Growth Of Human Mesenchymal Stem Cells Research Articles

Surface Decoration of Redox-Modulating Nanoceria on 3D-Printed Tissue Scaffolds Promotes Stem Cell Osteogenesis and Attenuates Bacterial Colonization.

Oxidative stress at the bone defect site delays the bone regeneration process. Increased level of reactive oxygen species (ROS) is the primary cause of oxidative stress at the damaged site. Bone tissue scaffolds that scavenge ROS offer a potential and yet unexplored route for faster bone healing. Cerium oxide (ceria) is known for its redox-modulating behavior. Three-dimensional (3D)-printed porous scaffolds fabricated from degradable polymers provide a physical microenvironment but lack the bioactivity for tissue regeneration. In this work, porous poly(lactic acid) (PLA) scaffolds were prepared by 3D printing and modified with poly(ethylene imine) and citric acid to decorate with ceria nanoparticles. Scanning electron micrographs revealed a macroporous architecture decorated with ceria particles. The compressive modulus of 27 MPa makes them suitable for trabecular bone. The scaffolds supported human mesenchymal stem cell growth, confirming cytocompatibility. The ability to scavenge ROS confirmed that surface functionalization with ceria could reduce oxidative stress levels in the cells. Stem cell osteogenesis was enhanced after ceria decoration of the PLA scaffolds. Transcriptional profiling studied by sequencing revealed changes in the expression of genes associated with inflammation and cell-material interactions. The ceria-functionalized scaffolds show enhanced antibacterial activity against both Gram-negative and Gram-positive bacterial strains. These results demonstrate that surface decoration with nanoceria offers a viable route for enhancing the bioactivity of 3D-printed PLA scaffolds for bone tissue regeneration with ROS scavenging and antibacterial capability.

3D-printed gelatin methacrylate (GelMA)/silanated silica scaffold assisted by two-stage cooling system for hard tissue regeneration.

Among many biomaterials, gelatin methacrylate (GelMA), a photocurable protein, has been widely used in 3D bioprinting process owing to its excellent cellular responses, biocompatibility and biodegradability. However, GelMA still shows a low processability due to the severe temperature dependence of viscosity. To overcome this obstacle, we propose a two-stage temperature control system to effectively control the viscosity of GelMA. To optimize the process conditions, we evaluated the temperature of the cooling system (jacket and stage). Using the established system, three GelMA scaffolds were fabricated in which different concentrations (0, 3 and 10 wt%) of silanated silica particles were embedded. To evaluate the performances of the prepared scaffolds suitable for hard tissue regeneration, we analyzed the physical (viscoelasticity, surface roughness, compressive modulus and wettability) and biological (human mesenchymal stem cells growth, western blotting and osteogenic differentiation) properties. Consequently, the composite scaffold with greater silica contents (10 wt%) showed enhanced physical and biological performances including mechanical strength, cell initial attachment, cell proliferation and osteogenic differentiation compared with those of the controls. Our results indicate that the GelMA/silanated silica composite scaffold can be potentially used for hard tissue regeneration.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

SummaryHuman mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

Evaluation of fibroblast growth factor-2 on the proliferation of osteogenic potential and protein expression of stem cell spheroids composed of stem cells derived from bone marrow.

