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Methods for promoting and controlling the differentiation of human mesenchymal stem cells (hMSCs) in vitro before in vivo transplantation are crucial for the advancement of tissue engineering and regenerative medicine. In this study, we developed poly(ethylene glycol) diacrylate (PEGDA) hydrogels with tunable mechanical properties, including elasticity and viscoelasticity, coupled with bioactivity achieved through the immobilization of a mixture of RGD and a mimetic peptide of the BMP-2 protein. Despite the key relevance of hydrogel mechanical properties for cell culture, a standard for its characterization has not been proposed, and comparisons between studies are challenging due to the different techniques employed. Here, a comprehensive approach was employed to characterize the elasticity and viscoelasticity of these hydrogels, integrating compression testing, rheology, and atomic force microscopy (AFM) microindentation. Distinct mechanical behaviors were observed across different PEGDA compositions, and some consistent trends across multiple techniques were identified. Using a photoactivated cross-linker, we controlled the functionalization density independently of the mechanical properties. X-ray photoelectrin spectroscopy and fluorescence microscopy were employed to evaluate the functionalization density of the materials before the culturing of hMSCs on them. The cells cultured on all functionalized hydrogels expressed an early osteoblast marker (Runx2) after 2 weeks, even in the absence of a differentiation-inducing medium compared to our controls. Additionally, after only 1 week of culture with osteogenic differentiation medium, cells showed accelerated differentiation, with clear morphological differences observed among cells in the different conditions. Notably, cells on stiff but stress-relaxing hydrogels exhibited an overexpression of the osteocyte marker E11. This suggests that the combination of the functionalization procedure with the mechanical properties of the hydrogel provides a potent approach to promoting the osteogenic differentiation of hMSCs.
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Regeneración Ósea , Elasticidad , Hidrogeles , Células Madre Mesenquimatosas , Polietilenglicoles , Humanos , Polietilenglicoles/química , Hidrogeles/química , Hidrogeles/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Regeneración Ósea/efectos de los fármacos , Viscosidad , Diferenciación Celular/efectos de los fármacos , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/farmacología , Oligopéptidos/química , Oligopéptidos/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Ingeniería de Tejidos/métodosRESUMEN
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.
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Adhesión Celular , Silanos , Silicio , Propiedades de Superficie , Adhesión Celular/efectos de los fármacos , Silicio/química , Silanos/química , Humanos , Oligopéptidos/química , Oligopéptidos/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacologíaRESUMEN
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.
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Células Madre Mesenquimatosas , Osteogénesis , Adulto , Humanos , Señales (Psicología) , Ligandos , Diferenciación Celular , Péptidos/química , Células MadreRESUMEN
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.
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Señales (Psicología) , Osteogénesis , Humanos , Diferenciación Celular , Huesos , OligopéptidosRESUMEN
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.
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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.
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Células Madre Mesenquimatosas , Osteogénesis , Diferenciación Celular , Células Cultivadas , Humanos , Hidrogeles/química , Hidrogeles/farmacología , OsteoblastosRESUMEN
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.
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Acrilamidas/farmacología , Hidrogeles/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteocitos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Andamios del Tejido , Acrilamidas/síntesis química , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/farmacología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Reactivos de Enlaces Cruzados/química , Humanos , Hidrogeles/síntesis química , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocitos/citología , Osteocitos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacología , Estrés Mecánico , Relación Estructura-ActividadRESUMEN
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.
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Materiales Biocompatibles/farmacología , Diferenciación Celular , Células Progenitoras Endoteliales/citología , Oligopéptidos/farmacología , Fosfato de Sitagliptina/farmacología , Materiales Biocompatibles/química , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Progenitoras Endoteliales/efectos de los fármacos , Humanos , Oligopéptidos/química , Fosfato de Sitagliptina/química , Propiedades de SuperficieRESUMEN
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.
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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.
