Validation of reference genes for real-time PCR of cord blood mononuclear cells, differentiating endothelial progenitor cells, and mature endothelial cells.

In the last ten years, endothelial progenitor cells (EPCs) have gained interest as an attractive cell population in regenerative medicine for vascular applications. This population is defined as the precursor of endothelial mature cells (ECs) through a process of differentiation. To our knowledge, no single marker can be used to discriminate them from mature ECs. To effectively study their differentiation kinetics, gene expression must be assessed. Quantitative real-time PCR (RT-qPCR) is widely used to analyze gene expression. To minimize the impact of variances from RT-qPCR, a rigorous selection of reference genes must be performed prior to any experiments due to variations in experimental conditions. In this study, CD34+ mononuclear cells were extracted from human cord blood and differentiated into EPCs after seeding for a maximum period of 21 days. To choose the best combinations of reference genes, we compared the results of EPCs, CD34+ mononuclear cells, and mature endothelial cells to ensure that the differentiation kinetics did not affect the expression of our selected reference genes. The expression levels of seven genes, namely, YWHAZ, GAPDH, HPRT1, RPLP0, UBC, B2M, and TBP were thus compared. The algorithms geNorm, NormFinder, BestKeeper, and the Comparative ΔCt method were employed to assess the expression of each candidate gene. Overall results reveal that the expression stability of reference genes may differ depending on the statistical program used. YWHAZ, GAPDH, and UBC composed the optimal set of reference genes for the gene expression studies performed by RT-qPCR in our experimental conditions. This work can thus serve as a starting point for the selection of candidate reference genes to normalize the levels of gene expression in endothelial progenitor cell populations.

Vancomycin Functionalized Nanoparticles for Bactericidal Biomaterial Surfaces.

In this paper, we describe a simple and powerful way to synthesize antibacterial biomaterials with applications as implants in orthopedic surgery. Such implants are obtained by covalently grafting onto the Ti90A16 V4 alloy surface with vancomycin-functionalized nanoparticles. Nanoparticles were produced by ring-opening metathesis polymerization of α-norbornenyl-ω-vancomycin poly(ethylene oxide) macromonomers. Vancomycin is an interesting candidate because of its use in the field of implant associated infection as it is a glycopeptide which acts on bacterial walls. As a consequence, vancomycin does not need to be released for it to be active. In the first part of this paper, the synthesis and the complete characterization of these materials are described. In a second part, the in vitro antibacterial behavior is analyzed and discussed.

Altered nanofeature size dictates stem cell differentiation.

The differentiation of stem cells can be modulated by physical factors such as the micro- and nano-topography of the extracellular matrix. One important goal in stem cell research is to understand the concept that directs differentiation into a specific cell lineage in the nanoscale environment. Here, we demonstrate that such paths exist by controlling only the micro- and nano-topography of polymer surfaces. Altering the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts, in the absence of osteogenic medium. Small (10 nm) depth patterns promoted cell adhesion without noticeable differentiation, whereas larger depth patterns (100 nm) elicited a collective cell organization, which induced selective differentiation into osteoblast-like cells. This latter response was dictated by stress through focal-adhesion-induced reorganization of F-actin filaments. The results have significant implications for understanding the architectural effects of the in vivo microenvironment and also for the therapeutic use of stem cells.

Comparison of the density of proteins and peptides grafted on silane layers and polyelectrolyte multilayers.

Immobilized proteins or peptides are of critical importance for applications such as biosensing or cell culture. We analyze the structure of layers of a large variety of proteins and peptides, grafted on silicon substrates by different routes differing in the nature of the intermediate layer linking the biomolecules to the substrate, either a silane monolayer, or a polyelectrolyte multilayer made from synthetic or natural polymers. The structural analysis is essentially performed by X-ray reflectometry, which proves to be an efficient methodology not requiring the use of tagged biomolecules, capable of evaluating consistently the amount of grafted biomolecules per surface area with estimated precisions ranging from 10 to 20%. The study provides a quantitative basis for selecting one among a series of well-proofed and sturdy grafting methodologies and underlines the potential of XRR for assessing the amount of grafted biomacromolecules without requiring the expensive tagging of molecules. Our results also show that, for the coupling route resting on synthetic polyelectrolytes, the grafting density is significantly lower than for direct coupling over a silane layer. In contrast, when performed over a cushion based on polysaccharides, the grafting density is well above the values found for a dense layer grafted on a silane monolayer, indicating partial penetration and swelling of the polysaccharide cushion.

