Human Neutrophil Elastase Responsive Delivery from Poly(ethylene glycol) Hydrogels

A novel enzyme-responsive hydrogel drug delivery system was developed with the potential to treat inflammation locally. Human neutrophil elastase (HNE) is a serine protease secreted by neutrophils which are the first cells recruited to inflammatory sites. We exploited this cell-secreted enzyme as a biological cue for controlled release. HNE sensitive peptide linkers were immobilized within poly(ethylene glycol) hydrogels using photopolymerization techniques. The kinetics of the enzyme reaction within the gel was tailored by varying the amino acid residues present in the P1 and P1 ′ substrate positions (immediately adjacent to cleavage location). A novel FRET-based hydrogel platform was designed to characterize the accessibility of the substrate within the cross-linked, macroscopic hydrogel. Lastly, a diffusion-reaction mathematical model with Michaelis-Menten kinetics was developed to predict the overall release profile and captured the initial 80 % of the experimentally observed release. The hydrogel platform presented shows highly controlled release kinetics with potential applications in cellular responsive drug delivery. 1

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al

Selective laser melting–enabled electrospinning: Introducing complexity within electrospun membranes

Additive manufacturing technologies enable the creation of very precise and well-defined structures that can mimic hierarchical features of natural tissues. In this article, we describe the development of a manufacturing technology platform to produce innovative biodegradable membranes that are enhanced with controlled microenvironments produced via a combination of selective laser melting techniques and conventional electrospinning. This work underpins the manufacture of a new generation of biomaterial devices that have significant potential for use as both basic research tools and components of therapeutic implants. The membranes were successfully manufactured and a total of three microenvironment designs (niches) were chosen for thorough characterisation. Scanning electron microscopy analysis demonstrated differences in fibre diameters within different areas of the niche structures as well as differences in fibre density. We also showed the potential of using the microfabricated membranes for supporting mesenchymal stromal cell culture and proliferation. We demonstrated that mesenchymal stromal cells grow and populate the membranes penetrating within the niche-like structures. These findings demonstrate the creation of a very versatile tool that can be used in a variety of tissue regeneration applications including bone healing

LEGO® bricks as building blocks for centimeter-scale biological environments

LEGO bricks are commercially available interlocking pieces of plastic that are conventionally used as toys. We describe their use to build engineered environments for cm-scale biological systems, in particular plant roots. Specifically, we take advantage of the unique modularity of these building blocks to create inexpensive, transparent, reconfigurable, and highly scalable environments for plant growth in which structural obstacles and chemical gradients can be precisely engineered to mimic soil

Spatially controlled cell adhesion on three-dimensional substrates

The microenvironment of cells in vivo is defined by spatiotemporal patterns of chemical and biophysical cues. Therefore, one important goal of tissue engineering is the generation of scaffolds with defined biofunctionalization in order to control processes like cell adhesion and differentiation. Mimicking extrinsic factors like integrin ligands presented by the extracellular matrix is one of the key elements to study cellular adhesion on biocompatible scaffolds. By using special thermoformable polymer films with anchored biomolecules micro structured scaffolds, e.g. curved and µ-patterned substrates, can be fabricated. Here, we present a novel strategy for the fabrication of µ-patterned scaffolds based on the “Substrate Modification and Replication by Thermoforming” (SMART) technology: The surface of a poly lactic acid membrane, having a low forming temperature of 60°C and being initially very cell attractive, was coated with a photopatterned layer of poly(L-lysine) (PLL) and hyaluronic acid (VAHyal) to gain spatial control over cell adhesion. Subsequently, this modified polymer membrane was thermoformed to create an array of spherical microcavities with diameters of 300 µm for 3D cell culture. Human hepatoma cells (HepG2) and mouse fibroblasts (L929) were used to demonstrate guided cell adhesion. HepG2 cells adhered and aggregated exclusively within these cavities without attaching to the passivated surfaces between the cavities. Also L929 cells adhering very strongly on the pristine substrate polymer were effectively patterned by the cell repellent properties of the hyaluronic acid based hydrogel. This is the first time cell adhesion was controlled by patterned functionalization of a polymeric substrate with UV curable PLL-VAHyal in thermoformed 3D microstructures

