Cell-IQ visualization of motility,cell mass,and osteogenic differentiation of multipotent mesenchymal stromal cells cultured with relief calcium phosphate coating

The Cell-IQ continuous surveillance system allowed us to establish the following changes in a 14- day culture in vitro: a twofold suppression of the directional migration of multipotent mesenchymal stromal cells of human adipose tissue (MMSC-AT) towards the samples with a microarc calcium phosphate (CP) coating from synthetic hydroxyapatite; a tenfold decrease in the cell mass on the interphase with the samples, which was accompanied by a slight reduction in the expression of membrane determinants of stromal stem cells; and an enhancement of their osteogenic differentiation (osteocalcin secretion and mineralized matrix formation) on the 21st day of the study. Calcium phosphate particles, but not the calcium and phosphorus ions, may trigger the phenotypic transformation of the MMSC-AT behavior in vitro.     

Morphofunctional changes of Jurkat T lymphoblasts upon short-term contact with a relief calcium phosphate surface

Human leukemic T lymphoblastoid cells (hereinafter, Jurkat T cells) have been used to model the morphofunctional reaction of T lymphocytes to 24-h in vitro contact with relief (roughness index Ra = 2.2–2.7 μm) pure titanium substrates (12 × 12 × 1 mm³) covered by a calcium phosphate (CP) bilateral coating prepared by the microarc method. Jurkat T cells placed in plastic multiwall plates (2D control of culture growth), as well as cells in contact for 24 h with oxide (TiO₂) microarc coating on pure titanium substrate (3D control), served as comparison tests. From 27 to 98% of immortalized cells in the 2D control culture had the CD3⁺CD4⁺CD71⁺CD45RA⁺ immunophenotype and secreted IL-2, IL-4, IL-8, IL-10, and TNFα, but not IL-1b and IL-6. Other markers of cell activation, differentiation, maturation, and death (CD8, CD16, CD56, CD25, and CD95) were found in 0–2.5% of cells. IL-8 release elevated by the microtextured CP surface by 183 and 160% relative to 2D- and 3D-control cultures, respectively. The CD4/CD8 ratio dropped to 9: 1 (13: 1 and 82: 1 in 2D and 3D controls, respectively) due to CD4⁺ cell depletion and an increase in CD8⁺ cell number. The total cell number (TCN) in Jurkat T cell culture after 24-h contact with CP coating was decreased to 88% compared to 2D control (p < 0.04), which indicated cell division suppression. A drop in total cell number was accompanied with accelerated IL-8 secretion (r = ?0.97, p < 0.00009). Low concentrations of IL-8 (pg/mL) induced apoptosis (r = 0.94, p < 0.0001). These results show that CP, rather than TiO₂, coating is a preferential surface material for endoprosthesis replacement and fracture osteosynthesis in patients suffering from hematological and bone malignancies.     

Hydroxyapatite from Cuttlefish Bone: Isolation,Characterizations, and Applications

Hydroxyapatite (HA), a bioceramic, is a widely utilized material for bone tissue repair and regeneration because of its excellent properties such as biocompatibility, exceptional mechanical strength, and osteoconductivity. HA can be obtained by both synthetic and natural means. Animal bones are often considered a promising natural resource for the preparation of pure HA for biological and biomedical applications. Cuttlefish bone, also called as cuttlebone, mainly consists of calcium carbonate, and pure HA can be produced by adding phosphoric acid or ammonium hydrogen phosphate to it. Recently, cuttlefish bone-derived HA has shown promising results in terms of bone tissue repair and regeneration. The synthesized cuttlefish bone-derived has shown excellent biocompatibility, cell proliferation, increased alkaline phosphate activity, and efficient biomineralization ability with mesenchymal stem cells and osteoblastic cells. To further improve the biological properties of cuttlefish bone-derived HA, bioglass, polycaprolactone, and polyvinyl alcohol were added to it, which gave better results in terms of cell proliferation and osteogenic differentiation. Cuttlefish bone-derived HA with polymeric substances provides excellent bone formation under in vivo conditions. The studies indicate that cuttlefish bone-derived HA, along with polymeric and, protein materials, will be promising biomaterials in the field of bone tissue regeneration.     