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Materiales Biocompatibles/química , Células Madre Mesenquimatosas/citología , Peptidomiméticos/química , Proteína Morfogenética Ósea 2/química , Línea Celular , Matriz Extracelular/química , Humanos , Oligopéptidos/química , Propiedades de SuperficieRESUMEN
Microvesicles (MVs) are used by various types of cells in the human body for intercellular communication, making them biomarkers of great potential for the early and non-evasive diagnosis of a spectrum of diseases. An integrated analysis including morphological, quantitative, and compositional studies is most desirable for the clinical application of MV detection; however, such integration is limited by the currently available analysis techniques. In this context, exploiting the phosphatidylserine (PS) exposure of MVs, we synthesized a series of dendritic molecules with PS-binding sites at the periphery. PS-dendron binding was studied at the molecular level using NMR approaches, whereas PS-containing membrane-dendron interaction was investigated in an aqueous environment using plasmon waveguide resonance spectroscopy. As a proof of concept, polyethylene terephthalate surface was functionalized with the synthetic dendrons, forming devices that can capture MVs to facilitate their subsequent analyses.
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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.
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Microscopía/métodos , Técnicas Fotoacústicas/métodos , Análisis de la Célula Individual , Línea Celular , Humanos , Relación Señal-Ruido , Factores de TiempoRESUMEN
Cell morphological analysis has long been used in cell biology and physiology for abnormality identification, early cancer detection, and dynamic change analysis under specific environmental stresses. This work reports on the remote mapping of cell 3D morphology with an in-plane resolution limited by optics and an out-of-plane accuracy down to a tenth of the optical wavelength. For this, GHz coherent acoustic phonons and their resonance harmonics were tracked by means of an ultrafast opto-acoustic technique. After illustrating the measurement accuracy with cell-mimetic polymer films we map the 3D morphology of an entire osteosarcoma cell. The resulting image complies with the image obtained by standard atomic force microscopy, and both reveal very close roughness mean values. In addition, while scanning macrophages and monocytes, we demonstrate an enhanced contrast of thickness mapping by taking advantage of the detection of high-frequency resonance harmonics. Illustrations are given with the remote quantitative imaging of the nucleus thickness gradient of migrating monocyte cells.
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Forma de la Célula , Imagenología Tridimensional , Fonones , Análisis de la Célula Individual , Acústica , Línea Celular Tumoral , Humanos , Macrófagos/patología , Monocitos/patología , Óptica y Fotónica , Osteosarcoma/diagnóstico por imagen , Osteosarcoma/patología , Polimetil Metacrilato/químicaRESUMEN
Nanotopography with length scales of the order of extracellular matrix elements offers the possibility of regulating cell behavior. Investigation of the impact of nanotopography on cell response has been limited by the inability to precisely control geometries, especially at high spatial resolutions and across practically large areas. In this paper, we demonstrate well-controlled and periodic nanopillar arrays of silicon and investigate their impact on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Silicon nanopillar arrays with critical dimensions in the range of 40-200 nm, exhibiting standard deviations below 15% across full wafers, were realized using the self-assembly of block copolymer colloids. Immunofluorescence and quantitative polymerase chain reaction measurements reveal clear dependence of osteogenic differentiation of hMSCs on the diameter and periodicity of the arrays. Further, the differentiation of hMSCs was found to be dependent on the age of the donor. While osteoblastic differentiation was found to be promoted by the pillars with larger diameters and heights independent of donor age, they were found to be different for different spacings. Pillar arrays with smaller pitch promoted differentiation from a young donor, while a larger spacing promoted those of an old donor. These findings can contribute for the development of personalized treatments of bone diseases, namely, novel implant nanostructuring depending on patient age.
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Nanoestructuras/química , Adulto , Anciano , Células de la Médula Ósea/citología , Diferenciación Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Microscopía Fluorescente , Osteogénesis , Osteopontina/genética , Osteopontina/metabolismo , Poliestirenos/química , Polivinilos/química , Piridinas/química , Silicio/química , Análisis de Matrices Tisulares/instrumentación , Análisis de Matrices Tisulares/métodosRESUMEN
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.