Universality of the network-dynamics of the cell nucleus at high frequencies.

The interior of the cell nucleus is comparable to a solid network bathed in an interstitial fluid. From the extrapolation of low frequency data, it is expected that such network should dictate the response of the nucleus to mechanical stress at high frequencies, described by unique elastic moduli. However, none of the existing techniques that can probe the mechanical properties of cells can exceed the kHz range, and the mechanics of the nuclear network remain poorly understood. We use laser-generated acoustic waves to probe remotely the stiffness and viscosity of nuclei in single cells in the previously unexplored GHz range with a ∼100 nm axial resolution. The probing of cells at contrasted differentiation stages, ranging from stem cells to mature cells originating from different tissues, demonstrates that the mechanical properties of the nuclear network are common across various cell types. This points to an asymptotically increasing influence of a solid meshwork of connected chromatin fibers.

Bioactive chemical nanopatterns impact human mesenchymal stem cell fate.

We present a method of preparing and characterizing nanostructured bioactive motifs using a combination of nanoimprint lithography and surface functionalization. Nanodots were fabricated on silicon surfaces and modified with a cell-adhesive RGD peptide for studies in human mesenchymal stem cell adhesion and differentiation. We report that bioactive nanostructures induce mature focal adhesions on human mesenchymal stem cells with an impact on their behavior and dynamics specifically in terms of cell spreading, cell-material contact, and cell differentiation.

Manipulating Stem Cell Fate with Disordered Bioactive Cues on Surfaces: The Role of Bioactive Ligand Selection.

The development of 2D or 3D bioactive platforms for rapidly isolating pure populations of cells from adult stem cells holds promise for advancing the understanding of cellular mechanisms, drug testing, and tissue engineering. Over the years, methods have emerged to synthesize bioactive micro- and nanostructured 2D materials capable of directing stem cell fate. We introduce a novel method for randomly micro- or nanopatterning any protein/peptide onto both 2D and 3D scaffolds via spray technology. Our goal is to investigate the impact of arranging bioactive micropatterns (ordered vs disordered) on surfaces to guide human mesenchymal stem cell (hMSC) differentiation. The spray technology efficiently coats materials with controlled, cost-effective bioactive micropatterns in various sizes and shapes. BMP-2 mimetic peptides were covalently grafted, individually or in combination with RGD peptides, onto activated polyethylene terephthalate (PET) surfaces through a spraying process, incorporating nano/microscale parameters like size, shape, and composition. The study explores different peptide distributions on surfaces and various peptide combinations. Four surfaces were homogeneously functionalized with these peptides (M1 to M4 with various densities of peptides), and six surfaces with disordered micro- and nanopatterns of peptides (S0 to S5 with different sizes of peptide patterns) were synthesized. Fluorescence microscopy assessed peptide distribution, followed by hMSC culture for 2 weeks, and evaluated osteogenic differentiation via immunocytochemistry and RT-qPCR for osteoblast and osteocyte markers. Cells on uniformly peptide-functionalized surfaces exhibited cuboidal forms, while those on surfaces with disordered patterns tended toward columnar or cuboidal shapes. Surfaces S4 and S5 showed dendrite-like formations resembling an osteocyte morphology. S5 showed significant overexpression of osteoblast (OPN) and osteocyte markers (E11, DMP1, and SOST) compared to control surfaces and other micropatterned surfaces. Notably, despite sharing an equivalent quantity of peptides with a homogeneous functionalized surface, S5 displayed a distinct distribution of peptides, resulting in enhanced osteogenic differentiation of hMSCs.

Silanization Strategies for Tailoring Peptide Functionalization on Silicon Surfaces: Implications for Enhancing Stem Cell Adhesion.

