the TPS homogeneously with nano-spikes (TPS+), creating a cell-compatible and antibacterial surface. The adherence and spreading of mesenchymal stem cells (MSC) were similar for TPS and TPS+ surfaces. However, MSC adherent to TPS+ expressed less and shorter pseudopodia. The induced osteogenic response of MSC was significantly increased in cells cultivated on TPS+ compared with TPS. In addition, Gram-negative bacteria (E. coli) adherent to the nano-spikes were partly destructed by a physico-mechanical mechanism; however, Gram-positive bacteria (S. aureus) were not significantly damaged.

Low-dose suspended graphene oxide nanosheets induce antioxidant response and osteogenic differentiation of bone marrow-derived mesenchymal stem cells via JNK-dependent FoxO1 activation.

Large bone defects caused by bone-related diseases and traumatic injuries can disrupt the self-healing potential of bone tissue. Mesenchymal stem cells (MSCs) are known as promising cell sources for bone tissue regeneration. Graphene oxide (GO), a derivative of graphene, has been recently used for controlling the differentiation of stem cells towards bone-forming cells. However, the effect of GO on the intracellular redox system in MSCs is still unknown. In this study, we found that low-dose GO nanosheets (0.1 μg mL-1) did not affect the viability and slightly increased the proliferation of BM-MSCs. Moreover, they could also maintain the redox balance by upregulating the antioxidant genes such as MnSOD and catalase during osteogenic differentiation. The osteoinductive and antioxidative effects of the low-dose GO nanosheets were regulated by the activation and nuclear localization of FoxO1, and its activation was dependent on the JNK activity. The blockade of JNK activity by SP600125 inhibited the nuclear translocation of FoxO1, and subsequently suppressed the osteogenic differentiation and antioxidant defense system of BM-MSCs. Overall, our results show that the osteoinductive and antioxidative effects of low-dose GO nanosheets occur through the activation of the JNK and FoxO1 signaling pathways.

Mesenchymal stem cell response to growth factor treatment and low oxygen tension in 3-dimensional construct environment

Mesenchymal stem cell response to growth factor treatment and low oxygen tension in 3-dimensional construct environment

Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation.

SummaryExogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

Quiescent Human Mesenchymal Stem Cells Are More Resistant to Heat Stress than Cycling Cells.

Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly, little is known about how quiescent cells respond to environmental challenges. The aim of the present study is to compare stress responses of cycling and quiescent mesenchymal stem cells (MSC). Human endometrial mesenchymal cells (eMSС) were employed as adult stem cells. eMSC quiescence was modeled by serum starvation. Sublethal heat shock (HS) was used as a stress factor. Both quiescent and cycling cells were heated at 45°C for 30 min and then returned to standard culture conditions for their recovery. HS response was monitored by DNA damage response, stress-induced premature senescence (SIPS), cell proliferation activity, and oxidative metabolism. It has been found that quiescent cells repair DNA more rapidly, resume proliferation, and undergo SIPS less than proliferating cells. HS-enforced ROS production in heated cycling cells was accompanied with increased expression of genes regulating redox-active proteins. Quiescent cells exposed to HS did not intensify the ROS production, and genes involved in antioxidant defense were mostly silent. Altogether, the results have shown that quiescent cells are more resistant to heat stress than cycling cells. Next-generation sequencing (NGS) demonstrates that HS-survived cells retain differentiation capacity and do not exhibit signs of spontaneous transformation.