Date of Award

7-2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Engineering and Sciences

First Advisor

Vipuil Kishore

Second Advisor

Venkat Keshav Chivukula

Third Advisor

Melissa Borgen

Fourth Advisor

Linxia Gu

Abstract

Bone tissue engineering (BTE) is a promising alternative approach to autografts and allografts when treating various bone diseases and disorders. BTE enables the fabrication of biocompatible, biomimetic, and osteo-stimulating constructs for the repair and regeneration of damaged bone tissue. The native bone environment primarily consists of anisotropic type 1 collagen (organic) and hydroxyapatite nanocrystals (inorganic). Composite biomimetic scaffolds fabricated by combining collagen with bioceramics have shown immense promise for BTE applications. Prior work has reported on different biofabrication methods to synthesize collagen-based composite scaffolds for use in BTE applications. However, to date, a customizable 3D composite scaffold that resembles the compositional (i.e., collagen and bioceramics) and microarchitectural (i.e., anisotropic collagen fibers) aspects of native bone tissue is elusive. Recent work in our laboratory has demonstrated that combining extrusion-based 3D printing with streptavidin coated magnetic particle (SMP) guided magnetic alignment approach in a novel 4D printing scheme enables the generation of 3D collagen scaffolds with high degree of collagen anisotropy. The goal of this study is to assess the impact of β-tricalcium phosphate (β-TCP) incorporation on the rheological properties of collagenous inks, collagen anisotropy, and print fidelity of 4D printed scaffolds. This work also explored the impact of β-TCP incorporation on Saos-2 cell morphology, metabolic activity, and osteogenic differentiation. The innovation of this work lies in examining the impact of β-TCP incorporation on the physicochemical properties of 4D printed collagen scaffolds and Saos-2 cell response. Experimental design entailed three different 4D printed collagen scaffolds: 1) 10% w/w β-TCP-collagen scaffolds, 2) 70% w/w β-TCP collagen scaffolds, and 3) collagen scaffolds with no β-TCP (control). Results showed that the addition of β-TCP maintained collagen ink viscosity and printability of collagen scaffolds. While the yield stress decreased upon β-TCP addition, all ink formulations exhibited shear thinning behavior, indicative of viscoelastic solid materials. Polarized light microscopy (PLM) and scanning electron microscopy (SEM) revealed that β-TCP incorporation disrupted SMP-guided alignment of collagen fibers, possibly due to hindrance in the mobility of SMP due to the presence of micron-sized β-TCP particulates. A significant increase (p < 0.05) in the stability of collagen scaffolds was observed upon β-TCP incorporation that correlated with significantly lower (p < 0.05) swelling ratio. Results from the cell studies showed that addition of β-TCP modulated Saos-2 cell morphology, promoting a more polygonal cell shape suggestive of osteoblastic differentiation. Although addition of β-TCP at 70% w/w composition resulted in significantly lower cell metabolic activity (p < 0.05), overall cell viability appeared well-maintained on all scaffolds. Increased alkaline phosphatase (ALP) activity was observed in Saos-2 cells cultured on 70% w/w β-TCP incorporated collagen scaffolds, albeit this finding was not statistically significant. Imaging of tetracycline staining showed increased formation of cell-mediated mineral-like deposits in 70% w/w β-TCP -collagen scaffolds compared to the control (no β-TCP). In conclusion, while β-TCP offers advantages in terms of scaffold stability and potentially promotes Saos-2 cell differentiation, further optimization is necessary to balance printability, desired collagen organization, and augmentation of osteogenic cell response for successful BTE applications.

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