Syntheses and Electrospinning of Poly(Amino Acid Ester) Phosphazene Biomaterials for Tissue Engineering Applications
Abstract
Despite advances made in the past decades to design biomaterials for tissue engineering, challenges remain. This thesis investigated the potential of electrospun poly(α-amino acid ester) phosphazenes (PαAPz) as novel biomaterials for vascular tissue engineering applications. As a class of biodegradable biomaterials, PαAPz provides biocompatibility and tunable properties and has gained attention as promising candidates for scaffolds in regenerative medicine, but their synthesis procedure is cumbersome due to the strict anhydrous environment and specialized equipment required for the thermal ring-opening reaction to produce the intermediate poly(dichlorophosphazene) (PDCP) product.
The research begins with the successful synthesis of PDCP using relatively simpler techniques using recrystallization and flame sealing with or without argon gas. The macromolecular substitution reaction to produce the final PαAPz was simplified using a one-step approach instead of the two-step conventional process. The PαAPz were tailored for vascular tissue engineering, focusing on the selection of α-amino acids (L-alanine, L-phenylalanine, and L-methionine) for their electrospinnability, biodegradability, and stem cell interaction properties. Following successful synthesis, electrospinning process parameters, such as polymer concentration, solvent selection, and electrospinning conditions, are systematically varied to fabricate beads-free fibrous mats with fiber diameters of 20nm to 700nm. Surface degradation studies showed PαAPz from L-phenylalanine degraded faster than those based on L-alanine, L-phenylalanine, and L-methionine. Atomic force microscopy (AFM) was used to evaluate the fiber mechanical characteristics and calculate its Young’s modulus, revealing it to closely mimic the stiffness of a natural extracellular matrix (ECM).
Mesenchymal stem cells derived from human induced pluripotent stem cells (iPSC), bone marrow-derived mesenchymal stem cells (BM-MSC) and primary human coronary artery smooth muscle cells (SMC) attached and well-spread on the fibers. Differentiation of iMSC to SMC was characterized by increased transcriptional levels of early to late-stage smooth muscle marker proteins on electrospun fibrous mats. Evaluation of mesenchymal multipotent 10T1/2 cell and mesenchymal stem cell (MSC) behavior on the scaffolds demonstrated significant cell viability, proliferation, and successful MSC differentiation into smooth muscle cells. Expression of collagen and elastin by MSCs on the fiber mats highlighted potential ECM formation during scaffold degradation. In addition, PαAPz from L-methionine served as a reactive oxygen species (ROS) scavenger, thus protecting cells from stress. In order to expand the utility of the synthesized PαAPz to bone tissue engineering, the effect of their degradation products of osteogenic differentiation of stem cells was studied. It was observed that the late-stage degradation product, such as phosphoric acid, can significantly influence the osteogenic differentiation of MSCs.
The data collectively presented in this thesis demonstrated the potential of PαAPz in vascular tissue engineering, showcasing their potential in functional tissue formation, MSC differentiation, and protection against oxidative stress.