Formation of highly porous biodegradable scaffolds for tissue engineering

In recent years, lack of donor organs has caused many to consider tissue engineering methods as means to replace diseased or damaged organs. This newly-emerging field uses tissue-specific cells in a three-dimensional organization, provided by a scaffolding material, to return functionality of the organ. For these applications, the choice of scaffolding material is crucial to the success of the technique. In addition to the chemical properties of the material, physical properties such as surface area for cell attachment are essential. Various methods of creating pores in these materials to increase surface area are reviewed here. Scaffolds formed using the different techniques, which include fiber bonding, solvent casting/particulate leaching, gas foaming and phase separation, are compared on the basis of porosity, pore size, and promotion of tissue growth

Effect of Selective Heparin Desulfation on Preservation of Bone Morphogenetic Protein‑2 Bioactivity after Thermal Stress

Bone morphogenetic protein-2 (BMP-2) plays an important role in bone and cartilage formation and is of interest in regenerative medicine. Heparin can interact electrostatically with BMP-2 and thus has been explored for controlled release and potential stabilization of this growth factor <i>in vivo</i>. However, in its natively sulfated state, heparin has potent anticoagulant properties that may limit its use. Desulfation reduces anticoagulant properties, but may impact heparin’s ability to interact and protect BMP-2 from denaturation. The goal of this study was to characterize three selectively desulfated heparin species (N-desulfated (Hep^–N), 6-O,N-desulfated (Hep^–N,–6O), and completely desulfated heparin (Hep^–)) and determine if the sulfation level of heparin affected the level of BMP-2 bioactivity after heat treatment at 65 °C. BMP-2 bioactivity was evaluated using the established C2C12 cell assay. The resulting alkaline phosphatase activity data demonstrated that native heparin maintained a significant amount of BMP-2 bioactivity and the effect appeared to be heparin concentration dependent. Although all three had the same molecular charge as determined by zeta potential measurements, desulfated heparin derivatives Hep^–N and Hep^–N,–6O were not as effective as native heparin in maintaining BMP-2 bioactivity (only ∼35% of original activity remained in both cases). These findings can be used to better select desulfated heparin species that exhibit low anticoagulant activity while extending the half-life of BMP-2 in solution and in delivery systems

Combination of Heparin Binding Peptide and Heparin Cell Surface Coatings for Mesenchymal Stem Cell Spheroid Assembly

Microtissues containing multiple cell types have been used in both <i>in vitro</i> models and <i>in vivo</i> tissue repair applications. However, to improve throughput, there is a need to develop a platform that supports self-assembly of a large number of 3D microtissues containing multiple cell types in a dynamic suspension system. Thus, the objective of this study was to exploit the binding interaction between the negatively charged glycosaminoglycan, heparin, and a known heparin binding peptide to establish a method that promotes assembly of mesenchymal stem cell (MSC) spheroids into larger aggregates. We characterized heparin binding peptide (HEPpep) and heparin coatings on cell surfaces and determined the specificity of these coatings in promoting assembly of MSC spheroids in dynamic culture. Overall, combining spheroids with both coatings promoted up to 70 ± 11% of spheroids to assemble into multiaggregate structures, as compared to only 10 ± 4% assembly when cells having the heparin coating were cultured with cells coated with a scrambled peptide. These results suggest that this self-assembly method represents an exciting approach that may be applicable for a wide range of applications in which cell aggregation is desired

Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering

We investigated the development of an injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) with encapsulated rabbit marrow mesenchymal stem cells (MSCs) and gelatin microparticles (MPs) loaded with transforming growth factor-β1 (TGF-β1) for cartilage tissue engineering applications. Rabbit MSCs and TGF-β1-loaded MPs were mixed with OPF, a poly(ethylene glycol)-diacrylate crosslinker and the radical initiators ammonium persulfate and N,N,N’,N’-tetramethylethylenediamine, and then crosslinked at 37°C for 8 min to form hydrogel composites. Three studies were conducted over 14 days in order to examine the effects of: 1) the composite formulation, 2) the MSC seeding density, and 3) the TGF-β1 concentration on the chondrogenic differentiation of encapsulated rabbit MSCs. Bioassay results showed no significant difference in DNA amount between groups, however, groups with MPs had a significant increase in glycosaminoglycan content per DNA starting at day 7 as compared to controls at day 0. Chondrocyte-specific gene expression of type II collagen and aggrecan were only evident in groups containing TGF-β1-loaded MPs and varied with TGF-β1 concentration in a dose dependent manner. Specifically, type II collagen gene expression exhibited a 161 ± 49 fold increase and aggrecan gene expression a 221 ± 151 fold increase after 14 days with the highest dose of TGF-β1 (16 ng/ml). These results indicate that encapsulated rabbit MSCs remained viable over the culture period and differentiated into chondrocyte-like cells, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for localized delivery of stem cells and bioactive molecules

Long-Term Spatially Defined Coculture Within Three-Dimensional Photopatterned Hydrogels

Spatially controlled coculture in three-dimensional environments that appropriately mimic in vivo tissue architecture is a highly desirable goal in basic scientific studies of stem cell physiological processes (e.g., proliferation, matrix production, and tissue repair) and in enhancing the development of novel stem-cell-based clinical therapies for a variety of ailments. This study describes a novel fabrication system for photopatterning and assembling cell-laden oligo(polyethylene glycol)-fumarate:poly(ethylene glycol)-diacrylate hydrogels with high spatial fidelity and thickness using a controlled, inert nitrogen environment without the need for expensive precision equipment. Cross-linking was performed using Irgacure-2959 photoinitiator and 365-nm light (∼7 mW/cm2) to form gels ranging from 0.9 to 3 mm in width. Employing a nitrogen environment increased gel thickness up to 240%, generating gels >1 mm thick before swelling. This technique was further applied for spatially controlled patterning of primary tendon/ligament fibroblasts and marrow stromal cells in a single 1.5-mm-thick laminated hydrogel construct. Cells encapsulated using this technique maintained viability over 14 days in culture. This system potentially enables better understanding of paracrine effects on a range of stem cell functions and therefore may be useful as an in vitro model system for a wide array of regenerative medicine applications