ABSTRACT
Collagen hydrogels are among âthe most well-studied platforms for drug delivery and in situ tissue engineering, thanks to their low cost, low immunogenicity, versatility, biocompatibility, and similarity to the natural extracellular matrix (ECM). Despite collagen being largely responsible for the tensile properties of native connective tissues, collagen hydrogels have relatively low mechanical properties in the absence of covalent cross-linking. This is particularly problematic when attempting to regenerate stiffer and stronger native tissues such as bone. Furthermore, in contrast to hydrogels based on ECM proteins such as fibronectin, collagen hydrogels do not have any growth factor (GF)-specific binding sites and often cannot sequester physiological (small) amounts of the protein. GF binding and in situ presentation are properties that can aid significantly in the tissue regeneration process by dictating cell fate without causing adverse effects such as malignant tumorigenic tissue growth. To alleviate these issues, researchers have developed several strategies to increase the mechanical properties of collagen hydrogels using physical or chemical modifications. This can expand the applicability of collagen hydrogels to tissues subject to a continuous load. GF delivery has also been explored, mathematically and experimentally, through the development of direct loading, chemical cross-linking, electrostatic interaction, and other carrier systems. This comprehensive article explores the ways in which these parameters, mechanical properties and GF delivery, have been optimized in collagen hydrogel systems âand examines their in vitro or in vivo biological effect. This article can, therefore, be a useful tool to streamline future studies in the field, by pointing researchers into the appropriate direction according to their collagen hydrogel design requirements.
ABSTRACT
A one-step microfluidic system is developed in this study which enables the encapsulation of stem cells and genetically engineered non-pathogenic bacteria into a so-called three-dimensional (3D) pearl lace-like microgel of alginate with high level of monodispersity and cell viability. The alginate-based microgel constitutes living materials that control stem cell differentiation in either an autonomous or heteronomous manner. The bacteria (Lactococcus lactis) encapsulated within the construct surface display adhesion fragments (III7-10 fragment of human fibronectin) for integrin binding while secreting growth factors (recombinant human bone morphogenetic protein-2) to induce osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. We concentrate on interlinked pearl lace microgels that enabled us to prototype a low-cost 3D bioprinting platform with highly tunable properties.
ABSTRACT
Cell migration is a fundamental process involved in a wide range of biological phenomena. However, how the underlying mechanisms that control migration are orchestrated is not fully understood. In this work, we explore the migratory characteristics of human fibroblasts using different organisations of fibronectin (FN) triggered by two chemically similar surfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA); cell migration is mediated via an intermediate layer of fibronectin (FN). FN is organised into nanonetworks upon simple adsorption on PEA whereas a globular conformation is observed on PMA. We studied cell speed over the course of 24 h and the morphology of focal adhesions in terms of area and length. Additionally, we analysed the amount of cell-secreted FN as well as FN remodelling. Velocity of human fibroblasts was found to exhibit a biphasic behaviour on PEA, whereas it remained fairly constant on PMA. FA analysis revealed more mature focal adhesions on PEA over time contrary to smaller FAs found on PMA. Finally, human fibroblasts seemed to remodel adsorbed FN more on PMA than on PEA. Overall, these results indicate that the cell-protein-material interface affects cell migratory behaviour. Analysis of FAs together with FN secretion and remodelling were associated with differences in cell velocity providing insights into the factors that can modulate cell motility.
Subject(s)
Cell Movement/drug effects , Cellular Microenvironment/drug effects , Fibronectins/pharmacology , Adsorption , Cell Adhesion/drug effects , Fibronectins/chemistry , Focal Adhesions/drug effects , Focal Adhesions/metabolism , HumansABSTRACT
Here we report on the development of a new type of hybrid fibrinogen-polylactic acid (FBG-PLA) nanofibres (NFs) with improved stiffness, combining the good mechanical properties of PLA with the excellent cell recognition properties of native FBG. We were particularly interested in the dorsal and ventral cell response to the nanofibres' organization (random or aligned), using human umbilical endothelial cells (HUVECs) as a model system. Upon ventral contact with random NFs, the cells developed a stellate-like morphology with multiple projections. The well-developed focal adhesion complexes suggested a successful cellular interaction. However, time-lapse analysis shows significantly lowered cell movements, resulting in the cells traversing a relatively short distance in multiple directions. Conversely, an elongated cell shape and significantly increased cell mobility were observed in aligned NFs. To follow the dorsal cell response, artificial wounds were created on confluent cell layers previously grown on glass slides and covered with either random or aligned NFs. Time-lapse analysis showed significantly faster wound coverage (within 12 h) of HUVECs on aligned samples vs. almost absent directional migration on random ones. However, nitric oxide (NO) release shows that endothelial cells possess lowered functionality on aligned NFs compared to random ones, where significantly higher NO production was found. Collectively, our studies show that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization. Copyright © 2016 John Wiley & Sons, Ltd.