Interaction of human mesenhymal stromal with immune cells

Multipotential mesenchymal stromal cells (MMSCs) are the subject of increasing scientific interest due to the key role of these cells in physiological renewal and repair. Allogeneic MMSC interaction with other components of tissue processes in the environment, in particular, with immune cells is one of the most intriguing questions of modern cell physiology. MMSCs possess pronounced immunomodulatory capabilities based on their immmunoprivilege properties and the ability to suppress an immune response. This review considers the state-of-the-art in the field of MMSC immunomodulatory effects and their mechanisms. The interaction between MMSCs and immune cells is a complex, multidirectional process governed by both direct cell-to-cell interactions and soluble factors (interferon-γ, tumor necrosis factor, prostaglandin E₂, hepatocyte growth factor, interleukins, etc.). The importance of physical environmental factors, primarily oxygen tension, for the characteristics of the interaction between MMSCs and immune cells is discussed.     

Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension

It has been shown that a decrease in oxygen tension during cultivation of multipotent mesenchymal stromal cells (MMSCs) caused a short-term decline in the proportion of CD73⁺ cells in the population, with no effect on the number of cells expressing the other constitutive surface markers (CD90, CD105). The heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable under conditions close to tissue oxygen levels (5% O₂). At lower oxygen concentration, it is gradually reduced from the third to fourth passages. The increase in proliferative activity was not accompanied by increased telomerase gene expression, which indicated that no cell transformation had occurred. Global gene expression analysis of MMSC gene expression revealed changes in expression of cyclins (CCND2, PCNA), regulatory subunit cyclin-dependent kinase (CKS2), and an inhibitor of cyclin-dependent kinases 4 and 6 (CDKN2C) regulating the cell cycle, which probably facilitated the proliferative activity of cells at lower oxygen tension.     

SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway

Background

Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms.

Methods

SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs.

Results

SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential.

Conclusions

The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

     

Human MMSC immunosuppressive activity at low oxygen tension: Direct cell-to-cell contacts and paracrine regulation

In recent years, multipotent mesenchymal stromal (stem) cells (MMSCs) were identified and isolated from many tissues and their immunoprivilege and immunosuppressive potential, along with high proliferative activity and multilineage differentiation, have been demonstrated. At the same time, there is an increasing body of evidence of the MMSC plasticity due to a wide range of microenvironmental factors: extracellular matrix, cell-to-cell interactions, oxygen tension, etc. In this study, direct cell-to-cell and paracrine effects of MMSCs on human phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (MNCs) at the standard (20%) and reduced (5%) O₂ concentrations in the culture medium have been compared. It has been shown that coculture with MMSCs decreases the proliferative activity of PHA-MNCs, the proportion of HLA-DR⁺ T cells, and the interleukin (IL)-6, IL-8, and tumor necrosis factor α (TNF-α) concentrations, and increases the IL-10 and interferon γ (IFN-γ) in the medium. A potentiating effect of low oxygen tension on the immunomodulating properties of MMSCs has been observed, which is of great importance to enchance immunosuppression.     

Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate

Background

The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol–gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO₂). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation.

Results

The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO₂ and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8–9 with an advantage for Ti SiO₂ implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO₂ implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO₂ implants.

Conclusions

The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO₂ scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFβ1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.

     

The Effect of Multipotent Mesenchymal Stromal Cell Derivatives on the Properties of Breast Cancer Cells in vitro and in vivo

The effects of multipotent mesenchymal stromal cell (MMSC) derivatives on breast cancer cells have been studied in vitro and in vivo. A cytostatic effect of substances produced by human bone marrow MMSCs on the tumor cell population and the inhibition of tumor cell migration into the suspension fraction in vitro were demonstrated. These phenomena were accompanied by upregulation of tumor-associated marker expression: cytokeratin and EpCAM expression was upregulated in 2D and 3D cell cultures and vimentin expression was upregulated in 3D cultures. A single injection of mouse bone marrow MMSC lysate into the experimental animals suppressed tumor growth in thigh muscles. Moreover, this treatment contributed to the preservation of muscle tissue integrity and the normalization of biochemical parameters of the blood in animals that received grafts of Ehrlich adenocarcinoma tumor cells.     

