Fibrinogen-Based Hydrogel Modulus and Ligand Density Effects on Cell Morphogenesis in Two-Dimensional and Three-Dimensional Cell Cultures.

There is a need to further explore the convergence of mechanobiology and dimensionality with systematic investigations of cellular response to matrix mechanics in 2D and 3D cultures. Here, a semisynthetic hydrogel capable of supporting both 2D and 3D cell culture is applied to investigate cell response to matrix modulus and ligand density. The culture materials are fabricated from adducts of polyethylene glycol (PEG) or PluronicF127 and fibrinogen fragments, formed into hydrogels by free-radical polymerization, and characterized by shear rheology. Control over the modulus of the materials is accomplished by changing the concentration of synthetic PEG-diacrylate crosslinker (0.5% w/v), and by altering the molecular length of the PEG (10 and 20 kDa). Control over ligand density is accomplished by changing fibrinogen concentrations from 3 to 12 mg mL-1 . In 2D culture, cell motility parameters, including cell speed and persistence time are significantly increased with increasing modulus. In both 2D and 3D culture, cells express vinculin and there is evidence of focal adhesion formation in the high stiffness materials. The modulus- and ligand-dependent morphogenesis response from the cells in 2D culture is contradictory to the same measured response in 3D culture. In 2D culture, anchorage-dependent cells become more elongated and significantly increase their size with increasing ligand density and matrix modulus. In 3D culture, the same anchorage-dependent cells become less spindled and significantly reduce their size in response to increasing ligand density and matrix modulus. These differences arise from dimensionality constraints, most notably the encapsulation of cells in a non-porous hydrogel matrix. These insights underscore the importance of mechanical properties in regulating cell morphogenesis in a 3D culture milieu. The versatility of the hydrogel culture environment further highlights the significance of a modular approach when developing materials that aim to optimize the cell culture environment.

Improving the Mechanical Rigidity of Hyaluronic Acid by Integration of a Supramolecular Peptide Matrix.

Hyaluronic acid (HA), a major component of the extracellular matrix, is an attractive material for various medical applications. Yet, its low mechanical rigidity and fast in vivo degradation hinder its utilization. Here, we demonstrate the reinforcement of HA by its integration with a low-molecular-weight peptide hydrogelator to produce a composite hydrogel. The formulation of HA with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide, one of the most studied self-assembling hydrogel-forming building blocks, showing notable mechanical properties, resulted in the formation of stable, homogeneous hydrogels. Electron microscopy analysis demonstrated a uniform distribution of the two matrices in the composite forms. The composite hydrogels showed improved mechanical properties and stability to enzymatic degradation while maintaining their biocompatibility. Moreover, the storage modulus of the FmocFF/HA composite hydrogels reached up to 25 kPa. The composite hydrogels allowed sustained release of curcumin, a hydrophobic polyphenol showing antioxidant, anti-inflammatory, and antitumor activities. Importantly, the rate of curcumin release was modulated as a function of the concentration of the FmocFF peptide within the hydrogel matrix. This work provides a new approach for conferring mechanical rigidity and stability to HA without the need of cross-linking, thus potentially facilitating its utilization in different clinical applications, such as sustained drug release.

A method for preparation of hydrogel microcapsules for stem cell bioprocessing and stem cell therapy.

A method for the preparation of suspension culture microcapsules used in the bioprocessing of human mesenchymal stem cells (hMSCs) is reported. The microcapsules are prepared from a semi-synthetic hydrogel comprising Pluronic®F127 conjugated to denatured fibrinogen. The Pluronic-fibrinogen adducts display a lower critical solubility temperature (LCST) at ∼30 °C, thus enabling mild, cell-compatible physical crosslinking of the microcapsules in a warm gelation bath. Cell-laden microgels were prepared from a solution of Pluronic-fibrinogen hydrogel precursor and hMSCs; these were cultivated for up to 15 days in laboratory-scale suspension bioreactors and harvested by reducing the temperature of the microcapsules to disassemble the physical polymer network. The viability, proliferation and cell recovery yields of the hMSCs were shown to be better than photo-chemically crosslinked microcapsules made from a similar material. The cell culture yields, which exceeded 300% after 15 days in suspension culture, were comparable to other microcarrier systems used for the mass production of hMSCs. The simplicity of this methodology, both in terms of the cell inoculation and mild recovery conditions, represent distinct advantages for stem cell bioprocessing with suspension culture bioreactors.

