Date of Award

8-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

LaBerge, Martine

Committee Member

Wen , Xuejun

Committee Member

Zhang , Ning

Committee Member

Yao , Hai

Committee Member

Kindy , Mark

Abstract

The ultimate goal of tissue regeneration is to replace damaged or diseased tissue with a cell-based or biomaterial-based tissue that accurately mimics the functionality, biology, mechanics, and cellular and extracellular matrix (ECM) composition of the native tissue. Specifically, the ability to control the architecture of tissue engineered constructs plays a vital role in all of these issues as scaffold architecture has an affect on function, biomechanics, and cellular behavior. Many tissue engineered scaffolds focus on the ability to mimic natural tissue by simulating the ECM due to the fact that in each distinct tissue, the ECM serves as a structural component by providing unique mechanical strength as well as regions for cellular attachment or the storage of a variety of biomolecules. Additionally, cellular behavior has the ability to be controlled based on the structure and composition of the ECM. More specifically, matrix has the ability to modulate a variety of cellular behaviors such as: adhesion, morphology, migration, proliferation, and differentiation while also controlling the ability of cells to produce and synthesize ECM with similar characteristics to that of surrounding tissue. Tissue matrix and structure plays an essential role during the process of tissue formation, remodeling, and regeneration.
The ability to mimic native tissue ECM using various biofabrication-based techniques has become an emerging concept in the realm of tissue regeneration. Biofabrication utilizes automated computer-aided-design (CAD) and computer-controlled technologies to create reproducible biomaterial and cell-based scaffolds that have the ability to imitate native tissue ECM. Of particular interest are strategies that employ biofabrication with the aim of improving the overall control over scaffold architecture and microstructure while also providing reproducibility.
Due to their versatility, a variety of promising biofabrication strategies exist, including rapid prototyping methods such as bioprinting and additive manufacturing, which rely on the deposition or extrusion of materials. Using these methods, a multitude of materials can be easily used to fabricate scaffold structures with various morphologies. However, the potential of many biofabrication methods in tissue engineering applications is limited by the potential resolution of the structures that can be created. It was our goal to investigate a unique biofabrication strategy with the aim of fabricating 3-D scaffolds at a high resolution with morphological, biological, and mechanical properties similar to those of natural intervertebral discs (IVDs).
Initially, a CAD-based biofabrication approach was developed and systematically optimized. This method was selected to utilize a custom-designed computer interface with 3-D motion control that allowed for greater resolution and precision of the fabricated scaffold architecture. Furthermore, we incorporated a temperature controlled polymer collection stage, which proved advantageous in enhancing the resolution of the biofabrication technique. By lowering the temperature of the collecting stage below the freezing point of the polymer solution, it was discovered that the extruded polymer solution could be solidified directly as it exited the micropipette extrusion tip through an increase in viscosity. Results from initial studies provided valuable clues towards determining the relationship between motor speeds, polymer solution temperatures, micropipette size, extrusion rate, and polymer solution viscosity. These results encouraged the investigation of the ability to use this method to precisely control scaffold spatial orientation for the fabrication of IVD scaffolds.
Since previous IVD scaffold fabrication methods have not effectively accounted for the inadequacies of spinal fusion and artificial disc replacement in the treatment of a degenerated disc, we addressed the significance of matching native tissue histology and biomechanics by using fabricated scaffolds that closely mimic natural IVD tissue. The annulus fibrosus (AF), or outer region of the IVD, was the focus of this project due to current and previous challenges in recreating its discrete tissue architecture, which is not an issue for the inner nucleus pulposus (NP) region, as it is more commonly mimicked with the use of a hydrogel-based biomaterial.
Multiple elastomeric materials, including biocompatible and biodegradable polyurethane (PU) and chitosan-gelatin (CS/GEL), were investigated to evaluate the usefulness of this biofabrication approach to create biomimetic IVD scaffolds utilizing various materials. It was determined that the biofabrication method enabled the use of multiple materials and that the fabricated scaffolds were able to mimic the kidney shaped structure of the IVD. Additionally, the scaffolds exhibited ideal concentric lamellar thickness and spacing, accurately mimicking the native structure of the AF in the human IVD. To the best of our knowledge, these accomplishments in recreating the native AF histological architecture within tissue engineered constructs have not been achieved elsewhere. Cells attached and aligned on the scaffolds in the direction of the concentric lamellar structure, emulating cell behavior comparable to the native AF. These 3-D scaffolds exhibited ideal elastic properties and did not experience permanent deformation under dynamic loading. Additionally, the scaffold mechanical properties showed no significant differences when compared with native human IVD tissue. The scaffolding promoted chondrocyte cell attachment and proliferation in alignment with the concentric lamellae, proving this method improves upon current IVD scaffold fabrication approaches, as it takes into account native tissue structure and cell response.
To expand upon these findings, the biomimetic IVD scaffolds were investigated to analyze the formation of 3-D cellularized tissue. 3-D multicellular spheroids formed from chondrocytes were incorporated within the scaffold to fully cellularize the void spacing within the IVD scaffold lamellae. The ability of this 3-D cellularized structure to emulate native IVD tissue was then further analyzed by evaluating the ability of the scaffolds to synthesize matrix that was structurally and compositionally similar to that of native tissue. Our studies indicate that the 3-D cellularized IVD constructs accurately mimic native IVD tissue and provide not only a scaffold, but a cellularized platform to promote tissue regeneration. Future studies will assess the biofabricted IVD structures for tissue regeneration and biostability using in vivo rodent subcutaneous animal models.

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