Subchronic nandrolone administration reduces cardiac oxidative markers during restraint stress by modulating protein expression patterns

lncRNAs are an emerging class of regulators involved in multiple biological processes. MEG3, an lncRNA, acts as a tumor suppressor, has been reported to be linked with osteogenic differentiation of MSCs. However, limited knowledge is available concerning the roles of MEG3 in the multilineage differentiation of hASCs. The current study demonstrated that MEG3 was downregulated during adipogenesis and upregulated during osteogenesis of hASCs. Further functional analysis showed that knockdown of MEG3 promoted adipogenic differentiation, whereas inhibited osteogenic differentiation of hASCs. Mechanically, MEG3 may execute its role via regulating miR-140-5p. Moreover, miR-140-5p was upregulated during adipogenesis and downregulated during osteogenesis in hASCs, which was negatively correlated with MEG3. In conclusion, MEG3 participated in the balance of adipogenic and osteogenic differentiation of hASCs, and the mechanism may be through regulating miR-140-5p.     

Community and hospital acquired methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> efficiently retain the <Emphasis Type="Italic">Van A</Emphasis> determinant

Dissemination of vancomycin resistance from hospital to community strains is a serious threat to public health. Our study aimed to provide evidence for transmission of Van A type resistance from the hospital to the community. Wild-type community and hospital associated methicillin resistant Staphylococcus aureus strains were studied in vitro and in a model that mimicked a natural environment to ascertain their ability to acquire and maintain the vancomycin resistance determinant (Van A gene) from vancomycin resistant Enterococcus faecalis. Fitness was assessed and the cost of Van A acquisition and retention was estimated. In vitro mating experiments were carried out using a filter mating technique and a model of a natural water body environment. Transfer of resistance was carried out in two different conditions: restricted and favorable. Transconjugants were confirmed by E test and PCR using specific primer sets. Growth kinetics and fitness measurements were done by spectrometric analysis. Using the in vitro filter mating technique, high transfer frequencies that ranged from 0.7 × 10^–3(0.0006) to 3.1 × 10^–4(0.00011) were recorded, with the highest transfer frequencies for CA MRSA (thermosensitively homogenous) (0.7 × 10^–3), and 1.2 × 10^–4 to 2.4 × 10^–6 in the model. HA MRSA (homogenous) showed a greater capacity (3.6 × 10^–4) to receive the Van A gene, while CA MRSA showed a reduced ability to maintain the gene after serial subcultures. CA and HA thermosensitively heterogeneous MRSA transconjugants exhibited higher growth rates. The present study provides evidence for the enhanced ability of CA and HA MRSA clones to acquire and maintain Van A type resistance.     

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells

Background

Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs.

Methods

Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide.

Results

We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs.

Conclusions

Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state.

General significance

These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy.     

Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells

In this study, adipose-derived mesenchymal stem cells (ADSCs) were isolated from adipose tissues of rats. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by the results of oil red O staining, alkaline phosphatase (ALP), and alcian blue staining, respectively. ADSCs at a density of 1 × 10⁶/cm² were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 d. The cell sheets were positive with von kossa staining. The transmission electron microscopy (TEM) result showed that needle-like calcium salt crystals were deposited on the ECM. These results suggested that the osteogenic cell sheets may have potential osteogenesis ability. ADSCs at a density of 1 × 10⁶/cm² were cultured in the endothelial cell growth medium-2 and the endothelial cell sheets can be formed after 16 d of culture. The TEM image confirmed that the Weibel-Palade corpuscle was seen in the cells. The expression of CD31 was positive, suggesting that the endothelial cell sheets may have a strong ability to form blood vessels. In this study, two types of cell sheets with the potential abilities of osteogenesis and blood vessels formation were obtained by induced culture of ADSCs in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defects.     