Matrix stiffness determines the fate of nucleus pulposus-derived stem cells.

Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposus-derived stem cells (NP-SCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three-dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G' = 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G' = 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate elucidation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions.

Fabrication of PEGylated fibrinogen: a versatile injectable hydrogel biomaterial.

Hydrogels are one of the most versatile biomaterials in use for tissue engineering and regenerative medicine. They are assembled from either natural or synthetic polymers, and their high water content gives these materials practical advantages in numerous biomedical applications. Semisynthetic hydrogels, such as those that combine synthetic and biological building blocks, have the added advantage of controlled bioactivity and material properties. In myocardial regeneration, injectable hydrogels premised on a semisynthetic design are advantageous both as bioactive bulking agents and as a delivery vehicle for controlled release of bioactive factors and/or cardiomyocytes. A new semisynthetic hydrogel based on PEGylated fibrinogen has been developed to address the many requirements of an injectable biomaterial in cardiac restoration. This chapter highlights the fundamental aspects of making this biomimetic hydrogel matrix for cardiac applications.

Seamless metallic coating and surface adhesion of self-assembled bioinspired nanostructures based on di-(3,4-dihydroxy-L-phenylalanine) peptide motif.

The noncoded aromatic 3,4-dihydroxy-L-phenylalanine (DOPA) amino acid has a pivotal role in the remarkable adhesive properties displayed by marine mussels. These properties have inspired the design of adhesive chemical entities through various synthetic approaches. DOPA-containing bioinspired polymers have a broad functional appeal beyond adhesion due to the diverse chemical interactions presented by the catechol moieties. Here, we harnessed the molecular self-assembly abilities of very short peptide motifs to develop analogous DOPA-containing supramolecular polymers. The DOPA-containing DOPA-DOPA and Fmoc-DOPA-DOPA building blocks were designed by substituting the phenylalanines in the well-studied diphenylalanine self-assembling motif and its 9-fluorenylmethoxycarbonyl (Fmoc)-protected derivative. These peptides self-organized into fibrillar nanoassemblies, displaying high density of catechol functional groups. Furthermore, the Fmoc-DOPA-DOPA peptide was found to act as a low molecular weight hydrogelator, forming self-supporting hydrogel which was rheologically characterized. We studied these assemblies using electron microscopy and explored their applicative potential by examining their ability to spontaneously reduce metal cations into elementary metal. By applying ionic silver to the hydrogel, we observed efficient reduction into silver nanoparticles and the remarkable seamless metallic coating of the assemblies. Similar redox abilities were observed with the DOPA-DOPA assemblies. In an effort to impart adhesiveness to the obtained assemblies, we incorporated lysine (Lys) into the Fmoc-DOPA-DOPA building block. The assemblies of Fmoc-DOPA-DOPA-Lys were capable of gluing together glass surfaces, and their adhesion properties were investigated using atomic force microscopy. Taken together, a class of DOPA-containing self-assembling peptides was designed. These nanoassemblies display unique properties and can serve as multifunctional platforms for various biotechnological applications.

Cellularized biosynthetic microhydrogel polymers for intravascular liver tissue regeneration therapy.

