Ph.D. Dissertation Defense by Blaise D. Porter
Monday, November 14, 2005

(Dr. Robert E. Guldberg, Chair)

" Development and Application of a 3-D Perfusion Bioreactor Cell Culture System for Bone Tissue Engineering"

Abstract

The need for improved clinical strategies to restore function to damaged or degenerated bone due to segmental defects, spine instability, acute trauma and fractures is well recognized. The current gold standard treatment to augment repair of bone defects involves harvesting autologous bone chips from the iliac crest of the patient. However, donor site morbidity and pain, lack of structural strength, and limited graft material volume are significant drawbacks. Tissue engineering strategies that combine porous biomaterial scaffolds with cells capable of osteogenesis or bioactive proteins have shown promise as effective bone graft substitutes. Attempts to culture bone tissue-engineering constructs thicker than 1mm in vitro often result in a shell of viable cells and mineralized matrix surrounding a necrotic core. To address this limitation, we have developed a perfusion bioreactor system that improves mass transport throughout large cell-seeded constructs. Perfusion resulted in a 140-fold increase in mineral deposition at the interior of 3 mm thick polymer scaffolds seeded with rat bone marrow stromal cells. Furthermore, we have established and validated 3-D computational methods to model flow and shear stresses within the microporosity of perfused constructs. Micro-CT scanning and analysis techniques were used to monitor mineral development over time in culture. After determining that repeated x-ray scans on the same construct did not inhibit mineral formation, mineral volume, spatial distribution, density, particle size and particle number were quantified on cell-seeded constructs in 5 different culture environments. The effect of time varying flow conditions was compared to continuous perfusion as well as two different control cell culture methods in an attempt to improve mineral construct development. Intermittent elevated perfusion and dynamic culture in an orbital rocker plate produced the greatest amount of mineral within 9mm long constructs compared to low continuous flow and high continuous flow cases. Mineral particle size distribution analysis indicated that increases in mineral volume were predominantly a function of increasing mineral particle size, not an increase in the number of particles for flow cultures.