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A nonfouling peptide grafted polymer was synthesized that can promote endothelial cell (EC) binding. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate, and CGRGDS peptide. The peptide was incorporated into the polymer system either by a chain transfer reaction or by coupling to an acrylate-PEG-N...
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... of peptides by N-Hydroxysuccinimide (NHS) chemistry In a two-step reaction, the PEG monomer was first combined with the peptide (Fig. 2). The average molecular weight of acrylate-PEG-N-hydroxysuccinimide (JenKem Technology, Allen, TX) was 2000. The reaction was conducted at a molar ratio of 1:1.2. To prevent the production of acryloyl-PEG-OH, the product of the competing hydrolysis reaction, anhydrous dimethylformamide (DMF) was used as the solvent. Unacry- lated ...
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The interest in dextran hydrogels for soft tissue engineering has increased due to their great biocompatibility and capability to reduce nonspecific protein adsorption and cell attachment. However, these properties are not sufficient for dextran hydrogels to fulfill their role to work as a tissue engineering scaffold candidate. In addition, the sca...
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... This is because our cellular experiments showed that PEG 2 -AQEE30 more strongly enhanced the binding to C3AR1, cAMP secretion, proliferation, and mineralization activities of osteoblasts than AQEE30. Actually, although the exact mechanism is unknown, it has been reported that PEGylation can regulate cell adhesion and growth 47,48 , and speci cally promoted the proliferation of human osteoblasts in vitro. ...
New therapeutic targets, especially those that stimulate bone formation in cortical bone, are needed to overcome the limitations of current anti-osteoporotic drugs. We previously demonstrated that factors secreted from megakaryocytes (MK) promote bone formation. In this study, we conducted a proteomic analysis to identify a novel bone-forming factor from MK secretions. We revealed that Vgf , a nerve growth factor-responsive gene, and its derived active peptide AQEE30 in MK-conditioned media, played an important role in osteoblast proliferation and in vitro bone formation. In both Vgf -deficient male and female mice, cortical bone mass was significantly decreased due to reduced osteoblast number and bone formation activity. AQEE30 stimulated intracellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity in osteoblasts, whereas an adenylyl cyclase inhibitor blocked the AQEE30-stimulated osteoblast proliferation and in vitro bone formation. Complement C3a receptor-1 (C3AR1) were expressed and interacted with AQEE30 in osteoblasts, and C3AR1 inhibitions blocked all AQEE30-induced changes, including stimulated proliferation, bone formation, and cAMP production, in osteoblasts. Injecting a mini-PEGylated AQEE30 into calvaria increased the number of osteocalcin-positive cells and new bone formation. In conclusion, this study reveals a novel role of VGF in bone formation, particularly in cortical bone, and shows that AQEE30, a VGF-derived peptide, mediates this role by activating cAMP-PKA signalling via the C3AR1 receptor in osteoblasts.
... Previous studies have demonstrated that laminin-associated motifs including arginineglycine-aspartic acid (RGD) and isoleucine-lysinevaline-alanine-valine (IKVAV) can create engineered microenvironments that mimic native cell-matrix interaction and support endothelial growth and tubulogenesis [13]. RGD in particular has been used to facilitate endothelial attachment in both 2D [14,15] and 3D [16] systems. The RGD motif binds to a wide array of integrin isoforms, perhaps the most well-characterized being αVβ3 [17], that are known to mediate the endothelial response to shear stress [18]. ...