INTRODUCTION: The liver is the natural microenvironment for hepatocytes transplantation but unfortunately engraftment efficiency is low. Cell-laden microhydrogels made of fibrinogen attached to poly(ethylene glycol) (PEG)-diacrylate side chains, were used as a cell carrier, for intravascular transplantation. This approach may reduce shear stress and immediate immunological pressure after intravascular transplantation and provide biomatrix for environmental support. AIMS: In vitro assessment of HuH-7 viability and function after polymerization within PEGylated fibrinogen-hydrogel. In vivo assessment of intraportal transplantation of cell-laden microhydrogels with rat adult parenchymal cells. METHODS: (1) In vitro assessment of HuH-7 cell viability and function, after cell-laden hydrogel (hydrogel volume 30 µL) fabrication, by propidium iodide (PI)/fluorescein diacetate (FDA), and MTT assays, albumin concentration and CYP1A activity. (2) Fabrication of cell-laden microhydrogels and their intraportal transplantion. Engraftment efficiency in vivo was evaluated by real-time qPCR of Y chromosome (SRY gene) and histology. RESULTS: The viability of cells in hydrogels in culture was comparable to viability of not embedded cells during the first 48 h. However, the viability of cells in hydrogels was reduced after 72 h compared with not embedded cells. Activity of CYP1A in hydrogel was comparable to that of not embedded cells (4.33±1 pmole/µg DNA/4 h vs. 5.13±1 pmole/µg DNA/4 h, respectively). Albumin concentration increased at day 3 in hydrogels to 1.4±0.6 µg/10(4)/24 h and was greater to that of free cells, 0.3±0.1 µg/10(4)/24 h. Cell-laden microhydrogels at a size of 150-150-600 µm (6×10(6) cells/rat) showed better engraftment efficiency at 21 days post-transplantation, compared with isolated cell transplantation (54.6%±5% vs. 1.8%±1.2%, p<0.001). CONCLUSIONS: The in vitro HuH-7 viability and function after polymerization in PEGylated fibrinogen hydrogel was comparable to cells without the hydrogel. Long-term survival and engraftment efficiency of intravascular transplanted adult hepatocytes is much better in within cell-laden microhydrogels compared with isolated cells. The overall efficiency of the procedure needs to be improved.

Reversible Ag(+)-crosslinked DNA hydrogels.

DNA hydrogels, consisting of Y-shaped nucleic acid subunits or of nucleic acid-functionalized acrylamide chains, undergo switchable gel-to-solution transitions. The Ag(+)-stimulated formation of cytosine-Ag(+)-cytosine complexes results in the crosslinking of the units to yield the hydrogels, while the cysteamine-induced elimination of the Ag(+) ions dissociates the hydrogels into a solution phase.

Fluorescent DNA hydrogels composed of nucleic acid-stabilized silver nanoclusters.

Y-shaped DNA units functionalized with Ag-nanoclusters are crosslinked by nucleic acids to yield fluorescent hydrogels with controlled luminescence properties.

Time-dependent cellular morphogenesis and matrix stiffening in proteolytically responsive hydrogels.

Mesenchymal stromal cells residing in proteolytically responsive hydrogel scaffolds were subjected to changes in mechanical properties associated with their own three-dimensional (3-D) morphogenesis. In order to investigate this relationship the current study documents the transient degradation and restructuring of fibroblasts seeded in hydrogel scaffolds undergoing active cell-mediated reorganization over 7days in culture. A semi-synthetic proteolytically degradable polyethylene glycol-fibrinogen (PF) hydrogel matrix and neonatal human dermal fibroblasts (NHDF) were used. Rheology (in situ and ex situ) measured stiffening of the gels and confocal laser scanning microscopy (CLSM) measured cell morphogenesis within the gels. The assumption that the matrix modulus systematically decreases as cells locally begin to enzymatically disassemble the PF hydrogel to become spindled in the material was not supported by the bulk mechanical property measurements. Instead, the PF hydrogels exhibited cell-mediated stiffening concurrent with their dynamic morphogenesis, as indicated by a four-fold increase in storage modulus after 1week in culture. Fibrin hydrogels, which were used as the control biomaterial, proved similarly adaptive to cell-mediated remodeling only in the presence of the exogenous serine protease inhibitor aprotinin. Acellular and non-viable hydrogels also served as control groups to verify that transient matrix remodeling was entirely associated with cell-mediated events, including collagen deposition, cell-mediated proteolysis, and the formation of multicellular networks within the hydrogel constructs. The fact that cell network formation and collagen deposition both paralleled transient stiffening of the PF hydrogels, further reinforces the notion that cells actively balance between proteolysis and ECM synthesis when remodeling proteolytically responsive hydrogel scaffolds.

Switchable catalytic acrylamide hydrogels cross-linked by hemin/G-quadruplexes.

Copolymer chains consisting of acrylamide units and guanine (G)-containing oligonucleotide-tethered acrylamide units undergo, in the presence of K(+) ions, cross-linking by G-quadruplexes to yield a hydrogel. The hydrogel is dissociated upon addition of 18-crown-6 ether that traps the K(+) ions. Reversible formation and dissociation of the hydrogel is demonstrated by the cyclic addition of K(+) ions and 18-crown-6 ether, respectively. Formation of the hydrogel in the presence of hemin results in a hemin/G-quadruplex-cross-linked catalytic hydrogel mimicking the function of horseradish peroxidase, reflected by the catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), ABTS(2-), by H2O2 to ABTS(·-) and by the catalyzed generation of chemiluminescence in the presence of luminol/H2O2. Cyclic "ON" and "OFF" activation of the catalytic functions of the hydrogel are demonstrated upon the formation of the hydrogel in the presence of K(+) ions and its dissociation by 18-crown-6 ether, respectively. The hydrogel is characterized by rheology measurements, circular dichroism, and probing its chemical and photophysical properties.