Biomaterial surface engineering and the integration of cell-adhesive ligands are crucial in biological research and biotechnological applications. The interplay between cells and their microenvironment, influenced by chemical and physical cues, impacts cellular behavior. Surface modification of biomaterials profoundly affects cellular responses, especially at the cell-surface interface. This work focuses on enhancing cellular activities through material manipulation, emphasizing silanization for further functionalization with bioactive molecules such as RGD peptides to improve cell adhesion. The grafting of three distinct silanes onto silicon wafers using both spin coating and immersion methods was investigated. This study sheds light on the effects of different alkyl chain lengths and protecting groups on cellular behavior, providing valuable insights into optimizing silane-based self-assembled monolayers (SAMs) before peptide or protein grafting for the first time. Specifically, it challenges the common use of APTES molecules in this context. These findings advance our understanding of surface modification strategies, paving the way for tailoring biomaterial surfaces to modulate the cellular behavior for diverse biotechnological applications.

Pericytes, stem-cell-like cells, but not mesenchymal stem cells are recruited to support microvascular tube stabilization.

An experimental model is introduced for the induction of endothelial cell (EC) tubulogenesis after 24 h of incubation on micropatterned polymer surfaces. Pericytes or mesenchymal stem cells are added separately to this system to evaluate their effect on tubular stabilization. In the absence of additional cells, the tubular structures are lost after 36 h. Addition of only pericytes, however, stabilizes the EC vasculogenic tubes.

Modulation of lumen formation by microgeometrical bioactive cues and migration mode of actin machinery.

How endothelial cells (ECs) express the particular filopodial or lamellipodial form of the actin machinery is critical to understanding EC functions such as angiogenesis and sprouting. It is not known how these mechanisms coordinately promote lumen formation of ECs. Here, adhesion molecules (RGD peptides) and inductor molecules (BMP-2 mimetic peptides) are micropatterned onto polymer surfaces by a photolithographic technique to induce filopodial and lamellipodial migration modes. Firstly, the effects of peptide microgeometrical distribution on EC adhesion, orientation and morphogenesis are evaluated. Large micropatterns (100 µm) promote EC orientation without lumen formation, whereas small micropatterns (10-50 µm) elicit a collective cell organization and induce EC lumen formation, in the case of RGD peptides. Secondly, the correlation between EC actin machinery expression and EC self-assembly into lumen formation is addressed. Only the filopodial migration mode (mimicked by RGD) but not lamellipodial migration mode (mimicked by BMP-2) promotes EC lumen formation. This work gives a new concept for the design of biomaterials for tissue engineering and may provide new insight for angiogenesis inhibition on tumors.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Rat Nucleus Pulposus Cells from Oxidative Stress.

BACKGROUND: Oxidative stress (OS) is mainly associated with the pathogenesis of intervertebral disc (IVD) degeneration; it causes nucleus pulposus cells (NPCs) to undergo senescence and triggers autophagy and apoptosis. This study aims to evaluate the regeneration potential of extracellular vesicles (EVs) derived from human umbilical cord-mesenchymal stem cells (hUC-MSCs) in an in vitro rat NPC-induced OS model. DESIGN: NPCs were isolated from rat coccygeal discs, propagated, and characterized. OS was induced by hydrogen peroxide (H2O2), which is confirmed by 2,7-dichlorofluorescein diacetate (H2DCFDA) assay. EVs were isolated from hUC-MSCs and characterized by analyzing the vesicles using fluorescence microscope, scanning electron microscope (SEM), atomic force microscope (AFM), dynamic light scattering (DLS), and Western blot (WB). The in vitro effects of EVs on migration, uptake, and survival of NPCs were determined. RESULTS: SEM and AFM topographic images revealed the size distribution of EVs. The phenotypes of isolated EVs showed that the size of EVs was 403.3 ± 85.94 nm, and the zeta potential was -0.270 ± 4.02 mV. Protein expression analysis showed that EVs were positive for CD81 and annexin V. Treatment of NPCs with EVs reduced H2O2-induced OS as evidenced by a decrease in reactive oxygen species (ROS) levels. Co-culture of NPCs with DiI-labeled EVs showed the cellular internalization of EVs. In the scratch assay, EVs significantly increased NPC proliferation and migration toward the scratched area. Quantitative polymerase chain reaction analysis showed that EVs significantly reduced the expression of OS genes. CONCLUSION: EVs protected NPCs from H2O2-induced OS by reducing intracellular ROS generation and improved NPC proliferation and migration.