Recent developments in digital light processing (DLP) can advance the structural and biochemical complexity of perfusable in vitro models of the blood-brain barrier. Here, we describe a strategy to functionalize complex, DLP-printed vascular models with multiple peptide motifs in a single hydrogel. Different peptides can be clicked into the walls of distinct topologies, or the peptide motifs lining channel walls can differ from those in the bulk of the hydrogel. The flexibility of this approach is used to both characterize the effects of various bioactive domains on endothelial coverage and tight junction formation, in addition to facilitating astrocyte attachment in the hydrogel surrounding the endothelialized vessel to mimic endothelial-astrocyte interaction. Peptides derived from proteins mediating cell-extracellular matrix (ECM) (e.g. RGD and IKVAV) and cell-cell (e.g. HAVDI) adhesions are used to mediate endothelial cell attachment and coverage. HAVDI and IKVAV-lined channels exhibit significantly greater endothelialization and increased ZO-1 localization to cell-cell junctions of endothelial cells, indicative of tight junction formation. RGD is then used in the bulk hydrogel to create an endothelial-astrocyte co-culture model of the blood-brain barrier that exceeds the limitations of previous platforms incapable of complex topology or tunable bioactive domains. This approach yields an adjustable, biofabricated platform to interrogate the effects of cell-matrix interaction on blood-brain barrier mechanobiology.
... The development of antifouling, protein repellent-based materials is relevant to reduce nonspecific binding and promote simultaneous endothelialization. Non-fouling CGRGD-grafted polymers were synthesized according to different procedures [53] . Acrylate-PEG-NHS was first reacted with NH 2 RGD peptide (1:1.2 ...
Nanofiber films produced by electrospinning currently provide a promising platform for different applications. Although nonfunctionalized nanofiber films from natural or synthetic polymers are extensively used, electrospun materials combined with peptides are gaining more interest. In fact, the selection of specific peptides improves the performance of the material for biological applications and mainly for tissue engineering, mostly by maintaining similar mechanical properties with respect to the simple polymer. The main drawback in using peptides blended with a polymer is the quick release of the peptides. To avoid this problem, covalent linking of the peptide is more beneficial. Here, we reviewed synthetic protocols that enable covalent grafting of peptides to polymers before or after the electrospinning procedures to obtain more robust electrospun materials. Applications and the performance of the new material compared to that of the starting polymer are discussed.
... This detailed mechanistic knowledge has resulted in the development of bioactive materials that are functionalized with polypeptide motifs or ligands that regulate cell adhesion through specific interactions with a cell's integrin receptors (Geiger et al., 2001;Humphries et al., 2006;. For nearly 30 years, researchers have functionalized synthetic materials with a variety of integrin-binding ligands such as the RGD, GFOGER, REDV, and CRRETAWAC polypeptides to promote specific, integrin-mediated cellular adhesion (Massia & Hubbell, 1991;Hersel et al., 2003;Wang et al., 2012;Cherny et al., 1993;Noel et al., 2015;Larsen et al., 2007;Knight et al., 2000;Wojtowicz et al., 2010;Bellis, 2011;Massia & Hubbell, 1992). ...
We have engineered biomaterials that display nanoclusters of ligands that bind both integrin and syndecan‐4 cell receptors. These surfaces regulate cell behaviours under static conditions including adhesion, spreading, actin stress fibre formation, and migration. The syndecan‐4 receptors are also critical mediators of cellular mechanotransduction. In this contribution we assess whether this novel class of materials can regulate the response of cells to applied mechanical stimulation, using the shear stress imparted by laminar fluid flow as a model stimulus. Specifically, we assess endothelial cell detachment due to flow, cell alignment due to flow, and cell adhesion from the flowing fluid. A high degree of cell retention was observed on surfaces containing integrin‐binding ligands or a mixed population of integrin‐ and syndecan‐binding ligands. However, the presence of both ligand types was necessary for the cells to align in the direction of flow. These results imply that integrin engagement is necessary for adhesion strength, but engagement of both receptor types aids in appropriate mechanotransduction. Additionally, it was found that surfaces functionalised with both ligand types were able to scavenge a larger number of cells from flow, and to do so at a faster rate, compared to surfaces functionalized with only integrin‐ or syndecan‐binding ligands. These results show that interfaces functionalized with both integrin‐ and syndecan‐binding ligands regulate a significant range of biophysical cell behaviours in response to shear stress.
