Cell Adhesive RGD Peptide Research Articles

Fully synthetic, tunable poly(α-amino acids) as the base of bioinks curable by visible light

Bioinks play a crucial role in tissue engineering, influencing mechanical and chemical properties of the printed scaffold as well as the behavior of encapsulated cells. Recently, there has been a shift from animal origin materials to their synthetic alternatives. In this context, we present here bioinks based on fully synthetic and biodegradable poly(α,L-amino acids) (PolyAA) as an alternative to animal-based gelatin methacrylate (Gel-Ma) bioinks. Additionally, we first reported the possibility of the visible light photoinitiated incorporation of the bifunctional cell adhesive RGD peptide into the PolyAA hydrogel matrix. The obtained hydrogels are shown to be cytocompatible, and their mechanical properties closely resemble those of gelatin methacrylate-based scaffolds. Moreover, combining the unique properties of PolyAA-based bioinks, the photocrosslinking strategy, and the use of droplet-based printing allows the printing of constructs with high shape fidelity and structural integrity from low-viscosity bioinks without using any sacrificial components. Overall, presented PolyAA-based materials are a promising and versatile toolbox that extends the range of bioinks for droplet bioprinting.

Aligned skeletal muscle assembly on a biofunctionalized plant leaf scaffold.

Decellularized plant scaffolds have drawn attention as alternative tissue culture platforms due to their wide accessibility, biocompatibility, and diversity of innate microstructures. Particularly, in this work, monocot leaves with innate uniaxial micropatterned topography were utilized to promote cell alignment and elongation. The leaf scaffold was biofunctionalized with poly(PEGMEMA-r-VDM-r-GMA) copolymer that prevented non-specific protein adsorption and was modified with cell adhesive RGD peptide to enable cell adhesion and growth in serum-free media. The biofunctionalized leaf supported the adhesion, growth, and alignment of various human cells including embryonic stem cells (hESC) derived muscle cells. The hESC-derived myogenic progenitor cells cultured on the biofunctionalized leaf scaffold adopted a parallel orientation and were elongated along the leaf topography. These cells showed significant early myogenic differentiation and muscle-like bundled myotube formation. The aligned cells formed compact myotube assemblies and showed uniaxial muscle contraction under chemical stimulation, a critical requirement for developing functional skeletal muscle tissue. Polymer-functionalized plant leaf scaffolds offer a novel human cell culture platform and have potential in human tissue engineering applications that require parallel alignment of cells. STATEMENT OF SIGNIFICANCE: Plant scaffolds are plentiful sources in nature and present a prefabricated construct to present topographical cues to cells. Their feature width is ideal for human cell alignment and elongation, especially for muscle cells. However, plant scaffolds lack proteins that support mammalian cell culture. We have developed a polymer coated leaf scaffold that enables cell adhesion and growth in serum-free media. Human muscle cells cultured on the biofunctionalized leaf, aligned along the natural parallel micro-patterned leaf topography, and formed muscle-like bundled myotube assemblies. These assemblies showed uniaxial muscular contraction, a critical requirement for developing functional skeletal muscle tissue. The biodiversity of the plant materials offers a novel human cell culture platform with potential in human tissue engineering.

Universal and One-Step Modification to Render Diverse Materials Bioactivation.

Bioactive materials that can support cell adhesion and tissue regeneration are greatly in demand in clinical applications. Surface modification with bioactive molecules is an efficient strategy to convert conventional bioinert materials into bioactive materials. However, there is an urgent need to find a universal and one-step modification strategy to realize the above transformation for bioactivation. In this work, we report a universal and one-step modification strategy to easily modify and render diverse materials bioactivation by dipping materials into the solution of dibutylamine-DOPA-lysine-DOPA (DbaYKY) tripeptide-terminated cell-adhesive molecules, β-peptide polymer, or RGD peptide for only 5 min. This strategy provides materials with a stable surface modification layer and does not cause an undesired surface color change like the widely used polydopamine coating. This one-step strategy can endow material surfaces with cell adhesion properties without concerns on nonspecific conjugation of proteins and macromolecules. This universal and one-step surface bioactivation strategy implies a wide range of applications in implantable biomaterials.