Subject(s)
Blood Vessels/physiology , Fibrinogen/pharmacology , Nanofibers/chemistry , Polyesters/pharmacology , Tissue Engineering/methods , Adhesiveness , Animals , Blood Vessels/drug effects , Cattle , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Microscopy, Atomic Force , Nanofibers/ultrastructure , Nitric Oxide/metabolismABSTRACT
Vascular basement membrane remodeling involves assembly and degradation of its main constituents, type IV collagen (Col IV) and laminin, which is critical during development, angiogenesis, and tissue repair. Remodeling can also occur at cell-biomaterials interface altering significantly the biocompatibility of implants. Here we describe the fate of adsorbed Col IV in contact with endothelial cells adhering on positively charged NH2 or hydrophobic CH3 substrata, both based on self-assembly monolayers (SAMs) and studied alone or mixed in different proportions. AFM studies revealed distinct pattern of adsorbed Col IV, varying from single molecular deposition on pure NH2 to network-like assembly on mixed SAMs, turning to big globular aggregates on bare CH3. Human umbilical endothelial cells (HUVECs) interact better with Col IV adsorbed as single molecules on NH2 surface and readily rearrange it in fibril-like pattern that coincide with secreted fibronectin fibrils. The cells show flattened morphology and well-developed focal adhesion complexes that are rich on phosphorylated FAK while expressing markedly low pericellular proteolytic activity. Conversely, on hydrophobic CH3 substrata HUVECs showed abrogated spreading and FAK phosphorylation, combined with less reorganization of the aggregated Col IV and significantly increased proteolytic activity. The later involves both MMP-2 and MMP-9, as measured by zymography and FITC-Col IV release. The mixed SAMs support intermediate remodeling activity. Taken together these results show that chemical functionalization combined with Col IV preadsorption provides a tool for guiding the endothelial cells behavior and pericellular proteolytic activity, events that strongly affect the fate of cardiovascular implants.
Subject(s)
Biocompatible Materials , Collagen Type IV/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Surface PropertiesABSTRACT
Conventional 2D substrates fail to represent the natural environment of cells surrounded by the 3D extracellular matrix (ECM). We have proposed sandwich-like microenvironments as a versatile tool to study cell behaviour under quasi-3D conditions. This is a system that provides a broad range of dorsal and ventral independent spatio-temporal stimuli. Here, we use this sandwich technology to address the role of dorsal stimuli in cell adhesion, cell proliferation and ECM reorganisation. Under certain conditions, dorsal stimuli within sandwich microenvironments prevent the formation of focal plaques as well as the development of the actin cytoskeleton, whereas α5versusαv integrin expression is increased compared to the corresponding 2D controls. Cell signaling is similarly enhanced after dorsal stimuli (measured by the pFAK/FAK level) for cells sandwiched after 3 h of 2D ventral adhesion, but not when sandwiched immediately after cell seeding (similar levels to the 2D control). Cell proliferation, studied by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay, was significantly reduced within sandwich conditions as compared to 2D substrates. In addition, these results were found to depend on the ability of cells to reorganise the dorsal layer of proteins at the material interface, which could be tuned by adsorbing FN on material surfaces that results in a qualitatively different conformation and distribution of FN. Overall, sandwich-like microenvironments switch cell behaviour (cell adhesion, morphology and proliferation) towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo.
ABSTRACT
Due to the large potential of electroactive materials in novel tissue engineering strategies, the aim of this work is to determine if the crystalline phase and/or the surface electrical charge of electroactive poly(vinylidene fluoride), PVDF, have influence on the biological response in monolayer cell culture. Non-polar α-PVDF and electroactive ß-PVDF were prepared. The ß-PVDF films were poled by corona discharge to show negative or positive electrical surface charge density. It has been concluded that hydrophilicity of the PVDF substrates depends significantly on crystalline phase and polarity. Furthermore, by means of atomic force microscopy and an enzyme-linked immunosorbent assay test, it has been shown that positive or negative poling strongly influences the behavior of ß-PVDF supports with respect to fibronectin (FN) adsorption, varying the exhibition of adhesion ligands of adsorbed FN. Culture of MC3T3-E1 pre-osteoeblasts proved that cell proliferation depends on surface polarity as well. These results open the viability of cell culture stimulation by mechanical deformation of a piezoelectric substrate that results in varying electrical charge densities on the substrate surface.
