Georgia Papavasiliou

MMP-sensitive PEG diacrylate hydrogels with spatial variations in matrix properties stimulate directional vascular sprout formation.

The spatial presentation of immobilized extracellular matrix (ECM) cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP)-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP). Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10–12 hours in the more crosslinked regions to 4–6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.

Three-Dimensional Patterning of Poly(Ethylene Glycol) Hydrogels Through Surface-Initiated Photopolymerization

Photopolymerizable hydrogels have been investigated extensively for biomedical applications, specifically in the area of tissue engineering. While fabrication approaches have shown promise in designing hydrogel scaffolds that guide cell function, the ability to spatially control localization in three-dimensions has been limited. We have developed a method for generating two-dimensional and three-dimensional (3D) patterns within multilayered poly(ethylene glycol) diacrylate (PEG-DA) hydrogels. Covalently attached hydrogel layers are formed using precursor solutions with a 10:1 mole ratio of PEG-DA to PEG-aminoacrylate (Acr-PEG-NH2). Upon illumination of the precursor with visible light (wavelength = 514 nm), a hydrogel layer forms with pendant amine groups induced by the presence of Acr-PEG-NH2 macromer. Pendant amine groups are further functionalized with free carboxyl groups present on the visible light photoinitiator eosin, allowing for the formation of subsequent hydrogel layers. Using noncontact photolithography, the prepolymer solution is polymerized through a photomask, resulting in hydrogel structures with distinct pattern formation in each layer. Unreacted regions immobilized with eosin can be subsequently filled with a different PEG hydrogel. The technique presented shows a great potential for tissue engineering applications, for biosensors, and in the formation of cell and protein patterning for biotechnology.

Strategies for Vascularization of Polymer Scaffolds

Biocompatible, degradable polymer scaffolds combined with cells or biological signals are being investigated as alternatives to traditional options for tissue reconstruction and transplantation. These approaches are already in clinical use as engineered tissues that enhance wound healing and skin regeneration. The continued enhancement of these material strategies is highly dependent on the ability to promote rapid and stable neovascularization (new blood vessel formation) within the scaffold. Whereas neovascularization therapies have shown some promise for the treatment of ischemic tissues, vascularization of polymer scaffolds in tissue engineering strategies provides a unique challenge owing to the volume and the complexity of the tissues targeted. In this article, we examine recent advances in research focused on promoting neovascularization in polymer scaffolds for tissue engineering applications. These approaches include the use of growth factors, cells, and novel surgical approaches to both enhance and control the nature of the vascular networks formed. The continued development of these approaches may lead to new tissue engineering strategies for the generation of skin and other tissues or organs.

Calculation of Molecular Weight Distributions in Non‐Linear Free‐Radical Polymerization Using the Numerical Fractionation Technique

Mathematical Modeling of non-linear polymerization systems subject to gel formation is a challenging endeavor. At the gel point, the second and higher molecular weight moments diverge to infinity making it impossible to obtain the molecular weight distribution (MWD). The numerical fractionation (NF) technique utilizes a refinement of the method of moments to model non-linear polymerization systems that form gel. Since the method of moments yields results in terms of average quantities, some information is lost when reconstructing the MWD using NF. As a consequence, a broad shoulder appears at the high chain length end of the MWD tail. This study demonstrates that the validity of the gamma distribution deteriorates for the broader branched polymer generations and evaluates the performance of various alternative model distributions. Proper selection of the model distribution enhances the NF-reconstructed MWD. Illustration of closure approximations derived from known distribution functions, and proposed closure for the direct solution.

FGF-1 and proteolytically mediated cleavage site presentation influence three-dimensional fibroblast invasion in biomimetic PEGDA hydrogels