Dynamic modulation of matrix adhesiveness induces epithelial-to-mesenchymal transition in prostate cancer cells in 3D

Synthetic matrices with dynamic presentation of cell guidance cues are needed for the development of physiologically relevant in vitro tumor models. Towards the goal of mimicking prostate cancer progression and metastasis, we engineered a tunable hyaluronic acid-based hydrogel platform with protease degradable and cell adhesive properties employing bioorthogonal tetrazine ligation with strained alkenes. The synthetic matrix was first fabricated via a slow tetrazine-norbornene reaction, then temporally modified via a diffusion-controlled method using trans-cyclooctene, a fierce dienophile that reacts with tetrazine with an unusually fast rate. The encapsulated DU145 prostate cancer single cells spontaneously formed multicellular tumoroids after 7 days of culture. In situ modification of the synthetic matrix via covalent tagging of cell adhesive RGD peptide induced tumoroid decompaction and the development of cellular protrusions. RGD tagging did not compromise the overall cell viability, nor did it induce cell apoptosis. In response to increased matrix adhesiveness, DU145 cells dynamically loosen cell-cell adhesion and strengthen cell-matrix interactions to promote an invasive phenotype. Characterization of the 3D cultures by immunocytochemistry and gene expression analyses demonstrated that cells invaded into the matrix via a mesenchymal like migration, with upregulation of major mesenchymal markers, and down regulation of epithelial markers. The tumoroids formed cortactin positive invadopodia like structures, indicating active matrix remodeling. Overall, the engineered tumor model can be utilized to identify potential molecular targets and test pharmacological inhibitors, thereby accelerating the design of innovative strategies for cancer therapeutics.

Mimicking Bone Extracellular Matrix: From BMP-2-Derived Sequences to Osteogenic-Multifunctional Coatings.

Cell-material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.

Anisotropic Nanoscale Presentation of Cell Adhesion Ligand Enhances the Recruitment of Diverse Integrins in Adhesion Structures and Mechanosensing‐Dependent Differentiation of Stem Cells

AbstractThe nanoscale anisotropic patterns of bioactive ligands in the extracellular matrix regulate cell adhesion behaviors. However, the mechanisms of such regulation remain unclear. Here, RGD‐bearing gold nanorods (AuNRs) are conjugated with different aspect ratios (ARs, from 1 to 7) on cell culture substrates to decouple the effect of nanoscale anisotropic presentation of cell adhesive RGD peptides on cell adhesion. Compared with AuNRs with small ARs, AuNRs with large ARs significantly promote cell spreading, the alignment of the basal cytoskeletal structure, and nanopodia attachment. Furthermore, both ‐β3 and ‐β1 class integrins are recruited to AuNRs with large ARs, thereby promoting the development of focal adhesion toward fibrillar adhesion, whereas the recruitment of diverse integrins and the development of cell adhesion structures are hindered by small ARs AuNRs. The anisotropic presentation of ligands by large AR AuNRs better activates mechanotransduction signaling molecules. These findings are confirmed both in vitro and in vivo. Hence the enhanced mechanotransduction promotes osteogenic differentiation in stem cells. These findings demonstrate the potential use of well‐controlled synthetic nanoplatforms to unravel the fundamental mechanisms of cell adhesion and associated signaling at the molecular level and to provide valuable guidance for the rational design of biomaterials with tailored bioactive functions.

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 µm in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Shape-Changing Photodegradable Hydrogels for Dynamic 3D Cell Culture.

Inspired by natural examples of swelling-actuated self-folding, we utilize photodegradable hydrogels as dynamically tunable, shape-changing scaffolds for culturing cells. Poly(ethylene glycol) diacrylate-based thin films incorporating ortho-nitrobenzyl (o-NB) moieties are transformed from flat 2D sheets to folded 3D structures by exposure to 365 nm UV light. As the UV light is attenuated through the thickness of the gel, a cross-link density gradient is formed. This gradient gives rise to differential swelling and a bending moment, resulting in gel folding. By tuning the UV light dose and the molar ratio of photodegradable to nondegradable species, both the initial degree of folding and the relaxation of tubular structures can be accurately controlled. These self-folding photodegradable gels were further functionalized with a cell-adhesive RGD peptide for both seeding and encapsulation of C2C12 mouse myoblasts. Light-induced folding of RGD functionalized hydrogels from flat sheets to tubular structures was demonstrated 1 or 3 days after C2C12 seeding. The C2C12s remained adhered on the inner walls of folded tubes for up to 6 days after folding. The minimum measured diameter of a tubular structure containing C2C12s was 1 mm, which is similar to the size of muscle fascicles. Furthermore, the viability of encapsulated C2C12s was not adversely affected by the UV light-induced folding. This is the first account of a self-folding material system that allows 2D-3D shape change in the presence of both seeded and encapsulated cells at a user-directed time point of choice.

Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings

Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. Statement of SignificanceIn the present work, we show for the first time that-an oriented and dense monolayer of flagellin molecules mimics the surface of the bacterial flagellar filament. Consequently, the fabricated layer is completely cell repellent.-By genetically modifying flagellin, we incorporate cell adhesion regulating functionalities into this anti-adhesive coating.-We can easily tune the adhesion of living cells from completely cell repellent to highly adhesive.

Swelling Behavior and Nanomechanical Properties of (Peptide-Modified) Poly(2-hydroxyethyl methacrylate) and Poly(poly(ethylene glycol) methacrylate) Brushes

Poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(poly(ethylene glycol) methacrylate) (PPEGMA) brushes represent a class of thin, surface-tethered polymer films that have been extensively used e.g. to generate non-biofouling surfaces or as model systems to study fundamental biointerfacial questions related to cell–surface interactions. As the properties of PHEMA and PPEGMA brushes depend on the hydration and swelling of these thin films, it is important to understand the influence of basic structural parameters such as the composition of the polymer brush, the film thickness, or grafting density on these phenomena. This article reports results of a series of experiments that were performed to investigate the swelling behavior and mechanical properties of a diverse library of PHEMA and PPEGMA brushes covering a range of film thicknesses and grafting densities. The swelling ratios of the PHEMA and PPEGMA brushes were determined by ellipsometry and neutron reflectivity experiments and ranged from ∼1.5 to ∼5.0. Decreasing the grafting density and decreasing the film thickness generally results in an increase in the swelling ratio. Modification of the PHEMA and PPEGMA brushes with the cell adhesive RGD peptide ligand was found to result in a decrease in the swelling ratio. The neutron reflectivity experiments further revealed that solvated PHEMA and PPEGMA brushes are best described by a two-layer model, consisting of a polymer-rich layer close to the substrate and a second layer that is swollen to a much higher degree at the brush–water interface.

Co-immobilization of adhesive peptides and VEGF within a dextran-based coating for vascular applications

Multifunctional constructs providing a proper environment for adhesion and growth of selected cell types are needed for most tissue engineering and regenerative medicine applications. In this context, vinylsulfone (VS)-modified dextran was proposed as a matrix featuring low-fouling properties as well as multiple versatile moieties. The displayed VS groups could indeed react with thiol, amine or hydroxyl groups, be it for surface grafting, crosslinking or subsequent tethering of biomolecules. In the present study, a library of dextran-VS was produced, grafted to aminated substrates and characterized in terms of degree of VS modification (%VS), cell-repelling properties and potential for the oriented grafting of cysteine-tagged peptides. As a bioactive coating of vascular implants, ECM peptides (e.g. RGD) as well as vascular endothelial growth factor (VEGF) were co-immobilized on one of the most suitable dextran-VS coating (%VS=ca. 50% of saccharides units). Both RGD and VEGF were efficiently tethered at high densities (ca. 1nmol/cm2 and 50fmol/cm2, respectively), and were able to promote endothelial cell adhesion as well as proliferation. The latter was enhanced to the same extent as with soluble VEGF and proved selective to endothelial cells over smooth muscle cells. Altogether, multiple biomolecules could be efficiently incorporated into a dextran-VS construct, while maintaining their respective biological activity. Statement of SignificanceThis work addresses the need for multifunctional coatings and selective cell response inherent to many tissue engineering and regenerative medicine applications, for instance, vascular graft. More specifically, a library of dextrans was first generated through vinylsulfone (VS) modification. Thoroughly selected dextran-VS provided an ideal platform for unbiased study of cell response to covalently grafted biomolecules. Considering that processes such as healing and angiogenesis require multiple factors acting synergistically, vascular endothelial growth factor (VEGF) was then co-immobilized with the cell adhesive RGD peptide within our dextran coating through a relevant strategy featuring orientation and specificity. Altogether, both adhesive and proliferative cues could be incorporated into our construct with additive, if not synergetic, effects.

