3D Printed Bioglass Incorporated Collagen Matrices for Musculoskeletal Tissue Engineering
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
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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
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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.