New strategies for the decellularization of biological tissues

The beginning of the 21st century has been marked by the rise of chronic diseases. This development has led to increased interest in the development of new therapies that focus on restoring normal tissue function through transplantation of injured tissue with biomedically engineered smart matrices. Decellularization, a process that focuses on the removal of immunogenic cellular material from a tissue or organ, has become an appealing methodology for the creation of functional and bioactive scaffolds. The present thesis focused on the creation of new methodologies for the decellularization of biological tissues. For this purpose, the author reviewed current decellularization literature and put forward a study that investigated the potential of three different decellularization protocols for porcine trabecular bone tissue using Tri(n-butyl) phosphate (TnBP), supercritical carbon dioxide (scCO2), and a combination of both. The use of TnBP as a decellularization agent, instead of harsh chemicals such as detergents, could lead to better preservation of the extracellular matrix (ECM), and better biochemical and mechanical properties to the resulting scaffold. As well, the use of supercritical fluids could lead to faster decellularization times, not only reducing the time tissues are exposed to potentially harmful agents, but also reducing the financial cost of the process. In total, five different protocols were implemented and examined: 1% (v/v) TnBP treatment for 48 hours, scCO2 treatment for 1 hour and 3 hours, and scCO2 treatment with 0.1% (w/v) TnBP for 1 hour and 3 hours. Due to the innovative nature of this work, time variants to protocols were implemented to investigate any possible harmful effects caused by prolonged exposure to scCO2 treatment. Results revealed that both TnBP and scCO2 led to the removal of DNA content, but this effect was more pronounced in treatments that used TnBP. Mechanical analysis of TnBP-treated samples revealed a higher ultimate strength and yield strain, suggesting some degree of crosslinking of collagen fibers occurred. Meanwhile, the use of scCO2 led to dehydration of samples, increasing values for Young’s Modulus and ultimate strength. The combined protocol of scCO2-TnBP led to a decrease in DNA content to about half of that measured for untreated samples, demonstrating the potential of this methodology and opening new possibilities for future optimizations that could achieve required decellularization levels.