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Células Progenitoras Endoteliales/metabolismo , Perfilación de la Expresión Génica , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , Algoritmos , Diferenciación Celular/genética , Células Cultivadas , Sangre Fetal/metabolismo , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Humanos , Leucocitos Mononucleares/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 µm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.
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Adhesión Celular , Microscopía Acústica , Humanos , Ingeniería de Tejidos , UltrasonidoRESUMEN
The commitment and differentiation of human mesenchymal stem cells (hMSCs) are guided by bioactive molecules within the extracellular matrix. Among the various approaches to design biomaterials, the functionalization of biomaterial surfaces with peptides from the sequence of proteins from the extracellular matrix is quite common. The purpose of this functionalization is to recruit hMSCs and promote their differentiation into the appropriate lineage. The aim of this work was to investigate the influence of RGD and FHRRIKA peptides and peptide sequences taken from bone morphogenic protein (BMP-2) and histone H4 (osteogenic growth peptide; OGP) either tethered alone or as a mixture on the surface of a model material and to also examine the level of hMSC osteogenic commitment without using a differentiation medium. Grafting of the different peptides was assessed by X-ray photoelectron spectroscopy (XPS), while their surface density was quantified by fluorescence microscopy, and their surface properties were assessed by atomic force microscopy (AFM) and contact angle (CA). The osteogenic commitment of hMSCs cultured on the different surfaces was characterized by immunohistochemistry using Runx-2 as an earlier osteogenic marker and OPN, a late osteogenic marker, and by RT-qPCR through the expression of ColI-a1, Runx-2, and ALP. Biological results show that the osteogenic commitment of the hMSCs was increased on surfaces tethered with a mixture of peptides. Results indicate that tethered peptides in the range of pmol mm-2 were indeed effective in inducing a cellular response after 2 weeks of cell culture without using an osteogenic media. These findings contribute to the research efforts to design biomimetic materials able to induce a response in human stem cells through tethered bioactive molecules for bone tissue engineering.
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Within the native microenvironment, extracellular matrix (ECM) components are thought to display a complex and heterogeneous distribution, spanning several length scales. Herein, the objective is to mimic, in vitro, the hierarchical organization of proteins and growth factors as well as their crosstalk. Photolithography technique was used to adjacently pattern geometrically defined regions of RGD and BMP-2 mimetic peptides onto glass substrates. These ECM-derived ligands are known to jointly regulate mesenchymal stem cells (MSCs) osteogenic differentiation. By manipulating the spatial distribution of dually grafted peptides, the extent of human MSCs osteogenic differentiation was significantly affected, depending on the shape of peptide micropatterns. Our data highlight the existence of a strong interplay between geometric cues and biochemical signals. Such in vitro systems provide a valuable tool to investigate mechanisms by which multiple ECM cues overlap to regulate stem cell fate, thereby contributing to the design of bioinspired biomaterials for bone tissue engineering applications.
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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.
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Antibacterianos/química , Materiales Biocompatibles Revestidos/síntesis química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Nanopartículas/química , Prótesis e Implantes/microbiología , Vancomicina/química , Antibacterianos/farmacología , Materiales Biocompatibles Revestidos/química , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Polietilenglicoles/química , Propiedades de Superficie , Titanio/química , Vancomicina/farmacologíaRESUMEN
Intravascular devices for continuous glucose monitoring are promising tools for the follow up and treatment of diabetic patients. Limiting the inflammatory response to the implanted devices in order to achieve better biocompatibility is a critical challenge. Herein we report on the production and the characterization of gold surfaces covalently derivatized with the peptide α-alpha-melanocyte stimulating hormone (α-MSH), with a quantifiable surface density. In vitro study demonstrated that the tethered α-MSH is able to decrease the expression of an inflammatory cytokine produced by endothelial cells.