Subject(s)
Cell Adhesion/drug effects , Cell Survival/drug effects , Coated Materials, Biocompatible/pharmacology , Fibronectins/pharmacology , Membranes, Artificial , Polyvinyls/chemistry , 3T3 Cells , Adsorption , Animals , Coated Materials, Biocompatible/chemistry , Fibronectins/chemistry , Materials Testing , Mice , Static Electricity , Surface PropertiesABSTRACT
Poly(ε-caprolactone)/poly(hydroxyethyl acrylate) networks have been investigated by thermally stimulated depolarization currents (TSDC) and differential scanning calorimetry (DSC). The introduction of hydrophilic units (HEA) in the system aiming at tailoring the hydrophilicity of the system results in a series of copolymer networks with microphase separation into hydrophobic/hydrophilic domains. Polycaprolactone (PCL) crystallization is prevented by the topological constraints HEA units imposed in such heterogeneous domains. Moreover, the mobility of the amorphous PCL chains is enhanced as revealed by the main relaxation process which becomes faster. The glass transition of PHEA-rich domains shifts to lower temperatures, as the total amount of PCL in the copolymer increases, due to the presence of PCL units within the same region. The behaviour of the copolymer networks swollen with different content of water has been investigated to analyze the interaction between water molecules and hydrophobic/hydrophilic domains and provide further insights into the molecular structure of the system.
Subject(s)
Biocompatible Materials/chemistry , Polyesters/chemistry , Polyhydroxyethyl Methacrylate/chemistry , Biophysics/methods , Calorimetry, Differential Scanning/methods , Crystallization , Glass , Hot Temperature , Kinetics , Materials Testing , Molecular Structure , Polymers/chemistry , Temperature , Water/chemistryABSTRACT
Considering the structural role of type IV collagen (Col IV) in the assembly of the basement membrane (BM) and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass) and hydrophobic trichloro(octadecyl)silane (ODS) surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50microg/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine - nearly single molecular size - network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC) attach less efficiently to the aggregated Col IV (on ODS), as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both alpha1 and alpha2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.
Subject(s)
Cell Communication/drug effects , Collagen Type IV/metabolism , Endothelial Cells/physiology , Tissue Engineering/methods , Blood Vessels/cytology , Cell Adhesion , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Glass , Humans , Hydrophobic and Hydrophilic Interactions , Protein Binding , Silanes/pharmacology , Tissue ScaffoldsABSTRACT
The isothermal cold-crystallization of the glass-former low-molecular-weight compound, ethylene glycol dimethacrylate (EGDMA), was monitored by real-time dielectric relaxation spectroscopy (DRS) and differential scanning calorimetry (DSC). The alpha-relaxation associated with the dynamic glass transition as detected by DRS was followed at different crystallization temperatures, T(cr), nearly above the glass transition temperature, 176 K (1.06 < or = T(cr)/T(g) < or = 1.12). It was found that the alpha-process depletes upon cold-crystallization with no significant changes in either shape or location. At advanced crystallization states, a new relaxation, alpha'-process, evolves that was assigned to the mobility of molecules lying adjacent to crystalline surfaces. From the time evolution of the normalized permittivity, it was possible to get kinetic information that was complemented with the calorimetric data. From DSC measurements that were also carried out under melt-crystallization, an enlarged temperature range was covered (up to T(cr)/T(g) = 1.24), allowing us to draw a diagram of time-temperature crystallization for this system. Dielectric relaxation spectroscopy proved to be a sensitive tool to probe the mobility in the remaining amorphous regions even at high crystallinities.