In vivo evaluation of biomimetic fluorosurfactant polymer-coated expanded polytetrafluoroethylene vascular grafts in a porcine carotid artery bypass model

The objective of this study was to evaluate the potential for biomimetic self-assembling fluorosurfactant polymer (FSP) coatings incorporating heptamaltose (M7-FSP) to block nonspecific protein adsorption, the cell adhesive RGD peptide (RGD-FSP), or the endothelial cell-selective CRRETAWAC peptide (cRRE-FSP) to improve patency and endothelialization in small-diameter expanded polytetrafluoroethylene (ePTFE) vascular graft implants. ePTFE vascular grafts (4mm in diameter, 5cm in length) were coated with M7-FSP, RGD-FSP, or cRRE-FSP by dissolving FSPs in distilled water and flowing solution through the graft lumen for 24hours. Coatings were confirmed by receding water contact angle measurements on the lumen surface. RGD-FSP and cRRE-FSP grafts were presodded invitro with porcine pulmonary artery endothelial cells (PPAECs) using a custom-designed flow system. PPAEC coverage on the lumen surface was visualized with epifluorescent microscopy and quantified. Grafts were implanted as carotid artery interposition bypass grafts in seven pigs for 33± 2days (ePTFE, n= 3; M7-FSP, n= 4; RGD-FSP, n= 3; cRRE-FSP, n= 4). Patency was confirmed immediately after implantation with duplex color flow ultrasound and at explantation with contrast-enhanced angiography. Grafts were sectioned for histology and stained: Movat pentachrome stain to outline vascular layers, immunofluorescent staining to identify endothelial cells (anti-von Willebrand factor antibody), and immunohistochemical staining to identify smooth muscle cells (anti-smooth muscle α-actin antibody). Neointima to lumen area ratio was determined to evaluate neointimal hyperplasia. Receding water contact angle measurements on graft luminal surfaces were significantly lower (P< .05) on FSP-coated ePTFE surfaces (M7-FSP, 40± 16 degrees; RGD-FSP, 25± 10 degrees; cRRE-FSP, 33± 16 degrees) compared with uncoated ePTFE (126± 2 degrees), confirming presence of the FSP layer. Invitro sodding of PPAECs on RGD-FSP and cRRE-FSP grafts resulted in a confluent monolayer of PPAECs on the luminal surface, with a similar cell population on RGD-FSP (1200± 187 cells/mm(2)) and cRRE-FSP (1134± 153 cells/mm(2)) grafts. All grafts were patent immediately after implantation, and one of three uncoated, two of three RGD-FSP, two of four M7-FSP, and two of four cRRE-FSP grafts remained patent after 1month. PPAEC coverage of the lumen surface was seen in all patent grafts. RGD-FSP grafts had a slightly higher neointima to lumen area ratio (0.53± 0.06) compared with uncoated (0.29±0.15), M7-FSP (0.20± 0.15), or cRRE-FSP (0.17± 0.09) grafts. Biomimetic FSP-coated ePTFE grafts can be used successfully invivo and have potential to support endothelialization. Grafts modified with the M7-FSP and cRRE-FSP showed lower intimal hyperplasia compared with RGD-FSP grafts.

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications.

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant’s biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO3 or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO3-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.

Delivery of bFGF for Tissue Engineering by Tethering to the ECM.

Delivery of growth factors to target cells is an important subject in tissue engineering. Towards that end, we have developed a growth factor-tethered extracellular matrix (ECM). Here, basic fibroblast growth factor (bFGF) was tethered to extracellular matrix noncovalently. The designed ECM was comprised of 12 repeats of the APGVGV peptide motif derived from elastin as a stable structural unit and included the well-known cell adhesive RGD peptide as an active functional unit. To bind bFGF to the ECM, an acidic amino acid-rich sequence was introduced at the C-terminus of the ECM protein. It consisted of 5 repeats of 4 aspartic acids and a serine, DDDDS. bFGF has a highly basic amino acid domain. Therefore, bFGF was tethered to the ECM protein by electrostatic interaction. Cells cultured on bFGF-tethered ECM were well attached to the ECM and induced proliferation without addition of soluble bFGF.

Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials.