Controlled scaffold degradation is a critical design criterion for the clinical success of tissue-engineered constructs. Here, we exploited a biomimetic poly(ethylene glycol) diacrylate (PEGDA) hydrogel system immobilized with tethered YRGDS as the cell adhesion ligand and with either single (SSite) or multiple (MSite) collagenase-sensitive domains between crosslinks, to systematically study the effect of proteolytic cleavage site presentation on hydrogel degradation rate and three-dimensional (3-D) fibroblast invasion in vitro. Through the incorporation of multiple collagenase-sensitive domains between cross-links, hydrogel degradation rate was controlled and enhanced independent of alterations in compressive modulus. As compared to SSite hydrogels, MSite hydrogels resulted in increased 3-D fibroblast invasion in vitro, which occurred over a wider range of compressive moduli. Furthermore, encapsulated soluble acidic fibroblast growth factor (FGF-1), a potent mitogen during processes such as vascularization and wound healing, was incorporated into SSite and MSite PEGDA scaffolds to determine its in vitro potential on fibroblast cell invasion. Hydrogels containing soluble FGF-1 significantly enhanced 3-D fibroblast invasion in a dose-dependent manner within the different types of PEG matrices investigated over a period of 15 days. The methodology presented provides flexibility in designing PEG scaffolds with desired mechanical properties, but with increased susceptibility to proteolytically mediated degradation. These results indicate that effective tuning of initial matrix stiffness and hydrogel degradation kinetics plays a critical role in effectively designing PEG scaffolds that promote controlled 3-D cellular behavior and in situ tissue regeneration.

Generation of Mechanical and Biofunctional Gradients in PEG Diacrylate Hydrogels by Perfusion-Based Frontal Photopolymerization

The spatial presentation of soluble growth factors, immobilized extracellular matrix molecules, as well as matrix rigidity, plays an important role in directed and guided cell migration. Synthetic hydrogel scaffolds offer the ability to systematically introduce gradients of these factors contributing to our understanding of how the 3D arrangement of biochemical and mechanical cues influence cell behavior. Using a novel photopolymerization technique, perfusion-based frontal photopolymerization (PBFP), we have engineered poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with gradients of mechanical properties and immobilized biofunctionality. The controlled delivery of a buoyant photoinitiator, eosin Y, through a glass frit filter results in the formation and subsequent propagation of a polymer reaction front that is self-sustained and able to propagate through the monomeric mixture. Propagation of this front results in monomer depletion, leading to variations in cross-linking, as well as spatial gradients of elastic modulus and immobilized concentrations of the YRGDS cell adhesion ligand within PEGDA hydrogels. Furthermore, the magnitudes of the resulting gradients are controlled through alterations in polymerization conditions. Preliminary in vitro cell-culture studies demonstrate that the gradients generated stimulate directed 2D cell growth on the surface of PEGDA hydrogels. By day 14, fibroblast aggregates spread roughly twice as far in the direction parallel to the slope of the gradient as compared to the perpendicular direction. The presented technique has great potential in controlling gradients of mechanical properties and immobilized biofunctionality for directing and guiding 3D cell behavior within tissue-engineered scaffolds.

Effective tuning of ligand incorporation and mechanical properties in visible light photopolymerized poly(ethylene glycol) diacrylate hydrogels dictates cell adhesion and proliferation

Cell behavior is guided by the complex interplay of matrix mechanical properties as well as soluble and immobilized biochemical signals. The development of synthetic scaffolds that incorporate key functionalities of the native extracellular matrix (ECM) for support of cell proliferation and tissue regeneration requires that stiffness and immobilized concentrations of ECM signals within these biomaterials be tuned and optimized prior to in vitro and in vivo studies. A detailed experimental sensitivity analysis was conducted to identify the key polymerization conditions that result in significant changes in both elastic modulus and immobilized YRGDS within visible light photopolymerized poly(ethylene glycol) diacrylate hydrogels. Among the polymerization conditions investigated, single as well as simultaneous variations in N-vinylpyrrolidinone and precursor concentrations of acryl-PEG3400-YRGDS resulted in a broad range of the hydrogel elastic modulus (81-1178 kPa) and YRGDS surface concentration (0.04-1.72 pmol cm(-2)). Increasing the YRGDS surface concentration enhanced fibroblast cell adhesion and proliferation for a given stiffness, while increases in the hydrogel elastic modulus caused decreases in cell adhesion and increases in proliferation. The identification of key polymerization conditions is critical for the tuning and optimization of biomaterial properties and the controlled study of cell-substrate interactions.

In situ generation of cell-laden porous MMP-sensitive PEGDA hydrogels by gelatin leaching.

