Article

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego.
Journal of Visualized Experiments 02/2013; DOI: 10.3791/50018
Source: PubMed

ABSTRACT Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9.
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

0 Followers
 · 
63 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ability of fibronectin to bind cells can be accounted for by the tetrapeptide L-arginyl-glycyl-L-aspartyl-L-serine, a sequence which is part of the cell attachment domain of fibronectin and present in at least five other proteins. This tetrapeptide may constitute a cellular recognition determinant common to several proteins.
    Nature 05/1984; 309(5963):30-3. DOI:10.1038/309030a0 · 42.35 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cartilage tissue engineering strategies generally result in homogeneous tissue structures with little resemblance to the native zonal organization of articular cartilage. The objective of this study was to use bilayered photopolymerized hydrogels to organize zone-specific chondrocytes in a stratified framework and study the effects of this three-dimensional coculture system on the properties of the engineered tissue. Superficial and deep zone chondrocytes from bovine articular cartilage were photoencapsulated in separate hydrogels as well as in adjacent layers of a bilayered hydrogel. Histology, mechanical testing, and biochemical analysis was performed after culturing in vitro. To evaluate the influence of coculture on tissue properties, the layers were separated and compared to constructs containing only superficial or deep cells. In the bilayered constructs, deep cells produced more collagen and proteoglycan than superficial cells, resulting in cartilage tissue with stratified, heterogeneous properties. Deep cells cocultured with superficial cells in the bilayered system demonstrated reduced proliferation and increased matrix synthesis compared to deep cells cultured alone. The bilayered constructs demonstrated greater shear and compressive strength than homogenous cell constructs. This study demonstrated that interactions between zone-specific chondrocytes affect the biological and mechanical properties of engineered cartilage. Strategies aimed to structurally organize zone-specific cells and encourage heterotypic cell interactions may contribute to improved functional properties of engineered cartilage.
    Tissue Engineering 03/2007; 13(2):405-14. DOI:10.1089/ten.2006.0068 · 4.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel method for the sequential formation of hydrogel multilayers is described. Formation of the first layer is based on surface initiated photopolymerization of hydrogel precursors on eosin derivatized surfaces. In order to attach subsequent layers it is necessary to be able to functionalize intermediate hydrogel layers with eosin. In the present work, this is accomplished by introducing poly(ethylene glycol) amino acrylate (NH2-PEG-Acr) along with other hydrogel precursors such as poly(ethylene glycol) diacrylate (PEG-DA) on the intermediate layers. The pendant amine groups allow functionalization of the intermediate layers with eosin for subsequent photopolymerization of new hydrogel layers. The process can be repeated sequentially to construct multilayered hydrogel membranes. The NH2-PEG-Acr monomer can be formed by coupling cysteamine to PEG-DA by a conjugate addition reaction. The approach to multilayer formation could allow the incorporation of specific functionalities or compositions within each hydrogel layer so that multifunctional membranes can be formed. It could also be implemented, through proper photopatterning procedures, for the formation of 3-D hydrogel structures. The mild photopolymerization conditions employed using visible (514 nm), rather than ultraviolet light would make this technique especially attractive for tissue engineering, drug delivery, biomaterials, biosensor development and other situations where the elements incorporated are sensitive to UV light exposure.
    Biomaterials 04/2006; 27(8):1209-15. DOI:10.1016/j.biomaterials.2005.08.025 · 8.31 Impact Factor

Preview

Download
1 Download
Available from