Fibroblast growth factor-2 (FGF-2) is reported to have various functions and is considered a key human mesenchymal stem cell mitogen, often supplemented to increase human mesenchymal stem cell growth rates. The purpose of this study was to evaluate the effects of FGF-2 on cellular viability and osteogenic differentiation using three-dimensional cell spheroids of stem cells. Three-dimensional cell spheroids were fabricated using concave silicon elastomer-based microwells in the presence of FGF-2 at concentrations of 0, 30, 60 and 90 ng/ml. Qualitative cellular viability was determined with a confocal microscope, and quantitative cellular viability was evaluated using a Cell Counting Kit-8 assay. Alkaline phosphatase activity and Alizarin Red S staining were used to assess osteogenic differentiation. Spheroids were well formed in silicon elastomer-based concave microwells on Day 1. The average spheroid diameters at Day 1 for FGF-2 at 0, 30, 60 and 90 ng/ml were 202.2±3.0, 206.6±22.6, 208.8±6.8 and 196.6±26.7 µm, respectively (P>0.05). The majority of the cells in the cell spheroids emitted green fluorescence. The relative Cell Counting Kit-8 assay values for FGF-2 at 0, 30, 60 and 90 ng/ml at Day 1 were 100.0±5.5, 101.8±8.8, 99.2±4.8 and 103.4±9.6% (P>0.05). The addition of FGF-2 at 60 ng/ml concentration produced the highest value for alkaline phosphatase activity. Mineralized extracellular deposits were evenly observed in each group, and the highest value was identified for FGF-2 groups at 60 ng/ml concentration for Alizarin Red S staining. Based on these findings, it was concluded that FGF-2 may increase alkaline phosphatase activity or Alizarin Red S staining, and further studies are needed to fully elucidate the mechanisms of FGF-2.

Mathematical modeling of oxygen transfer in porous scaffolds for stem cell growth: The effects of porosity, cell type, scaffold architecture and cell distribution

Abstract Oxygen plays a key role in human mesenchymal stem cell growth. Without adequate oxygen (hypoxic condition), cells are not able to survive, proliferate, and migrate. The objective of the present study is to investigate oxygen transfer through the cell-seeded scaffolds stored in static or dynamic bioreactors using a mathematical model. The effects of porosity, cell type, scaffold architecture and cell distribution as potential effective parameters on oxygen transfer kinetics were examined. The results suggest the substantial effect of porosity and cell type on the oxygen concentration within the scaffold compared to scaffold architecture (homogeneous vs. gradient). The obtained data show that the direction of oxygen transfer in deep regions with dead cells changes over time and reverse mass transfer allows the cells to nourish from both top and bottom layers. Finally, the extent of oxygen transfer in static bioreactors/cultures was compared to dynamic ones. The results show that dynamic bioreactors have a better performance and are more efficient for oxygen transfer.

Poly(3-hydroxybutyrate) graft copolymer dense membranes for human mesenchymal stem cell growth

Abstract Background The use of novel materials as an artificial extracellular matrix for stem cell growth is a current strategy of increasing interest for regenerative medicine. Here, we prepare thermal-remolded membrane scaffolds from poly(3-hydroxybutyrate) grafted with 2-amino-ethyl methacrylate hydrochloride. However, it is unclear whether these membranes are useful for tissue engineering. Results The mechanical properties, tribology, and morphology of the dense membranes were assessed. The results show that tensile strain at break and roughness of the compressed membrane decrease with increasing graft degree. Moreover, graft copolymer membranes showed lower resistance to scratching, greater degree of swelling and higher brittleness than un-grafted P(3HB) films. Thus, it effectively supports the growth of dermal fibroblast, as demonstrated by epifluorescence microscopy. Conclusions It is concluded that the developed membrane can be properly used in is the restoration of skin tissue. How to cite: Gonzalez-Torres M, Sanchez-Sanchez R, Solis-Rosales SG, et al. Poly(3-hydroxybutyrate) graft copolymer dense membranes for human mesenchymal stem cell growth. Electron J Biotechnol 2018, 34; https://doi.org/10.1016/j.ejbt.2018.05.007 .

Knitted 3D Scaffolds of Polybutylene Succinate Support Human Mesenchymal Stem Cell Growth and Osteogenesis

Polybutylene succinate (PBS) is a biodegradable polyester with better processability and different mechanical properties compared to polylactides (PLAs), the most commonly used synthetic polymers in tissue engineering (TE). Since only few studies have evaluated PBS-containing materials for bone TE, we prepared PLA-PBS blends and analyzed material properties as well as cell attachment, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs) on scaffolds. In addition to PLA, PBS, and PLA-PBS blends, PLA-polycaprolactone and PLA-poly(trimethylene carbonate) blends were evaluated. Polymer fibers were prepared using melt spinning. Pure PBS was observed to have the highest crystallinity and strain at break compared to the tougher PLA and PLA blends. No degradation occurred during the 4-week hydrolysis in either of the materials. Knitted and rolled scaffolds were manufactured, seeded with hMSCs, and cultured for 27 days. Human MSC viability was good on all the materials, but cell spreading along the fibers was only detected in PBS-containing scaffolds. They also induced the strongest proliferative response and osteogenic differentiation, which diminished with decreasing PBS content. Based on these results, PBS is superior to PLA with respect to hMSC attachment, proliferation, and osteogenesis. This encourages utilizing PBS-based biomaterials more widely in bone TE applications.