Controlling differentiation of stem cells via bioactive disordered cues.

Ideal bone tissue engineering is to induce bone regeneration through the synergistic integration of biomaterial scaffolds, bone progenitor cells, and bone-forming factors. Biomimetic scaffolds imitate the native extracellular matrix (ECM) and are often utilized in vitro as analogues of the natural ECM to facilitate investigations of cell-ECM interactions and processes. In vivo, the cellular microenvironment has a crucial impact on regulating cell behavior and functions. A PET surface was activated and then functionalized with mimetic peptides to promote human mesenchymal stem cell (hMSC) adhesion and differentiation into an osteogenic lineage. Spray technology was used to randomly micropattern peptides (RGD and BMP-2 mimetic peptides) on the PET surface. The distribution of the peptides grafted on the surface, the roughness of the surfaces and the chemistry of the surfaces in each step of the treatment were ascertained by atomic force microscopy, fluorescence microscopy, time-of-flight secondary ion mass spectrometry, Toluidine Blue O assay, and X-ray photoelectron spectroscopy. Subsequently, cell lineage differentiation was evaluated by quantifying the expression of immunofluorescence markers: osteoblast markers (Runx-2, OPN) and osteocyte markers (E11, DMP1, and SOST). In this article, we hypothesized that a unique combination of bioactive micro/nanopatterns on a polymer surface improves the rate of morphology change and enhances hMSC differentiation. In DMEM, after 14 days, disordered micropatterned surfaces with RGD and BMP-2 led to a higher osteoblast marker expression than surfaces with a homogeneous dual peptide conjugation. Finally, hMSCs cultured in osteogenic differentiation medium (ODM) showed accelerated cell differentiation. In ODM, our results highlighted the expression of osteocyte markers when hMSCs were seeded on PET surfaces with random micropatterns.

Impact of RGD nanopatterns grafted onto titanium on osteoblastic cell adhesion.

This work reports on the synthesis of titanium bone implants functionalized with nanoparticles (NPs) containing Arg-Gly-Asp-Cys peptide (RGDC) and shows the adhesion behavior of cells seeded on these materials. RGDC peptides were first conjugated to a norbornenyl-poly(ethylene oxide) macromonomer (Nb-PEO). Then, functional NPs with a size of ∼300 nm and constituted of polynorbornene core surrounded by poly(ethylene oxide) shell were prepared by ring-opening metathesis polymerization in dispersed medium. The grafting density of these NPs on the titanium surface is up to 2 NPs·µm(-2) (80 pmol of RGDC per cm(-2) of NP surface). Cell adhesion was evaluated using preosteoblast cells (MC3T3-E1). Results of cells cultured for 24 h showed that materials grafted with NPs functionalized with RGDC peptides enhance specific cell adhesion and can create filopodia-like structures among NP sites by stressing the cells.

Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration.

To control specific endothelial cell (EC) functions, cell adhesive RGDS, EC specific REDV and YIGSR peptides, and angiogenic SVVYGLR sequences were covalently immobilized onto polyethylene terephthalate (PET) surfaces for the purpose of cell culture. X-ray photoelectron spectroscopy, atomic force microscopy, fluorescence microscopy and contact angle measurement were employed for characterization of surface modifications. The peptide density on PET surfaces was evaluated by fluorescence microscopy. The surfaces immobilized with peptides were exposed to human umbilical vein endothelial cells to study their specific effects onto EC functions. The results showed that the surface functionalized by these peptides enhanced the EC adhesion, spreading and migration as compared with native PET surfaces. Specifically, the RGDS peptides induced more cell adhesion than other peptides. The YIGSR and SVVYGLR sequences induced more cell spreading and cell migration, represented by intense focal adhesion at the leading edges of cell spreading and migration. The bi-functionalization of RGDS and SVVYGLR peptides (MIX) combined the advantages of both peptides and induced significant EC adhesion, spreading and migration. Our study indicates that the surface functionalization by peptides specific for ECs, especially the combination of RGDS with SVVYGLR or YIGSR peptides, has potential applications in promoting endothelialization of vascular prostheses and for construction of vascularized tissues in tissue engineering.

