Date of Award

12-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering and Sciences

First Advisor

Vipuil Kishore

Second Advisor

Chris Bashur

Third Advisor

Venkat Keshav Chivukula

Fourth Advisor

Csaba Palotai

Abstract

Musculoskeletal injuries impose a significant global burden, affecting millions and generating an economic toll exceeding $14 billion. Tissue engineering has gained traction as a promising strategy offering potential for functional restoration of damaged musculoskeletal tissues. This research aims to advance biofabrication techniques for generation of biomimetic tissue scaffolds, by optimizing scaffold design and modulating fabrication process parameters to generate scaffolds for musculoskeletal regeneration with application-specific tailored properties. In this realm, collagen-based biomaterials have been extensively investigated for tendon and ligamentous applications, and in combination with bioceramics incorporated into the collagen framework to produce composite scaffolds that better recapitulate the native bone microenvironment. The overarching goal of this dissertation was to develop advanced biofabrication methodologies towards the development of biomimetic tissue scaffolds with the essential biophysical (i.e., collagen anisotropy) and biochemical (i.e., bioceramics) cues to guide tissue-specific cell response for musculoskeletal tissue engineering and regeneration applications. Specifically, to create collagen-based scaffolds with tailored properties, advanced biofabrication techniques such as electrochemical alignment of collagen and 3D printing were employed. In addition, a novel 4D printing approach was developed to allow for the incorporation of biomimetic cues into 3D printed scaffolds by real-time matrix remodeling.

While bioceramics hold significant promise for bone tissue engineering (BTE), few studies have directly compared the osteogenic potential of these materials. In the first aim, three bioceramics – Bioglass 45S5 (BG), Laponite XLG (LAP), and β-Tricalcium Phosphate (β-TCP) incorporated into methacrylated collagen (CMA) hydrogels, were compared on the physical properties (such as swelling, stability, compressive modulus, and bone bioactivity). Additionally, the impact of these bioceramics on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) encapsulated within CMA hydrogels was assessed. Furthermore, the study examined how different bioceramic materials influenced the osteogenic differentiation of hMSCs cultured in two different media: osteoconductive (without Dexamethasone) and osteoinductive (with Dexamethasone). Results from bone bioactivity and alkaline phosphatase (ALP) activity suggest that β-TCP demonstrates the greatest potential for osteogenesis in osteoconductive media showing applicability of a material-directed approach for scaffold generation for BTE.

In addition to bioceramics, essential biophysical cues (i.e., surface topography, stiffness) are essential for guiding ell morphology, proliferation, and differentiation. While the impact of biomaterial-directed cues on cell response has been extensively documented, there is a lack of studies that have attempted to decouple these effects to gain a deeper understanding of how different physicochemical factors influence tissue-specific cell function. In the second aim of this work, β-TCP was introduced into both electrochemically aligned collagen (ELAC) and random collagen threads. The goal of the study was to explore the individual and combined effects of collagen alignment (biophysical) and bioceramic incorporation (biochemical) on osteoblast cell morphology, proliferation, differentiation, and mineralization. Results showed that alignment enhances cell-mediated mineralization which is further augmented by β-TCP incorporation.

Recent advancements have demonstrated that extrusion-based printing utilizing collagenous inks can produce 3D scaffolds with precise geometry and print fidelity. However, these scaffolds often lack collagen anisotropy. In the last aim, extrusion-based 3D printing was combined with a magnetic alignment approach in an innovative 4D printing scheme to create multilayered collagen scaffolds with a high degree of collagen fiber alignment. Specifically, the 4D printing process parameters—including the collagen (Col): xanthan gum (XG) ratio, streptavidin-coated magnetic particle concentration, and print flow speed were modulated. The effects of these components were assessed on rheological properties, print fidelity, and degree of collagen alignment. Additionally, the impact of collagen anisotropy on human mesenchymal stem cell (hMSC) morphology, metabolic activity, and differentiation towards a ligamentous lineage was evaluated. Results showed that 4D printing is a viable strategy to generate 3D anisotropic collagen scaffolds, and collagen alignment triggers cell orientation and ligamentous differentiation of hMSCs.

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