Bioactive magnetic near Infra-Red fluorescent core-shell iron oxide/human serum albumin nanoparticles for controlled release of growth factors for augmentation of human mesenchymal stem cell growth and differentiation.

BackgroundIron oxide (IO) nanoparticles (NPs) of sizes less than 50 nm are considered to be non-toxic, biodegradable and superparamagnetic. We have previously described the generation of IO NPs coated with Human Serum Albumin (HSA). HSA coating onto the IO NPs enables conjugation of the IO/HSA NPs to various biomolecules including proteins. Here we describe the preparation and characterization of narrow size distribution core-shell NIR fluorescent IO/HSA magnetic NPs conjugated covalently to Fibroblast Growth Factor 2 (FGF2) for biomedical applications. We examined the biological activity of the conjugated FGF2 on human bone marrow mesenchymal stem cells (hBM-MSCs). These multipotent cells can differentiate into bone, cartilage, hepatic, endothelial and neuronal cells and are being studied in clinical trials for treatment of various diseases. FGF2 enhances the proliferation of hBM-MSCs and promotes their differentiation toward neuronal, adipogenic and osteogenic lineages in vitro.ResultsThe NPs were characterized by transmission electron microscopy, dynamic light scattering, ultraviolet–visible spectroscopy and fluorescence spectroscopy. Covalent conjugation of the FGF2 to the IO/HSA NPs significantly stabilized this growth factor against various enzymes and inhibitors existing in serum and in tissue cultures. IO/HSA NPs conjugated to FGF2 were internalized into hBM-MSCs via endocytosis as confirmed by flow cytometry analysis and Prussian Blue staining. Conjugated FGF2 enhanced the proliferation and clonal expansion capacity of hBM-MSCs, as well as their adipogenic and osteogenic differentiation to a higher extent compared with the free growth factor. Free and conjugated FGF2 promoted the expression of neuronal marker Microtubule-Associated Protein 2 (MAP2) to a similar extent, but conjugated FGF2 was more effective than free FGF2 in promoting the expression of astrocyte marker Glial Fibrillary Acidic Protein (GFAP) in these cells.ConclusionsThese results indicate that stabilization of FGF2 by conjugating the IO/HSA NPs can enhance the biological efficacy of FGF2 and its ability to promote hBM-MSC cell proliferation and trilineage differentiation. This new system may benefit future therapeutic use of hBM-MSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0090-8) contains supplementary material, which is available to authorized users.

Effects of surface topography on growth and osteogenic differentiation of human mesenchymal stem cells

The clinical success of an endosseous artificial implant is related to the quality of its osseointegration with the surrounding living bone. To achieve a stable anchorage, mesenchymal cells, migrating to the implant surface from the surrounding tissue, must differentiate towards mature osteoblasts rather than connective tissue cell types. It is well known that the cell response is affected by the physicochemical parameters of the biomaterial surface, such as surface energy, surface charges or chemical composition. Topography seems to be one of the most crucial physical cues for cells (1). In particular, interactions between mesenchymal stem cells (MSCs) and surfaces with specific micro and nano patterns can stimulate MSCs to produce bone mineral in vitro (2). Herein, stamps reporting different micro and nano features were fabricated in order to obtain several corresponding replicas in a short time through microinjec- tion molding. Then, the effects of the substrate topography on human bone marrowderived MSC adhesion, proliferation, and osteogenic differentiation were investigated in the absence of inductive growth factors. Collectively, our data show that both micro- and nano-structured surfaces possess osteoinductive properties. A relationship between dimensional feature of surface topography and differentiative potential was noted. On the contrary, cell adhesion and proliferation seemed to be unaffected. Further in vivo studies will be carried out to confirm the osteoinductive properties of selected surface geometries.

