Optimizing a Scaffoldless Approach for Cartilage Tissue Engineering

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ProQuest, 2009 - 229 pages
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Articular cartilage has a poor intrinsic healing response, so tissue engineering provides a promising approach for cartilage regeneration. The major objective of this proposal was to enhance the self-assembling process, used in articular cartilage tissue engineering, by investigating the effects of construct confinement, hydrostatic pressure application, and growth factor addition. First, the effects of construct confinement in different directions and at different times were investigated. It was demonstrated that construct confinement resulted in enhanced biomechanical properties in the direction orthogonal to the confinement surface, either by enhancing collagen organization or by increasing collagen production. Next, the effects of hydrostatic pressure at different timepoints, magnitudes, and frequencies on the biomechanical and biochemical properties of self-assembled constructs were determined. It was demonstrated that the application of static hydrostatic pressure, at 10 MPa, for 1 h/day, from days 10-14 days led to significant increases in compressive and tensile properties, accompanied by significant increases in GAG and collagen content, respectively. To our knowledge, this was the first study to demonstrate increases in the biomechanical properties of tissue from pure HP application. Furthermore, the effects of exogenous application of growth factors, at varying concentrations, dosages, and combinations, with and without hydrostatic pressure, were assessed on the biochemical and biomechanical properties of engineered constructs. A systematic approach was used to determine the effects of BMP-2, IGF-I, and TGF-beta1, alone and in combination, on the functional properties of engineered constructs. This was the first study to demonstrate significant increases in both compressive and tensile biomechanical properties as a result of growth factor treatment. Also, for the first time, synergistic and additive effects on construct biomechanical and biochemical properties were found when combining growth factor treatment with hydrostatic pressure application. Finally, the effects of various decellularization treatments were examined, and it was determined that it was possible to remove cells while maintaining construct functional properties. The results presented in this thesis are exciting, as they have allowed for a better understanding of the self-assembling process, and have allowed the self-assembled constructs to mature into functional articular cartilage, as evidenced by biomechanical and biochemical properties spanning native tissue values.
  

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Contents

Paradigms of Tissue Engineering with Applications to Cartilage
6
The Use of Hydrostatic Pressure in Articular Cartilage Tissue
42
Effects of Confinement on the Mechanical Properties of Self
64
Effects of Temporal Hydrostatic Pressure on Tissue Engineered Bovine Articular Cartilage Constructs
85
ABSTRACT
86
INTRODUCTION
88
MATERIALS AND METHODS
91
RESULTS
97
ABSTRACT
145
INTRODUCTION
146
MATERIALS AND METHODS
147
RESULTS
152
DISCUSSION
155
Conclusions
160
References
169
Figures
191

DISCUSSION
101
Systematic Assessment of Growth Factor Treatment on Biochemical and Biomechanical Properties of Engineered Articular Cartilage Constructs
106
ABSTRACT
107
INTRODUCTION
109
METHODS
111
RESULTS
117
DISCUSSION
120
Synergistic and Additive Effects of Hydrostatic Pressure and Growth Factors on Tissue Formation
126
ABSTRACT
127
INTRODUCTION
129
MATERIALS AND METHODS
131
RESULTS
135
DISCUSSION
137
Evaluating Five Extraction Techniques for the Decellularization of Scaffoldless Tissue Engineered Articular Cartilage Constructs
144
Zonal arrangement of cartilage
192
Tissue engineering paradigm
193
A Hydrostatic pressure chamber B Direct compression device
194
Illustration of a chondrocyte exposed to HP
195
Representative HP bioreactor design
196
HP mechanotransduction
197
The experimental design
198
Histology of 2wk and 4wk confined constructs at 4 wks
199
Mechanical properties of constructs in radial confinement study
200
Mechanical properties of constructs in passive axial compression study
201
Histological and immunohistochemical images representative of all selfassembled constrcuts at 24 and 8 wks
202
Biomechanical and biochemical properties of selfassembled constructs in phase I
203
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