A Direct-Write Three-Dimensional Bioassembly Tool for Regenerative Medicine

by Smith, Cynthia Miller.

Tissue loss and end-stage organ failure caused by disease or injury are two of the most costly problems encountered in modern medicine. To combat these problems, a relatively new field, called tissue engineering, has emerged. This field combines the medical and engineering fields in hopes of establishing an effective method to restore, maintain, or improve damaged tissue. In order to best replace the diseased tissue, many approaches to fabricating new tissue have focused on trying to replicate native tissue. The overall hypothesis of this dissertation is that a direct-write, BioAssembly Tool (BAT) can be utilized to fabricate viable constructs of cells and matrix that have a specified spatial organization and are truly three-dimensional (3D). The results of the studies within this dissertation demonstrate that the BAT can generate viable, spatially organized constructs comprised of cells and matrix by carefully controlling the environmental parameters of the system. A joint hypothesis associated with this dissertation is that 3D microscopy and image processing techniques can be combined to generate accurate representative stacks of images of the tissue within 3D, tissue engineered constructs. The results of the studies examining this hypothesis demonstrate that by taking into account the attenuation with depth in the imaged construct as well as by looking at the intensity and gradient of each voxel, accurate and reproducible thresholding can be achieved. Furthermore, this tool can be utilized to aid in the characterization of 3D tissue engineered constructs. Based on these studies, 3D microscopy and image processing shows promise in accurately representing the cellular volume within a tissue. More importantly, 3D, direct-write technology, specifically the BioAssembly Tool, could be used in the fabrication of viable, 13 spatially organized constructs that can then be implanted into a patient to provide healthy tissue in the place of diseased or damaged tissue. 14 1. INTRODUCTION Two of the most detrimental and costly issues in modern medicine are tissue loss and end-stage organ failure caused by disease or injury. Three main surgical approaches have been used to remedy these problems: whole organ transplantation, surgical reconstruction, and replacement with a mechanical device such as a prosthesis or dialysis machine. While these techniques have caused substantial improvements in health care, none of these approaches is perfect. Organ transplantation is severely limited by donor shortages, which worsen each year. Also, patients who have undergone an organ transplant must be on life-long immunosuppression. Surgical reconstruction, which involves replacing a diseased tissue with healthy tissue, can result in long-term problems. And, mechanical devices are not able to perform the full physiological function of the replaced tissue. Plus, they are limited to adult patients, since the metal devices cannot expand and grow with an immature patient. (Langer & Vacanti, 1993; Shieh & Vacanti, 2005) In order to overcome these limitations, a new field called tissue engineering (TE) emerged. TE is an interdisciplinary field that utilizes the practices of engineering and sciences with the primary goal of developing biological alternatives that are capable of restoring, maintaining, or improving tissue function (Langer & Vacanti, 1993). The term “tissue engineering” was first coined in 1987 at a National Science Foundation meeting. Since that time, this field of research has greatly expanded and made an impact on industry. As more is learned about molecular and cell biology, TE promises to bring about a new generation of tissue and organ implants. And, it is estimated that by the year 15 2020, TE has the potential to be a $20 billion industry. It has been hypothesized that the success of TE is contingent upon it closely imitating nature (Lanza, et al., 2000). Some key observations have been made in the quest to better imitate natural tissue, namely that most tissues undergo remodeling; under favorable in vitro conditions, cells tend to assemble into the appropriate structures; and by providing a template, cells can be directed to organize into a specific architecture (Langer & Vacanti, 1993).