Proteolytically degradable poly(ethylene) glycol (PEG) hydrogels have been investigated as tissue engineering scaffolds; however, cell invasion and tissue regeneration are limited by the rate of cell-mediated degradation due to the small mesh size of the resultant crosslinked network. Gelatin leaching is combined with photopolymerization to form porous matrix-metalloproteinase (MMP)-sensitive PEG scaffolds under cytocompatible conditions in the presence of cells. Gelatin leaching allows control over pore size and porosity through selectivity of gelatin bead particle size and porogen loading, respectively. Increases in porogen loading lead to increased porosity, decreased compressive modulus and degradation time, and enhanced proliferation of encapsulated vascular smooth muscle cells.

Synthetic PEG Hydrogels as Extracellular Matrix Mimics for Tissue Engineering Applications

In recent years the field of tissue engineering, or regenerative medicine, has developed from the need to replace damaged and/or diseased tissues and organs by combining biomaterial scaffolds, biological signaling molecules, and cells. The regeneration of tissues may be achieved using either one of two principle approaches: 1) the in vitro construction or 2) the in vivo induction of tissue. In the first approach, biomaterial scaffolds are combined with biofunctional signaling molecules and cells and a fully functional tissue is grown in vitro which can then be implanted into the host. In the second approach, scaffolds are tailored with the desired biochemical composition as well as physical and mechanical properties of the target tissue, implanted into the host, and the body is used as a bioreactor to regenerate the tissue of interest. Therefore, biomaterial scaffolds play a central role in regenerative medicine as physical and biochemical milieus that dictate cell behavior, function, and tissue regeneration (Lutolf & Hubbell, 2005). While both natural and synthetic biomaterials have been extensively explored as scaffolds for tissue regeneration, polymeric materials from synthetic sources are advantageous due to their tunable mechanical properties and ability to systematically and selectively incorporate biological signals of the natural extracellular matrix (ECM) enabling controlled study of cell-substrate interactions. Over the last several decades synthetic crosslinked hydrogels of poly(ethylene) glycol have been extensively investigated for numerous biomedical applications including drug delivery, immunoisolation, and as matrices for engineering tissues. PEG hydrogels are biocompatible, hydrophilic polymers composed of 3D interstitial crosslinks that swell extensively in aqueous environments with water content similar to soft tissues. These biomaterials are inherently resistant to non-specific cell adhesion and protein adsorption, thus providing a blank slate upon which ECM-derived signals can be systematically introduced as well as spatially and temporally manipulated to control cell behavior and tissue regeneration. The continued enhancement of PEG-based biomaterial strategies towards the rational design of scaffolds is highly dependent on their ability to independently control the incorporation of multiple biofunctional signaling molecules from alterations in hydrogel degradation kinetics and mechanical properties, to temporally and spatially tune the presentation of mechanical and biofunctional signals, and to promote rapid and guided neovascularization (new blood vessel formation) prior to complete material degradation. The combination of the above-

Evaluation of MMP substrate concentration and specificity for neovascularization of hydrogel scaffolds

Controlled vascular response in scaffolds following implantation remains a significant clinical challenge. A critical biomaterial design criterion is the synchronization of the rates of scaffold degradation and vascularized tissue formation. Matrix metalloproteinases (MMPs) are key enzymes that regulate neovascularization and extracellular matrix remodelling. Synthetic protease-sensitive hydrogels offer controllable environments for investigating the role of matrix degradation on neovascularization. In this study, PEG hydrogels containing MMP-sensitive peptides with increased catalytic activity for MMPs expressed during neovascularization were investigated. Scaffolds were functionalized with MMP-2-, MMP-14- or general collagenase-sensitive peptides and with varying peptide concentration using crosslinkers containing one (SSite) or multiple (TSite) repeats of each protease-sensitive sequence. Increasing peptide concentration enhanced the degradation kinetics of scaffolds functionalized with MMP-specific sequences while 80% of the collagenase-sensitive scaffolds remained upon exposure to MMP-2 and MMP-14. In vitro neovascularization was consistent with in vivo tissue invasion with significantly increased invasion occurring within SSite MMP-specific as compared to collagenase-sensitive hydrogels and with further invasion in TSite as compared to SSite hydrogels regardless of peptide specificity. All scaffolds supported in vivo neovascularization; however, this was not dependent on peptide specificity. These findings demonstrate that peptide concentration and specificity regulate in vivo scaffold degradation, neovascularization and matrix remodelling.