Bone regeneration from mesenchymal stem cells (MSCs) and compact bone-derived MSCs as an animal model

Mesenchymal stem cells (MSCs) have emerged as a promising alternative to traditional surgical techniques. The purpose of this review is to document the current state of research and identify future research directions. At present, no specific markers have been shown to specifically identify MSCs. The most commonly reported positive markers are CD105, CD90, CD44, CD73, CD29, CD13, CD34, CD146, CD106, CD54 and CD166. The most frequently reported negative markers are CD34, CD14, CD45, CD11b, CD49d, CD106, CD10 and CD31. Regarding the source of MSCs, bone marrow-derived MSCs are the most frequently studied MSCs in bone regeneration; however, no reports have demonstrated advantages of bone marrow-derived MSCs over other types of MSCs in bone regeneration. For the purpose of clinical use, serum-free media is recommended to avoid risks connected with the use of animal products. Attempts have been made to develop defined serum-free media for animal and human MSC growth; however, most products have demonstrated only limited performance. Tumorigenesis is the other major problem in MSC regeneration. It is strongly recommended to prepare MSCs for tissue regeneration at early passages to avoid potential chromosomal abnormalities.

A “Twist box” Code of p53 Inactivation: Twist box:p53 Interaction Promotes p53 Degradation

Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we found that Twist1 acts as a mechanism alternative to TP53 mutation and MDM2 overexpression to inactivate p53 in mesenchymal tumors. We provide evidence that Twist1 binds p53 C terminus through the Twist box. This interaction hinders key posttranslational modifications of p53 and facilitates itsMDM2-mediated degradation. Our study suggests the existence of a Twist box code of p53 inactivation and provides the proof of principle that targeting the Twist box:p53 interaction might offer additional avenues for cancer treatment.

Expansion of human mesenchymal stem cells on microcarriers

The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 microcarriers were not damaged when held at the minimum speed, N(JS), for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8-10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the microcarriers in the flasks were 3,000 microcarriers ml(-1) (ca. 1 g dry weight l(-1)), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at N(JS) (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov's theory of isotropic turbulence.

High glucose concentration in cell culture medium does not acutely affect human mesenchymal stem cell growth factor production or proliferation.

Optimizing the function and proliferative capacity of stem cells is essential to maximize their therapeutic benefits. High glucose concentrations are known to have detrimental effects on many cell types. We hypothesized that human mesenchymal stem cells (hMSCs) cultured in high glucose-containing media would exhibit diminished proliferation and attenuated production of VEGF, hepatocyte growth factor (HGF), and FGF2 in response to treatment with TNF-alpha, LPS, or hypoxia. hMSCs were plated in medium containing low (5.5 mM) and high (20 mM or 30 mM) glucose concentrations and treated with TNF-alpha, LPS, or hypoxia. Supernatants were collected at 24 and 48 h and assayed via ELISA for VEGF, HGF, and FGF2. In addition, hMSCs were cultured on 96-well plates at the above glucose concentrations, and proliferation at 48 h was determined via bromo-2'-deoxy-uridine (BrdU) incorporation. At 24 and 48 h, TNF-alpha, LPS, and hypoxia-treated hMSCs produced significantly higher VEGF, HGF, and FGF2 compared with control. Hypoxia-induced VEGF production by hMSCs was the most pronounced change over baseline. At both 24 and 48 h, glucose concentration did not affect production of VEGF, HGF, or FGF2 by untreated hMSCs and those treated with TNF-alpha, LPS, or hypoxia. Proliferation of hMSCs as determined via BrdU incorporation was unaffected by glucose concentration of the media. Contrary to what has been observed with other cells, hMSCs may be resistant to the short-term effects of high glucose. Ongoing efforts to characterize and optimize ex vivo and in vivo conditions are critical if the therapeutic benefits of MSCs are to be maximized.

Electrical characterization of human mesenchymal stem cell growth on microelectrode

To support the development of stem cell therapies with in vitro assays, non-destructive methods are required for the quality control of stem cell culture. In this article, it was investigated wheth...

