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    3D Printed Bioglass Incorporated Collagen Matrices for Musculoskeletal Tissue Engineering

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    KAJAVE-DISSERTATION-2021.pdf (3.325Mb)
    Date
    2021-12
    Author
    Kajave, Nilabh Sunil
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    Abstract
    Musculoskeletal injuries are a major cause of disability affecting millions of people around the world and resulting in a significant economic burden of over $14 billion. Surgical intervention using autografts and allografts is the current gold standard used in the clinic for the repair and regeneration of musculoskeletal injuries, but these treatment options are associated with major limitations such as donor-site morbidity, need for multiple surgeries, immune-related complications, and risk for disease transmission. Over the past three decades, the development and application of tissue engineering strategies for restoration and reconstruction of damaged or diseased musculoskeletal tissues have gained considerable momentum as a promising alternative treatment option. In this realm, collagen and Bioglass (BG) materials have been often combined to generate tissue-mimicking composite scaffolds for use in bone tissue engineering (BTE) and interface tissue engineering (ITE) applications. Some of the most common biofabrication methodologies used for the generation of these composite scaffolds include freeze-drying and iv plastic compression, but these techniques offer little control over scaffold shape and yield scaffolds with weak mechanical properties. Extrusion-based 3D printing is a rapidly evolving layer-by-layer technique that enables the generation of custom-designed 3D scaffolds with welldefined complex geometries and precise spatial distribution of biological material for use in tissue engineering applications. The overarching goal of this dissertation was to develop BG incorporated collagen inks for 3D printing of biomimetic tissue constructs that resemble the compositional make-up of native tissue (i.e., bone, ACL enthesis) to provide the essential biochemical cues to control and direct cell function. Specifically, tissue-mimicking constructs were developed in this work to achieve material-directed tissue-specific cellular response in the absence of any external factors (e.g., growth factors, drugs). Recent work has shown that the addition of methacrylate groups to the collagen structure allows for photochemical crosslinking of collagen hydrogels while retaining the basic characteristics of native collagen. This in situ photo-crosslinking capability of methacrylated collagen (CMA) allows its use as a bioink for 3D printing of collagen constructs. In the first aim, a dual crosslinking strategy was developed to improve the mechanical properties, stability, and cell viability of 3D printed cell-laden CMA constructs by first photopolymerizing the CMA hydrogel followed by chemical crosslinking with genipin. Results showed that use of a dual crosslinking strategy with lower amounts of genipin yields mechanically superior, more stable, printable, and cell compatible CMA constructs. In the second aim, 3D printing was employed for the first time to generate finely controlled biomimetic BG incorporated collagen constructs for BTE applications. Results from this work demonstrated that BG incorporation enhances the stability, yield stress, percent recovery, and in vitro bone bioactivity of 3D printed CMA v constructs. Further, BG incorporation enhances osteogenic differentiation of human MSCs as evidenced by an increase in ALP activity and cell-mediated calcium deposition on BG-CMA constructs. In the third aim, Raman spectral mapping and 3D printing were coupled together for the first time for an innovative ‘design-build-validate’ strategy to develop a continuous biomimetic Bioglass gradient-integrated collagen matrix (BioGIM) for use in ACL reconstruction. First, Raman spectroscopy was used to generate high-resolution 3D biochemical compositional maps for modeling the mineral-collagen distribution of the native rabbit ACL enthesis. Next, a continuous biomimetic BioGIM was 3D printed using the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) approach to attain a BG gradient construct that mimics the native ACL enthesis mineral-collagen composition. Finally, Raman spectroscopy was used to validate the replication fidelity of the printed BioGIMs. Preliminary studies using human MSCs cultured on BioGIMs showed differences in cell morphology along the length of BioGIM. Results for this work demonstrate that ‘design-build-validate’ strategy is a promising approach to generate biomimetic tissue constructs for use at the ends of synthetic grafts to enable enthesis regeneration and improve the clinical outcomes of ACL reconstruction.
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    http://hdl.handle.net/11141/3481
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