Interplay of matrix stiffness and stress relaxation in directing osteogenic differentiation of mesenchymal stem cells.

The aim of this study is to investigate the impact of the stiffness and stress relaxation of poly(acrylamide-co-acrylic acid) hydrogels on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Varying the amount of the crosslinker and the ratio between the monomers enabled the obtainment of hydrogels with controlled mechanical properties, as characterized using unconfined compression and atomic force microscopy (AFM). Subsequently, the surface of the hydrogels was functionalized with a mimetic peptide of the BMP-2 protein, in order to favor the osteogenic differentiation of hMSCs. Finally, hMSCs were cultured on the hydrogels with different stiffness and stress relaxation: 15 kPa - 15%, 60 kPa - 15%, 140 kPa - 15%, 100 kPa - 30%, and 140 kPa - 70%. The cells on hydrogels with stiffnesses from 60 kPa to 140 kPa presented a star-like shape, typical of osteocytes, which has only been reported by our group for two-dimensional substrates. Then, the extent of hMSC differentiation was evaluated by using immunofluorescence and by quantifying the expression of both osteoblast markers (Runx-2 and osteopontin) and osteocyte markers (E11, DMP1, and sclerostin). It was found that a stiffness of 60 kPa led to a higher expression of osteocyte markers as compared to stiffnesses of 15 and 140 kPa. Finally, the strongest expression of osteoblast and osteocyte differentiation markers was observed for the hydrogel with a high relaxation of 70% and a stiffness of 140 kPa.

Evaluating Poly(Acrylamide-co-Acrylic Acid) Hydrogels Stress Relaxation to Direct the Osteogenic Differentiation of Mesenchymal Stem Cells.

The aim of this study is to investigate polyacrylamide-based hydrogels stress relaxation and the subsequent impact on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different hydrogels are synthesized by varying the amount of cross-linker and the ratio between the monomers (acrylamide and acrylic acid), and characterized by compression tests. It has been found that hydrogels containing 18% of acrylic acid exhibit an average relaxation of 70%, while pure polyacrylamide gels show an average relaxation of 15%. Subsequently, hMSCs are cultured on two different hydrogels functionalized with a mimetic peptide of the bone morphogenetic protein-2 to enable cell adhesion and favor their osteogenic differentiation. Phalloidin staining shows that for a constant stiffness of 55 kPa, a hydrogel with a low relaxation (15%) leads to star-shaped cells, which is typical of osteocytes, while a hydrogel with a high relaxation (70%) presents cells with a polygonal shape characteristic of osteoblasts. Immunofluorescence labeling of E11, strongly expressed in early osteocytes, also shows a dramatically higher expression for cells cultured on the hydrogel with low relaxation (15%). These results clearly demonstrate that, by fine-tuning hydrogels stress relaxation, hMSCs differentiation can be directed toward osteoblasts, and even osteocytes, which is particularly rare in vitro.

Directing hMSCs fate through geometrical cues and mimetics peptides.

The native microenvironment of mesenchymal stem cells (hMSCs)-the extracellular matrix (ECM), is a complex and heterogenous environment structured at different scales. The present study aims at mimicking the hierarchical microorganization of proteins or growth factors within the ECM using the photolithography technique. Polyethylene terephthalate substrates were used as a model material to geometrically defined regions of RGD + BMP-2 or RDG + OGP mimetic peptides. These ECM-derived ligands are under research for regulation of mesenchymal stem cells osteogenic differentiation in a synergic manner. The hMSCs osteogenic differentiation was significantly affected by the spatial distribution of dually grafted peptides on surfaces, and hMSCs cells reacted differently according to the shape and size of peptide micropatterns. Our study demonstrates the presence of a strong interplay between peptide geometric cues and stem cell differentiation toward the osteoblastic lineage. These tethered surfaces provide valuable tools to investigate stem cell fate mechanisms regulated by multiple ECM cues, thereby contributing to the design of new biomaterials and improving hMSCs differentiation cues.

