Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering.

摘要

Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering.; The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and further reduces hypertrophy of differentiating MSCs. On the other hand, although cells in HABPep-conjugated scaffolds produced less ECM components, they survived and proliferated significantly more than those in control, resulting in overall increase in ECM contents per scaffold. Once implanted in vivo, HABPep-conjugated constructs increased GAG and type II collagen contents further, compared to those of the control hydrogel. These results showed that enhanced interaction between HA and scaffold via HABPep improved the in vitro culture expansion of MSCs and further ECM production in vivo.; Effects of cell-secreted bioactive factors via cell-cell communication on stem cell differentiation were also investigated in 3D bilayer system. First, when mesenchymal progenitor cells (MPCs) were cocultured with ES-derived cells (ESDC), morphogenetic factors secreted by ESDCs showed a potential to improve MPC chondrogenesis in both control and chondrogenic medium by increasing not only MPC's chondrogenic gene expression, but also ECM production. Moreover, the effect of ESDC cell-mediated chondrogenesis of MSC could not be mimicked by chondrogenic medium supplemented with TGF-beta1 and dexamethasone. Secondly, coculturing hepatic cells enhanced specific chondrogenic differentiation of ES cells in the 3D bilayer system. These studies demonstrated that cell-secreted soluble factors can be used to guide stem cell differentiation.