Subject(s)
Glass/chemistry , Methacrylates/chemistry , Calorimetry, Differential Scanning , Crystallization , Kinetics , Transition Temperature , X-Ray DiffractionABSTRACT
We have synthesized methacrylate-endcapped caprolactone networks with tailored water sorption ability, poly(CLMA-co-HEA), in the form of three-dimensional (3D) scaffolds with the same architecture but exhibiting different hydrophilicity character (x(HEA)=0, 0.3, 0.5), and we investigated the interaction of goat bone marrow stromal cells (GBMSCs) with such structures. For this purpose, GBMSCs were seeded and cultured for 3, 7, 14, 21, and 28 days onto the developed scaffolds. Cells have proliferated throughout the whole scaffold volume. Cell adhesion and morphology were analyzed by SEM, whereas cell viability and proliferation was assessed by MTS test and DNA quantification concluding that numbers of cells increased as a function of the culturing time (until day 14) and also with the hydrophobic content in the samples (from 50 to 100% of CLMA). No significant difference between samples with 100% and 70% of CLMA were detected in some cases. Osteoblastic differentiation was followed by assessing the alkaline phosphatase activity of cells, as well as type I collagen and osteocalcin expressions levels until day 21. The three markers were positive at days 14 and 21 when cells were cultured in 100% CLMA substrates which suggests osteoblastic differentiation of mesenchymal stem cells within these scaffolds. On the other hand, when the CLMA content decreases (until 50%), type I collagen and osteocalcin were positive but ALP was negative indicating that the differentiation process is affected by hydrophilic content. We suggest that such system may be useful to extract information on the effect of materials' wettability on the corresponding biological performance in a 3D environment. Such general insights may be relevant in the context of biomaterials selection for tissue engineering strategies.
Subject(s)
Bone Marrow Cells/physiology , Cell Differentiation/physiology , Cell Proliferation , Goats , Stromal Cells/physiology , Tissue Scaffolds , Animals , Biocompatible Materials/chemistry , Biomarkers/metabolism , Bone Marrow Cells/cytology , Caproates/chemistry , Cell Adhesion/physiology , Cell Culture Techniques , Cells, Cultured , Lactones/chemistry , Materials Testing , Methacrylates/chemistry , Osteoblasts/cytology , Osteoblasts/physiology , Polymers/chemistry , Porosity , Stromal Cells/cytology , Surface Properties , Wettability , X-Ray MicrotomographyABSTRACT
The aim of this work is to explore the consequences on the kinetics of structural relaxation of considering a glass-forming system to consist of a series of small but macroscopic relaxing regions that evolve independently from each other towards equilibrium in the glassy state. The result of this assumption is a thermorheologically complex model. In this approach each relaxing zone has been assumed to follow the Scherer-Hodge model for structural relaxation (with the small modification of taking a linear dependence of configurational heat capacity with temperature). The model thus developed contains four fitting parameters. A least-squares search routine has been used to find the set of model parameters that fit simultaneously four DSC thermograms in PVAc after different thermal histories. The computer-simulated curves are compared with those obtained with Scherer-Hodge model and the model proposed by Gómez and Monleón. The evolution of the relaxation times during cooling or heating scans and also during isothermal annealing below the glass transition has been analysed. It has been shown that the relaxation times distribution narrows in the glassy state with respect to equilibrium. Isothermal annealing causes this distribution to broaden during the process to finally attain in equilibrium the shape defined at temperatures above Tg.
ABSTRACT
The enthalpy relaxation of polymer-silica nanocomposites prepared by simultaneous polymerization of poly(2-hydroxyethyl methacrylate) (PHEMA) and tetraethyloxysilane, TEOS, a silica precursor, is investigated. Both the glass transition temperature, Tg, and the temperature interval of the glass transition, DeltaTg , increase as the silica content in the sample does. Structural relaxation experiments show that the temperature interval in which conformational motions take place broadens as the silica content in the hybrid increases. A phenomenological model based on the evolution of the configurational entropy during the structural relaxation process, the SC model, has been used for determining the temperature dependence of the relaxation times during the process. The results show an increase of the fragility of the polymer as the silica content increases, a feature that can be related to the broadening of the distribution of relaxation times characterized by the beta parameter of the stretched exponential distribution. On another hand the silica content increase produces a significant change of the relaxation times in the glassy state.
Subject(s)
Nanocomposites/chemistry , Polyhydroxyethyl Methacrylate/chemistry , Silicon Dioxide/chemistry , Calorimetry, Differential Scanning , Chemistry, Inorganic/methods , Chemistry, Organic/methods , Entropy , Glass , Kinetics , Models, Statistical , Nanotechnology/methods , Polymers/chemistry , Temperature , ThermodynamicsABSTRACT
Human articular chondrocytes were cultured in vitro on poly(L-lactic) acid, PLLA, substrates. Influence of the surface topography on cell morphology was found. Different surface microtopographies were obtained on PLLA by crystallizing at 120 degrees C after nucleation treatments that include isothermal stages at temperatures just below (55 degrees C) and just above (75 degrees C) the glass transition temperature (T(g) = 65 degrees C). Isothermal crystallization from the melt gave rise to big spherulites (approx. 50 microm diameter) with approx. 1 microm depth. Crystallization after nucleation treatments results in smaller (approx. 5 microm)-difficult to distinguish-spherulites. Cell viability was excellent and not affected by the surface roughness. Cell population on the nucleated samples resembles the result of culture on the reference tissue culture polystyrene (TCPS). However, cells cultured on big spherulites (PLLA isothermally crystallized without nucleation treatment) show a peculiar morphology, with a more isolated disposition and growth oriented in a characteristic direction.
