Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Chemical and Biochemical Engineering

Supervisor

Kibret Mequanint

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.

Summary for Lay Audience

Every year, cardiovascular diseases (CVDs) are claiming the lives of 20.5 million people globally. Among these CVDs, coronary artery disease is the leading cause of death. Bypass grafting is currently one of the most common interventions. Many patients lack suitable donor sites, and donor grafts can trigger immune reactions due to foreign body responses. At the same time, the current synthetic graft has poor behavior in medium/small arteries. To address these issues, scientists are turning to tissue engineering, a field where engineering principles meet biological sciences. This approach involves building organs or tissues using a patient’s own cells and supportive scaffolds, much like constructing a building with steel and bricks. Using the patient’s own cells eliminates the problems of immune rejection and donor shortages. But building such a scaffold and maturing cells on this scaffold remains a problem.

This study focuses on finding a suitable synthetic material for creating scaffolds in vascular tissue engineering. Exploration was conducted on a polymer named poly [(α-amino acid ester) phosphazenes] (PαAPz), whose degradation products are mainly composed of buffering components. These buffering degradation products are believed to be beneficial to vascular cells, suggesting such material can have strong potential in vascular tissue engineering. After synthesizing this polymer, it is made into a fiber mat with a similar structure to mask cloth but a much smaller fiber diameter. This mat simulates the three-dimensional structure of cell growth within the body. Subsequently, various measurements were conducted to assess the properties of this mat, test its ability to support cell growth and differentiation, and evaluate its potential for vascular tissue engineering applications.

In conclusion, this research introduced and investigated the potential of natural α- amino acid-based phosphazene for the application of vascular tissue engineering.

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