Electrical characterization of human mesenchymal stem cell growth on microelectrode

To support the development of stem cell therapies with in vitro assays, non-destructive methods are required for the quality control of stem cell culture. In this article, it was investigated whether an electrode based chip with electrical impedance spectroscopy can be used to characterize the growth of human mesenchymal stem cells on electrodes without any chemical marker. From finite element method simulations, the electrical characteristics of cell layer under weak alternating electric fields were investigated with respect to the different cell/cell or cell/substrate gap, and modelled as an equivalent circuit. The impedance spectra were measured during the long-term cultivation of human mesenchymal stem cells on platinum electrodes. By fitting analyses the equivalent circuit to measured spectra, an extra cellular resistance reflecting the cell growth was extrapolated.

Reconstruction for local radiation injuries and proposed regeneration therapy for acute systemic radiation injuries

Skin and soft tissue radiation injuries are life-threatening as well as of esthetic concern in local tissues. As for local radiation injuries, clinically developed procedures such as local flaps and free vascularized flaps result in better and less-invasive reconstructive outcomes for victims of therapeutic radiation of malignant tumors, prolonged fluoroscopic procedures for cardiovascular diseases or possibly direct contact with radioactive waste, while systemic radiation injuries should find other solutions. In systemic radiation injuries, there is a good chance to save lives when the initial treatment is efficient, although donors of the skin grafting after debridement of the radiation-damaged tissue removal are unrealistically autogenous. Thus, exceeding certain extent and degree, systemic radiation injury-induced burns were not the medical objectives. Recent understanding and application of use in adult stem cells, however, create a new insight for severe systemic radiation injuries. Among the adult stem cells, the human mesenchymal stem cells are able to be freeze-dried, easily thawed and able to be cultured in vitro. A basic fibroblast growth factor is already of clinical use and efficient for the human mesenchymal stem cell growth both in vitro and in vivo. An artificial dermis is useful for temporal coverage of the raw surface of severe bedsores. The combined use of these successfully regenerates both dermis and epidermis after 20 Gy whole body irradiation in a nude rat model. Therefore, even severe radiation injuries, local tissue management and therapy are well-handled and systemic radiation injury may be hopeful with understanding and the specialized practice of regenerative medicine.

Bacterial Endotoxin LPS but Not LTA Can Enhance Osteogenic Differentiation of Human Mesenchymal Stem Cells.

Background & Objective: Dental implant requires osseointegration for anchoring and human's oral cavity has plenty of bacterial oral flora. Whether these bacteria have any effects on the human mesenchymal Stem Cells (MSCs) that can differentiate into osteoblasts remains unknown. We therefore investigated the effect of bacterial endotoxins commonly found in the oral cavity and gastrointestinal tract, namely lipopolysaccharides (LPS, Escherichia coli) and lipoteichoic acid (LTA, Streptococcus pyogenes), on the proliferation and osteogenic differentiation of MSCs.Methods: Human MSCs are derived from bone marrow (BM) of normal healthy donors. The culture condition, immunophenotyping determination and tests of differentiating functions of the human MSCs were similar to what we reported previously (Li J, Br J Haematol 2004). The proliferation of MSCs under either a 3-day or a prolonged 7-day endotoxins challenge was evaluated by XTT assay. The extent of osteogenic differentiation was examined under microscopy and measured by the increase in alkaline phosphatase (ALP) activity at day 10 and the calcium mineralization/deposition per unit volume of protein at day 14.Results: There was no significant effect of LPS and LTA on the growth and proliferation of MSCs, even under a relatively high dose. However, continued LPS challenge on MSCs under osteogenic culture condition was shown to increase the ALP activity and calcium deposition in a dose-dependent manner (100ng/ml, 1 ug/ml, 10ug/ml). No such phenomenon can be identified when LTA challenge was used.Conclusions: LPS and LTA did not show any significant effect on the proliferation and growth of human MSCs. However, LPS enhanced the osteogenic differentiation of MSCs in a dose-dependent manner. Our finding suggests that the endotoxin from bacteria commonly found in the oral cavity and gut does not have any negative impact on MSCs induced osteogenesis.