A Cellular Potts Model simulating cell migration on and in matrix environments

Cell migration on and through extracellular matrix plays a critical role in a wide variety of physiological and pathological phenomena, and in scaffold-based tissue engineering. Migration is regulated by a number of extracellular matrix- or cell-derived biophysical parameters, such as matrix fiber orientation, gap size, and elasticity, or cell deformation, proteolysis, and adhesion. We here present an extended Cellular Potts Model (CPM) able to qualitatively and quantitatively describe cell migratory phenotype on both two-dimensional substrates and within three-dimensional environments, in a close comparison with experimental evidence. As distinct features of our approach, the cells are represented by compartmentalized discrete objects, differentiated in the nucleus and in the cytosolic region, while the extracellular matrix is composed of a fibrous mesh and of a homogeneous fluid. Our model provides a strong correlation of the directionality of migration with the topological ECM distribution and, further, a biphasic dependence of migration on the matrix density, and in part adhesion, in both two-dimensional and three-dimensional settings. Moreover, we demonstrate that the directional component of cell movement is strongly correlated with the topological distribution of the ECM fibrous network. In the three-dimensional networks, we also investigate the effects of the matrix mechanical microstructure, observing that, at a given distribution of fibers, cell motility has a subtle bimodal relation with the elasticity of the scaffold. Finally, cell locomotion requires deformation of the cell's nucleus and/or cell-derived proteolysis of steric fibrillar obstacles within rather rigid matrices characterized by small pores, not, however, for sufficiently large pores. In conclusion, we here propose a mathematical modeling approach that serves to characterize cell migration as a biological phenomen in health, disease and tissue engineering applications. The research that led to the present paper was partially supported by a grant of the group GNFM of INdA

Fabrication of endothelial cell-laden carrageenan microfibers for microvascularized bone tissue engineering applications

ecent achievements in the area of tissue engineering (TE) have enabled the development of three-dimensional (3D) cell-laden hydrogels as in vitro platforms that closely mimic the 3D scenario found in native tissues. These platforms are extensively used to evaluate cellular behavior, cell-cell interactions, and tissue-like formation in highly defined settings. In this study, we propose a scalable and flexible 3D system based on microsized hydrogel fibers that might be used as building blocks for the establishment of 3D hydrogel constructs for vascularized bone TE applications. For this purpose, chitosan (CHT) coated κ-carrageenan (κ-CA) microfibers were developed using a two-step procedure involving ionotropic gelation (for the fiber formation) of κ-CA and its polyelectrolyte complexation with CHT (for the enhancement of fiber stability). The performance of the obtained fibers was assessed regarding their swelling and stability profiles, as well as their ability to carry and, subsequently, promote the outward release of microvascular-like endothelial cells (ECs), without compromising their viability and phenotype. Finally, the possibility of assembling and integrating these cell-laden fibers within a 3D hydrogel matrix containing osteoblast-like cells was evaluated. Overall, the obtained results demonstrate the suitability of the microsized κ-CA fibers to carry and deliver phenotypically apt microvascular-like ECs. Furthermore, it is shown that it is possible to assemble these cell-laden microsized fibers into 3D heterotypic hydrogels constructs. This in vitro 3D platform provides a versatile approach to investigate the interactions between multiple cell types in controlled settings, which may open up novel 3D in vitro culture techniques to better mimic the complexity of tissues.Authors thank the Portuguese Foundation for Science and Technology (FCT) for the personal grants SFRH/BD/42968/2008 through the MIT-Portugal Program (SMM) and SFRH/BD/64070/2009 (EGP). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS and MIT/ECE/0047/2009 project

Regulation of Epithelial Cell Morphology and Functions Approaching To More In Vivo-Like by Modifying Polyethylene Glycol on Polysulfone Membranes

Cytocompatibility is critically important in design of biomaterials for application in tissue engineering. However, the currently well-accepted “cytocompatible" biomaterials are those which promote cells to sustain good attachment/spreading. The cells on such materials usually lack the self-assembled cell morphology and high cell functions as in vivo. In our view, biomaterials that can promote the ability of cells to self-assemble and demonstrate cell-specific functions would be cytocompatible. This paper examined the interaction of polyethylene glycol (PEG) modified polysulfone (PSf) membranes with four epithelial cell types (primary liver cells, a liver tumor cell line, and two renal tubular cell lines). Our results show that PSf membranes modified with proper PEG promoted the aggregation of both liver and renal cells, but the liver cells more easily formed aggregates than the renal tubular cells. The culture on PEG-modified PSf membranes also enhanced cell-specific functions. In particular, the cells cultured on F127 membranes with the proper PEG content mimicked the in vivo ultrastructure of liver cells or renal tubules cells and displayed the highest cell functions. Gene expression data for adhesion proteins suggest that the PEG modification impaired cell-membrane interactions and increased cell-cell interactions, thus facilitating cell self-assembly. In conclusion, PEG-modified membrane could be a cytocompatible material which regulates the morphology and functions of epithelial cells in mimicking cell performance in vivo