The rheological and structural properties of Fmoc-peptide-based hydrogels: the effect of aromatic molecular architecture on self-assembly and physical characteristics.

Biocompatible hydrogels are of high interest as a class of biomaterials for tissue engineering, regenerative medicine, and controlled drug delivery. These materials offer three-dimensional scaffolds to support the growth of cells and development of hierarchical tissue structures. Fmoc-peptides were previously demonstrated as attractive building blocks for biocompatible hydrogels. Here, we further investigate the biophysical properties of Fmoc-peptide-based hydrogels for medical applications. We describe the structural and thermal properties of these Fmoc-peptides, as well as their self-assembly process. Additionally, we study the role of interactions between aromatic moieties in the self-assembly process and on the physical and structural properties of the hydrogels.

Photopolymerization of cell-encapsulating hydrogels: crosslinking efficiency versus cytotoxicity.

Cell-encapsulating hydrogels used in regenerative medicine are designed to undergo a rapid liquid-to-solid phase transition in the presence of cells and tissues so as to maximize crosslinking and minimize cell toxicity. Light-activated free-radical crosslinking (photopolymerization) is of particular interest in this regard because it can provide rapid reaction rates that result in uniform hydrogel properties with excellent temporal and spatial control features. Among the many initiator systems available for photopolymerization, only a few have been identified as suitable for cell-based hydrogel formation owing to their water solubility, crosslinking properties and non-toxic reaction conditions. In this study, three long-wave ultraviolet (UV) light-activtied photoinitiators (PIs) were comparatively tested in terms of cytotoxicity, crosslinking efficiency and crosslinking kinetics of cell-encapsulating hydrogels. The hydrogels were photopolymerized from poly(ethylene glycol) (PEG) diacrylate or PEG-fibrinogen precursors using Irgacure® PIs I2959, I184 and I651, as well as with a chemical initiator/accelerator (APS/TEMED). The study specifically evaluated the PI type, PI concentration and UV light intensity, and how these affected the mechanical properties of the hydrogel (i.e. maximum storage modulus), the crosslinking reaction times and the reaction's cytotoxicity to encapsulated cells. Only two initiators (I2959 and I184) were identified as being suitable for achieving both high cell viability and efficient crosslinking of the cell-encapsulating hydrogels during the photopolymerization reaction. Optimization of PI concentration or irradiation intensity was particularly important for achieving maximum mechanical properties; a sub-optimal choice of PI concentration or irradiation intensity resulted in a substantial reduction in hydrogel modulus. Cytocompatibility may be compromised by unnecessarily prolonging exposure to cytotoxic free radicals or inadvertently enhancing the instantaneous dose of radicals in solution, both of which are dependent on the PI type/concentration and irradiation intensity. In the absence of a radical initiator, the short exposures to long-wave UV light irradiation (up to 5 min, 20 mW cm(-2), 365 nm) did not prove to be cytotoxic to cells. Therefore, it is important to understand the relationship between PIs, light irradiation conditions and crosslinking when attempting to identify a suitable hydrogel formation process for cell encapsulating hydrogels.

Self-assembled Fmoc-peptides as a platform for the formation of nanostructures and hydrogels.

Hydrogels are of great interest as a class of materials for tissue engineering, axonal regeneration, and controlled drug delivery, as they offer 3D interwoven scaffolds to support the growth of cells. Herein, we extend the family of the aromatic Fmoc-dipeptides with a library of new Fmoc-peptides, which include natural and synthetic amino acids with an aromatic nature. We describe the self-assembly of these Fmoc-peptides into various structures and characterize their distinctive molecular and physical properties. Moreover, we describe the fabrication of the bioactive RGD sequence into a hydrogel. This unique material offers new opportunities for developing cell-adhesive biomedical hydrogel scaffolds, as well as for establishing strategies to modify surfaces with bioactive materials.