Regenerative Engineering in the Bone Microenvironment: Towards Reestablishing Homeostasis and Function Using Implantable Biomaterial Scaffolds

Abstract

Healthy bone is critically important to systemic and oral health. Therefore, it is essential to develop methods for predictable bone regeneration, by facilitating the development of highly cellularized, vascularized, and dynamic bone tissue. Tissue engineering is a promising approach to repairing and regenerating skeletal defects from trauma or disease. Biomaterial scaffolds serve as an artificial extracellular matrix to organize cells and engineered tissue in three dimensions and rely on endogenous or exogenous sources of stem cells and inductive factors to facilitate regeneration. Texture and porosity of scaffolds are generally recognized as critical design features to improve biocompatibility and facilitate tissue integration. To usher in the next generation of biomaterials-mediated tissue regeneration strategies, this dissertation seeks to develop an intimate understanding of the cell-biomaterial (scaffold) interface, accounting for the cellular heterogeneity and complexity of the bone microenvironment. The bone microenvironment involves complex cellular crosstalk and diverse cell types. Together, these cells maintain stem cell activities critical to tissue growth and repair throughout life, including a balance between maintenance of stemness and differentiation to a desired functional phenotype. Engineering the cell-specific trajectory of these diverse cell types through novel physical, mechanical, and biochemical strategies must be considered in developing new therapies for replacing damaged tissue and maintaining skeletal health. The overall goal of this dissertation is to elucidate the fundamental interactions and engineering criteria at the cell-biomaterial interface to robustly regenerate mineralized bone and a skeletal stem cell niche:

Aim 1 determined the role of scaffold macropore design in modulating differentiation or maintenance of stemness of skeletal MSCs. We elucidated a critical minimum pore size threshold required for scaffold angiogenesis and osteogenesis, and maximum pore size threshold that enables maintenance of stemness. Principle curvature modulates YAP/TAZ signaling based on pore size, explaining this phenomenon. A multi-compartment scaffold is engineered and validated to maintain a stem cell niche in vivo using lineage tracing and transgenic approaches. We further demonstrated this technology as a proof-of-concept for re-establishing the cranial suture stem cell niche in a mouse model of BMP-mediated midline craniosynostosis.

Aim 2 determined how scaffold texture and porosity modulate osteoclast behavior. Nanofibrous synthetic substrates mimic the texture of resorbed bone and apply pressure towards a regenerative osteoclast phenotype; their secreted factors induce osteogenesis. We developed a novel rhodamine-based probe for determining extracellular cellular acidification within the biomaterial environment. A smooth biomaterial induces osteoclast resorption and actin ring formation, consistent with an inflammatory phenotype. Pore size does not affect osteoclast behavior in vitro; however, pore-mediated modulation of angiogenesis regulates osteoclasts' ability to persist in the perivascular niche.

Aim 3 takes advantage of our macroporous scaffolds' high degree of internal surface area to develop a novel delivery system for the controlled release of extracellular vesicles (EVs) to catalyze in situ bone regeneration in vivo. Extracellular vesicles represent a unique delivery challenge due to their biological complexity. We developed a self-assembling biodegradable polymer to encapsulate EVs through droplet formation. EVs maintain their biological integrity and function after release and induce calvarial bone healing in a critical-size defect without transplanting exogenous cells.

Guiding cell fate within the skeletal microenvironment is critically essential in designing regenerative therapeutics for craniofacial regeneration, both in terms of maintenance of stemness and directed differentiation. We also demonstrate proof-of-concept data for future work in precision tissue engineering.