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... The cells were analyzed deliberately after a relative short incubation time of eight hours, because the focus of this study was the initial phase of cell adherence. HUVEC adhered within few hours, markedly more HUVEC on the surface with RGD-ligands [55]. Under static conditions the initial adherence of HUVEC could be significantly improved on PEI-films by the aptamer-cRGD functionalization in both cell culture systems. ...
Implantation of synthetic small-diameter vascular bypass grafts is often associated with an increased risk of failure, due to thrombotic events or late intimal hyperplasia. As one of the causes an insufficient hemocompatibility of the artificial surface is discussed. Endothelialization of synthetic grafts is reported to be a promising strategy for creating a self-renewing and regulative anti-thrombotic graft surface. However, the establishment of a shear resistant cell monolayer is still challenging. In our study, cyto- and immuno-compatible poly(ether imide) (PEI) films were explored as potential biomaterial for cardiovascular applications. Recently, we reported that the initial adherence of primary human umbilical vein endothelial cells (HUVEC) was delayed on PEI-films and about 9 days were needed to establish a confluent and almost shear resistant HUVEC monolayer. To accelerate the initial adherence of HUVEC, the PEI-film surface was functionalized with an aptamer-cRGD peptide based endothelialization supporting system. With this functionalization the initial adherence as well as the shear resistance of HUVEC on PEI-films was considerable improved compared to the unmodified polymer surface. The in vitro results confirm the general applicability of aptamers for an efficient functionalization of substrate surfaces.
... Usually, the endothelialization of implant materials is evaluated in in vitro studies [1,[5][6][7][8][9][10][11][12][13]. For this purpose, endothelial cells are seeded and subsequently the adherence of the endothelial cells until monolayer formation is investigated in terms of endothelial cell density, morphology, viability and cell function. ...
The endothelialisation of synthetic surfaces applied as cardiovascular implant materials is an important issue to ensure the anti-thrombotic quality of a biomaterial. However, the rapid and constant development of a functionally-confluent endothelial cell monolayer is challenging. In order to investigate the compatibility of potential implant materials with endothelial cells several in vitro studies are performed. Here, glass and tissue culture plates (TCP) are often used as reference materials for in vitro pre-testing. However, a direct comparison of both substrates is lacking.Therefore, a comparison of study results is difficult, since results are often related to various reference materials. In this study, the endothelialisation of glass and TCP was investigated in terms of adherence, morphology, integrity, viability and function using human umbilical vein endothelial cells (HUVEC).On both substrates an almost functionally confluent HUVEC monolayer was developed after nine days of cell seeding with clearly visible cell rims, decreased stress fiber formation and a pronounced marginal filament band. The viability of HUVEC was comparable for both substrates nine days after cell seeding with only a few dead cells. According to that, the cell membrane integrity as well as the metabolic activity showed no differences between TCP and glass. However, a significant difference was observed for the secretion of IL-6 and IL-8. The concentration of both cytokines, which are associated with migratory activity, was increased in the supernatant of HUVEC seeded on TCP. This result matches well with the slighty increased number of adherent HUVEC on TCP.In conclusion these findings indicate that both reference materials are almost comparable and can be used equivalently as control materials in in vitro endothelialisation studies.
... This in turn can affect cell morphology and migration rates ( Fig. 16.10). Mostly these ligands are adhesion sites found in the native ECM (e.g., the RGD tri-peptide found in fibronectin), 80 but researchers have also performed analyses to discover unique receptors present on their cell type of choice. For example, a phage display library was screened in order to identify ligands specific to an outgrowth of endothelial progenitor cells but not other commonly occurring cell types. ...
... For fibrinogen adsorption study, 50 mL of a 50 mg mL À1 solution of fluorescently conjugated fibrinogen (Fibrinogen from human plasma, Alexa Flour s 488 conjugated, ThermoFisher Scientific) was added to each well and allowed to interact with the surface for 40 min at 37 1C as described previously. 35,36 The solution was aspirated and the wells were washed 5 times with PBS to remove reversibly adsorbed fibrinogen. 50 mL of PBS was added to each well, and the fluorescence intensity was measured using a plate reader at excitation and emission wavelength of 480 nm and 525 nm, respectively. ...