The impact of oxygen in physiological regulation of human multipotent mesenchymal cell functions

Significant progress in studying cellular mechanisms of tissue homeostasis and physiological remodeling has been made in recent decades. Undifferentiated cells, such as multipotent mesenchymal stromal (stem) cells (MMSCs), play an important role in these processes. MMSCs were found in practically all organs occupying specific tissue niches associated with the perivascular spaces. The main characteristic of MMSCs is their ability, on the one hand, to provide structural integrity of tissues and, on the other hand, to respond to paracrine stimuli and migrate to damaged target tissues, which promotes tissue reparation. A low partial oxygen tension is the main feature of the physiological and regeneration microenvironment, which may significantly modify stromal cell properties. This review analyzes the recent data on MMSC tissue niches in terms of the integration of these cells into a comprehensive system of physiological and reparative tissue remodeling and the role of partial oxygen pressure in the fulfillment of the MMSC potential.     

MicroRNA-215 enhances invasion and migration by targeting retinoblastoma tumor suppressor gene 1 in high-grade glioma

Objective

To elucidate the molecular mechanism of microRNA-215 (miR-215) in the migration and invasion of high grade glioma.

Results

42 Patients were analysed for clinicopathological characteristics. qRT-PCR showed that miR-215 was up-regulated in glioma tissues compared with non-neoplastic brain tissues (P < 0.05). The up-regulated miR-215 was closely associated with high grade glioma (P < 0.01) and poor overall survival (P < 0.01). Transwell assay showed that re-expression of miR-215 enhanced migration and invasion of glioma cells. miR-215 also down-regulated retinoblastoma tumor suppressor gene 1 (RB1) expression by targeting its 3′-UTR. Reversely, re-expression of RB1 inhibited partial effect of miR-215 on migration and invasion in vitro.

Conclusions

Re-expression of miR-215 promoted cell migration and invasion of glioma by targeting RB1. miR-215 can thus be used as a biomarker for tumor progression and prognosis in human high grade glioma.

     

Impact of Metalloproteinase 1 Deficiency Induced by Specific Small Hairpin RNA on the Physiological Effects of Tumor Necrosis Factor

Matrix metalloproteinases play an important role in the pathogenesis of psoriasis. The aim of this paper was to explore the influence of MMP1 silencing with a specific shRNA on migration and proliferation of epidermal keratinocytes exposed to tumor necrosis factor, as well as changes in the expression of genes involved in their terminal differentiation. Changes in gene expression were analyzed by real-time PCR. The cell proliferation was assessed by comparative analysis of the growth curves. The cell migration was explored by scratch assay. To quantify cell migration, the representative areas of cell cultures were photographed in the equal periods of time and compared to each other. The obtained results demonstrated that an exposure of control cell line to tumor necrosis factor caused changes in the expression of several genes similar to ones that were previously observed in lesional psoriatic skin. Particularly, the expression of MMP9, IVL and KRT16 increased whereas the expression of LOR, KRT1 and-10—decreased. In contrast, MMP1-deficient cells treated with tumor necrosis factor exhibited higher levels of LOR, KRT1 and -10, as well as lower levels KRT16 and -17 compared to control cells treated with the same cytokines. Moreover, MMP1-deficient cells exhibited a lower level of CCNА2 and higher level of CCND1. In this respect, knocking MMP1 down resulted in a lower cell proliferation and migration rates of TNF-treated epidermal keratinocytes. In conclusion, this study demonstrated that MMP1 silencing with specific shRNA can be beneficial for psoriasis. We found that knocking MMP1 down has an antiproliferative effect on epidermal keratinocytes and partially normalizes the expression of cyclins CCNA2, and -D1, as well as the genes involved in the terminal differentiation of this kind of cells (LOR, KRT1, -10, -16 and -17).