Date

August 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

Translating cartilage engineering from a research concept to a clinical therapy is presently hindered by an inability to create a neocartilaginous tissue that can functionally replicate native articular cartilage. The main challenges are promoting the formation of a tissue with the phenotypic and mechanical characteristics of articular cartilage in a clinically relevant format. Tissue engineering technology utilizes a three-dimensional scaffold for the delivery of cells and bioactive components in a coordinated fashion to promote tissue formation. The best combination and delivery mechanism for these three components has not yet been determined. The overall goal of my research was to incorporate the biological principles of cartilage development into novel systems that could improve the functional outcome of tissue engineered cartilage. One objective was to address the biomechanical inferiority of neocartilage constructs relative to articular cartilage. The operating hypothesis for this work was that non-native assembly of the extracellular matrix in neocartilage constructs is a major factor contributing to their lack of mechanical integrity. I took two separate experimental approaches to promote an improved ultrastructural assembly of the cartilage matrix. The first was to develop a bioresponsive scaffold with degradation specifically tailored to chondrogenesis and matrix elaboration from the encapsulated cells. By characterizing chondrogenesis in poly(ethylene glycol) diacrylate scaffolds I identified matrix metalloproteinase-7 (MMP-7) as a candidate enzyme for modulating degradation. Mesenchymal stem cells (MSCs) encapsulated in scaffolds with MMP-7 degradable peptides produced a more extensive collagen II matrix that resulted in an increased dynamic compressive modulus. Furthermore, during the development of this bioresponsive scaffold, I validated a visible light photoinitiator system that facilitated faster and more complete formation of scaffolds using a technique that offers clinical advantages over ultraviolet photoinitiators. The second approach was to apply mechanical stimuli to the hydrogel constructs during MSC chondrogenesis to drive an anisotropic assembly of the extracellular matrix. Preliminary evidence from this research indicated inhibition of MSC chondrogenesis with compressive stimuli during development and the project was not pursued in depth. In parallel, I also tried to identify methods to promote a more hyaline cartilage phenotype from encapsulated mesenchymal stem cells following chondrogenic differentiation. This work involved evaluating the temporal significance of bioactive factors in maximizing production of the correct types of cartilage matrix molecules. I found that transforming growth factor-β (TGF-β) was essential to initiating MSC differentiation in scaffolds, and that its inclusion was required for at least three weeks to maximize collagen biosynthesis. However, dexamethasone, previously considered essential to MSC chondrogenesis, was dispensable in this culture format. Furthermore, excluding dexamethasone from the medium promoted a more hyaline cartilage phenotype in the neocartilage constructs. These data were used to design a bioactive scaffold with TGF-β tethered directly to the scaffold in a system that would be amenable to in vivo cartilage engineering. In a separate approach, I also developed a coculture system containing both MSCs and articular chondrocytes to investigate whether these cells would influence chondrogenesis. I found coculture promoted a synergistic relationship between the encapsulated cells and resulted in a neocartilage constructs with a hyaline cartilage phenotype. Taken together the work presented in this thesis provides the foundation for novel approaches to improve cartilage engineered constructs. Bioresponsive and bioactive scaffolds, such as those detailed in this research, aim to incorporate aspects of developmental biology into system design to improve functional outcome of engineered cartilage. Furthermore, by modulating the culture conditions through coculture and temporal regulation of bioactive factors I have found ways to promote MSC-derived neocartilage constructs with a hyaline cartilage phenotype.

Identifier

doi:10.6083/M4BK19BC

School

School of Medicine

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