Development of 3D hydrogel culture systems with on-demand cell separation.

Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations.

Modulation of mesenchymal stem cell shape in enzyme-sensitive hydrogels is decoupled from upregulation of fibroblast markers under cyclic tension.

Our laboratory has developed a tensile culture bioreactor as a system for understanding mesenchymal stem cell (MSC) differentiation toward a tendon/ligament fibroblast phenotype in response to cyclic tensile strain. In this study, we investigated whether increased degradability of the biomaterial carrier would induce changes in MSC morphology and subsequent upregulation of tendon/fibroblast markers under tensile strain. Degradability of a synthetic poly(ethylene glycol) hydrogel was introduced by incorporating either fast- or slow-degrading matrix metalloproteinase (MMP)-sensitive peptide sequences into the polymer backbone. Although a decline in cellularity was observed over culture in all sample groups, at 14 days, MSCs were significantly more spread in fast-cleaving gels (84%±8%) compared with slow-cleaving gels (59%±4%). Cyclic tensile strain upregulated tendon/ligament fibroblast-related genes, such as collagen III (3.8-fold vs. 2.1-fold in fast-degrading gels) and tenascin-C (2.5-fold vs. 1.7-fold in fast-degrading gels). However, few differences were observed in gene expression between different gel types. Immunostaining demonstrated increased collagen III deposition in dynamically strained gels at day 14, as well as increased collagen I and tenascin-C deposition at day 14 in all groups. Results suggest that cell spreading may not be a major factor controlling MSC response to cyclic strain in this system over 14 days. However, these findings provide key parameters for the design of future biomaterial carriers and strain regimens to prime stem cells to a tendon/ligament phenotype prior to release and use in vivo.

Engineering orthopedic tissue interfaces.

While a wide variety of approaches to engineering orthopedic tissues have been proposed, less attention has been paid to the interfaces, the specialized areas that connect two tissues of different biochemical and mechanical properties. The interface tissue plays an important role in transitioning mechanical load between disparate tissues. Thus, the relatively new field of interfacial tissue engineering presents new challenges--to not only consider the regeneration of individual orthopedic tissues, but also to design the biochemical and cellular composition of the linking tissue. Approaches to interfacial tissue engineering may be distinguished based on if the goal is to recreate the interface itself, or generate an entire integrated tissue unit (such as an osteochondral plug). As background for future efforts in engineering orthopedic interfaces, a brief review of the biology and mechanics of each interface (cartilage-bone, ligament-bone, meniscus-bone, and muscle-tendon) is presented, followed by an overview of the state-of-the-art in engineering each tissue, including advances and challenges specific to regenerating the interfaces.

Degradative properties and cytocompatibility of a mixed-mode hydrogel containing oligo[poly(ethylene glycol)fumarate] and poly(ethylene glycol)dithiol.

Our laboratory is currently exploring synthetic oligo(poly(ethylene glycol)fumarate) (OPF)-based biomaterials as a means to deliver fibroblasts to promote regeneration of central/partial defects in tendons and ligaments. In order to further modulate the swelling and degradative characteristics of OPF-based hydrogels, OPF crosslinking via a radically initiated, mixed-mode reaction involving poly(ethylene glycol) (PEG)-diacrylate and PEG-dithiol was investigated. Results demonstrate that mixed-mode hydrogels containing OPF can be formed and that the presence of 20 wt.% PEG-dithiol increases swelling and decreases degradation time vs. 10 wt.% PEG-dithiol and non-thiol-containing hydrogels (20% thiol fold swelling 28.7+/-0.8; 10% thiol fold swelling 11.6+/-1.4; non-thiol 8.7+/-0.2; 20% thiol-containing hydrogels degrade within 15 days in vitro). After encapsulation, tendon/ligament fibroblasts remained largely viable over 8 days of static culture. While the presence of PEG-dithiol did not significantly affect cellularity or collagen production within the constructs over this time period, image analysis revealed that the 20% PEG-dithiol gels did appear to promote cell clustering, with greater values for aggregate area observed by day 8. These experiments suggest that mixed-mode OPF-based hydrogels may provide an interesting alternative as a cell carrier for engineering a variety of soft orthopedic tissues, particularly for applications when it is important to encourage cell-cell contact.