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have been recently realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

PVDF film tethered with RGD-click-poly(glycidyl methacrylate) brushes by combination of direct surface-initiated ATRP and click chemistry for improved cytocompatibility

To enhance the cytocompatibility of polyvinylidene fluoride (PVDF) films, arginine–glycine–aspartic acid (RGD) peptide-click-poly(glycidyl methacrylate) (PGMA) polymer brushes were grafted onto the PVDF surface by the combination of surface-initiated atom transfer radical polymerization (ATRP) and click reaction. The direct initiation of the secondary fluorine atoms of PVDF backbone allowed for grafting of the PGMA brushes containing reactive epoxy groups. Subsequent introduction of the azide groups onto the side chain of PGMA brushes was achieved by the ring-open reaction of the epoxy groups with sodium azide. The cell adhesive RGD peptide was finally conjugated onto the PGMA brushes via alkyne-azide click reaction. Kinetic studies revealed that the PGMA chain growth from the PVDF surface was consistent with a “controlled” process, and that the amount of immobilized RGD peptide on the PGMA brushes increased with the concentration of the pendant azide groups. The specificity of cellular interactions of adipose tissue-derived stem cells (ASCs) on the functionalized PVDF films was investigated. Results demonstrated that cell adhesion and proliferation of ASCs were significantly improved on the RGD-immobilized PVDF substrates, and this improvement was positively correlated with the surface concentration of covalently-bonded RGD peptide. With the inherent superior chemical and mechanical properties of PVDF films and the biocompatible nature of cell-adhesive peptides, surface functionalized PVDF films are potentially useful for biomedical and tissue engineering applications.

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.

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation *

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.

Enzymatic, physicochemical and biological properties of MMP-sensitive alginate hydrogels

Protease-sensitive hydrogels that recapitulate the mechanisms of cell-driven enzymatic remodelling of the natural extracellular matrix (ECM) have been gaining popularity as artificial 3D cell-microenvironments. Here, the matrix metalloproteinase (MMP)-sensitive peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG) was double-end grafted to alginate forming water-soluble PVGLIG–alginate conjugates. The PVGLIG peptide was synthesized as a Fluorescence Resonance Energy Transfer (FRET) sensor and showed to be a good substrate for MMP-2, MMP-9, MMP-13 and MMP-14. After demonstrating that human MSC (hMSC) expressed both MMP-2 and MMP-14 under basal and osteogenic in vitro conditions, the effect of 3D-culture within MMP-sensitive alginate hydrogels on hMSC behaviour was addressed. In situ-forming alginate hydrogels containing only cell-adhesive RGD peptides (RGD–alginate, MMP-insensitive) or both peptides (PVGLIG/RGD–alginate, MMP-sensitive) were used. Cell–matrix and cell–cell interactions were enhanced in hMSC-laden MMP-sensitive alginate hydrogels, as compared to MMP-insensitive hydrogels with identical viscoelastic and microstructural properties. hMSC underwent osteogenic differentiation in both types of matrices. However, the presence of PVGLIG stimulated the secretion of proteases (most likely MMP-2) by hMSC, in both undifferentiated and differentiated cultures. By using the FRET sensor, it was possible to demonstrate that the cocktail of hMSC-secreted MMPs was effectively active in cleaving the PVGLIG motif. Protease-sensitive alginates can be used to create cell-responsive 3D microenvironments and offer promise as injectable carriers for therapeutic hMSC-delivery.

Covalent Grafting of the RGD‐Peptide onto Polyetheretherketone Surfaces via Schiff Base Formation

In recent years, the synthetic polymer polyetheretherketone (PEEK) has increasingly been used in a number of orthopedic implementations, due to its excellent mechanical properties, bioinertness, and chemical resistance. For in vivo applications, the surface of PEEK, which does not naturally support cell adhesion, has to be modified to improve tissue integration. In the present work we demonstrate a novel wet-chemical modification of PEEK to modify the surface, enabling the covalent grafting of the cell-adhesive RGD-peptide. Modification of the polymer surface was achieved via Schiff base formation using an aliphatic diamine and subsequent crosslinker-mediated immobilization of the peptide. In cell culture experiments with primary osteoblasts it was shown that the RGD-modified PEEK not only significantly promoted cellular adhesion but also strongly enhanced the proliferation of osteoblasts on the modified polymer surface.