Microchannel Molding Combined with Layer-by-Layer Approach for the Formation of Three-Dimensional Tube-like Structures by Endothelial Cells.

The development of a functional in vitro model for microcirculation is an unresolved challenge, with major impact for the creation and regeneration of organs in the tissue engineering. The absence of prevascularized engineered tissues limits enormously their efficacy and integration. Therefore, in this study, the in vitro formation of tubular-like structures with human umbilical vein endothelial cells (HUVECs) is investigated thanks to three-dimensional polycarbonate (PC) microchannel (µCh) scaffolds, surface biofunctionalized with hyaluronic acid/chitosan (HA/CHI) layer-by-layer (LbL) films grafted with adhesive (RGD) and angiogenic (SVV and QK) peptides, alone and in combination. The importance of this work lies in the formation of capillaries in the order of tens of µm, developing spontaneous microvessels, without the complexity of microfluidic approaches, and in a short time-scale. Ellipsometry, confocal laser scanning microscopy, and fluorospectrometry are used to characterize the biofunctionalized microchannels. PC-µCh scaffolds functionalized with (HA/CHI)12.5 film (PC-LbL) and further grafted with RGD and QK peptides (PC-RGD+QK) or with RGD and SVV peptides (PC-RGD+SVV) are then tested for in vitro blood vessel formation. These assays evidence a rapid formation of tubular-like structures after 2 h of incubation. Moreover, a coculture system involving HUVECs and human pericytes derived from placenta (hPCs-PL) stabilizes the tubes for a longer time.

Bioactive micropatterning of biomaterials for induction of endothelial progenitor cell differentiation: Acceleration of in situ endothelialization.

Synthetic grafts do not provide an appealing surface for endothelial cells to adhere and colonize the inner surface. To promote in situ endothelialization the following aspect has to be taken into account, endothelial progenitor cells (EPCs) needs to be mobilized on the surface of the graft. The surface of the graft has to be sufficiently biocompatible to create a prone environment for the EPCs to adhere, proliferate and, differentiate to form a layer and subsequently improve graft patency. In this work, two active molecules GRGDS and sitagliptin, were chosen for their abilities to recruit, enhance adhesion and induce differentiation of endothelial progenitor cells. They were grafted on PET surfaces in order to provide restrained cues triggering cell alignment and evaluate the influence of such structuration on EPCs fate. We then analyze cell behavior onto functionalized biomaterials. Their abilities to control EPCs fate were demonstrated via RT-qPCR, immunofluorescence, and enzymatic tests. The GRGDS/sitagliptin 100 × 10 surface enables to reduce the stemness phenotype on EPCs and induce the expression of endothelial lineage markers. These results highlight the importance of spatial patterning cues in guiding EPCs organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.

Label-free multi-parametric imaging of single cells: dual picosecond optoacoustic microscopy.

Advances in microscopy with new visualization possibilities often bring dramatic progress to our understanding of the intriguing cellular machinery. Picosecond optoacoustic micro-spectroscopy is an optical technique based on ultrafast pump-probe generation and detection of hypersound on time durations of picoseconds and length scales of nanometers. It is experiencing a renaissance as a versatile imaging tool for cell biology research after a plethora of applications in solid-state physics. In this emerging context, this work reports on a dual-probe architecture to carry out real-time parallel detection of the hypersound propagation inside a cell that is cultured on a metallic substrate, and of the hypersound reflection at the metal/cell adhesion interface. Using this optoacoustic modality, several biophysical properties of the cell can be measured in a noncontact and label-free manner. Its abilities are demonstrated with the multiple imaging of a mitotic macrophage-like cell in a single run experiment.