... This observation is also supported by previously published literature from our group and others. 35,36,38 Additionally, the RAFT synthesis conditions were tailored to produce polymer chains of high molecular weight (M n 4 50 kDa) and low PDI. Polymers synthesized from pure MMA or copolymers of MMA/PEGMA had narrow 1.13); however, as the mole percent of acrylate-PEG-peptide macro-monomer increased, we observed a slight increase in PDI, up to 1.36. ...
Blood contacting devices are commonly used in today’s medical landscape. However, such devices (including small diameter vascular grafts) are limited by poor blood compatibility and may fail due to thrombosis. An attractive strategy for improving the blood compatibility of such devices is to generate biomaterials that foster a confluent and functioning endothelial cell layer. Synthesizing materials that display integrin-binding peptide ligands is a common way to promote endothelialization. However, in addition to integrin-ligand binding, integrin clustering is necessary to achieve intracellular signaling events that influence cellular phenotype. In this study, we explored the impact of nano-scale clustering of integrin-binding ligands on endothelial cell functions by designing novel materials that promote the clustering of integrin receptors. RGD-functionalized copolymers were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization and used for the preparation of random and nano-clustered surfaces spanning a range of global and local RGD densities (global densities 0.4-1.9 µg of peptide/mg of polymer and local densities of 1-2.4 ligands/nano-cluster). The adhesion and migration of endothelial cells was improved on nano-clustered surfaces compared to random surfaces. The highest adhesion and migration speed of endothelial cells and the rapid development of the endothelial monolayer were observed on surfaces with the highest local and global peptide density. These results indicate that the nano-clustering of peptide ligands is a promising strategy for next generation cardiovascular biomaterials, especially for small diameter vascular graft applications where the development of a confluent and functioning endothelium holds the potential to prevent device failure due to thrombosis.
... Numerous fabrication strategies have been developed to enable the production of materials with a variety of micro-to nano-scale topographies including particle leaching, phase inversion, micromolding, etching, cryogelation, and electrospinning [47][48][49][50][51][52][53][54][55]. These topographies can have significant impact on the behaviour of adherent mammalian cells [56]. ...
The field of biomaterials science has exhibited astounding growth during the last half century. This growth can be attributed to a more thorough understanding of cellular behaviours, new mechanistic insights into cell–materials interactions, advances in polymer synthesis, and the development of innovative fabrication techniques. This progress has enabled biomaterials to transition from materials that fill in for damaged or diseased tissues to implants that actively work with biology to aid in the healing process. In this review, we will discuss several of these advances including emerging knowledge on mechanotransduction, the use of substrate topography in modulating cellular behaviour, and the incorporation of bioactive molecules to foster specific cellular adhesion, promote tissue morphogenesis, and enable biologically driven degradation. We will then consider future challenges and directions in the field.
... Biomaterials science has illustrated that various cues are present in the extracellular environment that drive the behaviour of adherent cells. These extracellular stimuli include physical factors such as substrate stiffness and substrate topography, as well as the biochemical composition of the substrate [38][39][40][41]. Due to the heterogeneity between the dECM substrates it seems likely that primary MSCs would exhibit differential behaviour when adhered to these substrates. ...
... Biomaterials science and basic biological research provide evidence that various cues present in extracellular scaffolding drive the behaviour of adherent cells. These extracellular stimuli include physical factors such as substrate stiffness and substrate topography, as well as the biochemical composition of the substrate [38][39][40][41]. In addition to the obvious use of these materials for primary MSC expansion, more extensive analyses of these dECM materials will result in improved knowledge of primary MSC biology through the identification of the novel attributes of the dECM-DMSC23 materials that promote the proliferation and differentiation of primary MSCs. ...
Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.
