Tissue engineering is a multidisciplinary field incorporating both biological and engineering principles to treat the loss of a tissue or organ. Disadvantages of current bone grafting techniques can be overcome using tissue engineering methods by inducing patient-derived cells to produce autologous bone upon a scaffold. Therefore, the focus of this dissertation is a novel approach to the production of a mechanically competent autologous trabecular bone substitute. In this approach, osteoblasts deposit a mineralized matrix upon a scaffold, enhancing biological and mechanical properties of the construct.

Cell-mediated bone deposition upon a trabeculated hydroxyapatite scaffold significantly increased the mechanical properties the construct. Electron probe microanalysis and nanoindentation confirmed the presence of mineral with Ca/P, modulus, and hardness similar to that of natural trabecular bone tissue. These results establish the feasibility of the production of an autologous hydroxyapatite-based trabecular bone substitute with properties enhanced through in vitro bone deposition.

An integral component of this investigation is the development of specialized tools for in vitro cell culture and histological evaluation of tissue engineered bone. A novel flow perfusion system was designed to allow long-term culture o f osteoblasts upon trabeculated hydroxyapatite scaffolds. This system facilitates long-term mineralization studies in which the effects of perfused flow on mineral quantity and quality can be assessed. To enable three-dimensional evaluation of tissue engineered specimens, a technique for volumetric fluorescence histology was developed. This technique produces three-dimensional reconstructions of fluorochrome labeled components within large biological specimens at high resolutions (on the order of 1 micron in-plane, 8 microns out-of-plane). Operator time and technical challenges are minimized by the incorporation of entirely automated components. Data produced by this technique allow the com putation of three-dim ensional measures of fluorescent signals previously approximated with two-dimensional information. Application of this system to the imaging of a tissue engineered trabecular bone substitute enabled the quantitative evaluation of cell and tissue density and distribution within the scaffold.

In summary, the work described herein provides a basis for the production of a biomechanically functional autologous trabecular bone substitute as well as powerful tools to aid in the development of tissue engineered implants.