Abstract
How to effectively restore bone defects is a universal clinical predicament. Bone tissue engineering, which requires the combination of tissue-specific cells and supporting scaffolds, has been considered as a promising approach to solvingthis problem. However, currently we are still in great need of a robust bone issue engineering system. Therefore, the overall research goal of this study is to identify the optimal combinations of productive cells and instructive scaffolds.
In particular, because of the self-renewal capability and ability to differentiate into all types of tissues, pluripotent stem cells (PSCs), specifically mouse embryonic stem cells (mESCs) were used in a novel differentiation protocol to reliably produce lineage committed osteoprogenitors at a high frequency. These cells were combined with specific hydrogels, which are high-water content polymeric materials that structurally resemble natural extracellular matrix (ECM), and used as supportive scaffolds for osteogenic differentiation in this study. In addition to biochemical signals, hydrogels provide mechanical cues that tremendously influence cellular activities, including stem cell differentiation. This study therefore aims at identifying the optimal components and parameters that have the maximum combinatory effects for materials-based osteogenic differentiation of PSC-derived osteoprogenitors.
Using a novel PSC-induction protocol, mESCs were induced to either osteochondro- or osteo-progenitors through the formation of primitive streak and mesoderm-like populations through the addition of BMP4 during a defined time window that converted osteochondro- progenitors to enriched osteoprogenitors of high purity. Culture of these PSC-derived osteoprogenitor cells on two-dimensional polyacrylamide hydrogels of different stiffnesses demonstrated that high stiffness (49 kPa) surprisingly failed to support normal osteogenic differentiation. In pursuit of a more stimulative three-dimensional biomaterial, two approaches were utilized. First, a self-healing polyethylene glycol (PEG) hydrogel system that utilizes ionic interaction was first refined and expanded, showing that while the PEG system can support the proliferation of cancer cells, it was less ideal for normal stem cell expansion and differentiation. Following this, a viscoelastic alginate hydrogel system was set up as the carrier for induced mESCs, where spheroids were identified as the optimal structures for encapsulation, and the results demonstrated that such hydrogels significantly stimulated the osteogenic differentiation of encapsulated cells. Finally, mechanistic analysis found that the universal mechanotransducer, yes-associated protein (YAP), was sensitive to the biophysical and environmental changes cells perceive, and was responsible for binding to and activating the osteogenic transcription factor gene Runx2 and downstream genes.
This study has identified a competent bone tissue engineering system composed of BMP4-induced mESC-derived osteoprogenitors and ionically crosslinked stress-relaxing alginate hydrogels. Yap has been identified as the regulating 3D mechanotransducer that can directly mediate target gene transcription and cell differentiation. This study offers a robust bone tissue engineering system that reliably produces bone-like matrix, and that will guide future design of bone regeneration platforms.
| Date of Award | 1 Apr 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Agamemnon Grigoriadis (Supervisor), Karen Liu (Supervisor) & Ricardo M. P. da Silva (Supervisor) |