Subject(s)
Biocompatible Materials/chemistry , Cell Culture Techniques/methods , Chondrocytes/cytology , Chondrocytes/physiology , Chondrogenesis/physiology , Polyesters/chemistry , Tissue Engineering/methods , Cell Proliferation , Cell Size , Cell Survival , Cells, Cultured , Humans , Materials Testing , Surface PropertiesABSTRACT
A series of polymeric biomaterials including poly (methyl acrylate) (PMA), chitosan (CHT), poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate and hydroxyethyl acrylate monomeric units were tested in vitro as culture substrates and compared for their impact on the proliferation and expansion of Schwann cells (SCs). Immunocytochemical staining assay and scanning electron microscopy techniques were applied to perform a quantitative analysis to determine the correct maintenance of the cultured glial cells on the different biomaterials. The results strongly suggest that cell attachment and proliferation is influenced by the substrate's surface chemistry, and that hydrophobic biomaterials based on PMA, PEA, and the copolymers PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and proliferation of SCs in vitro.
Subject(s)
Schwann Cells/cytology , Animals , Cell Adhesion , Cell Differentiation , Cell Proliferation , Cell Survival , Cells, Cultured , Rats , Rats, Wistar , Schwann Cells/ultrastructure , Substrate Specificity , Surface Tension , Water/metabolismABSTRACT
The dielectric relaxation spectrum of polycaprolactone (PCL) networks hydrophilized with different amounts of 2-hydroxyethyl acrylate (HEA) is investigated. PCL is a semicrystalline polyester with a complex relaxation spectrum that includes the main alpha relaxation and two secondary modes (beta, gamma) at lower temperatures. The overlapping of the different relaxational modes was split by using several Havriliak-Negami functions. Crosslinking the material modifies the dynamics of the main relaxation process as reflected by the parameters that characterize the Vogel behavior of the process and the dynamic fragility. The incorporation of HEA units in the network results in a material with microphase separation: two alpha processes are detected, the one corresponding to the PCL chains and the new one associated to nanometric regions that contain different amount of both comonomers. The incorporation of the HEA units in the system involves the presence of a new beta(sw) relaxation due to the link of two side chains by water molecules through hydrogen bonding.
Subject(s)
Acrylates/chemistry , Biocompatible Materials/chemistry , Polyesters/chemistry , Calorimetry, Differential Scanning , Computer Simulation , Cross-Linking Reagents/chemistry , Hydrogen Bonding , Models, Chemical , Spectrum Analysis , TemperatureABSTRACT
The crystallization kinetics of poly(l-lactide), PLLA, is slow enough to allow a quasi-amorphous polymer to be obtained at low temperature simply by quenching from the melt. The PLLA crystallization process was followed by differential scanning calorimetry and optical microscopy after nucleation isothermal treatments at temperatures just below (53 degrees C) and just above (73 degrees C) the glass transition temperature. The crystallization exotherm shown in the heating thermograms shifts toward lower temperatures as the annealing time at 73 degrees C increases. The same effect is shown to a lesser extent when the sample nucleates at 53 degrees C, showing the ability to nucleate in the glassy state, already shown in other polymers. The shape of the DSC thermograms is modeled by using Avrami's theory and allows an estimation of the number of crystallization germs formed. The results of optical microscopy are converted to thermograms by evaluating the average gray level of the image recorded in transmission mode as a function of temperature and calculating its temperature derivative. The shape of such optical thermograms is quite similar to that of the DSC traces but shows some peculiarities after long nucleation treatments. Atomic force microscopy was used to analyze the crystal morphology and is an additional proof of the effect of nucleation in the glassy state. The crystalline morphology observed in samples crystallized after nucleation in the glassy state is qualitatively different from that of samples nucleated above the glass transition temperature, and the number of crystals seems to be much greater than what could be expected from the crystallization kinetics.