Date of Award

Fall 2022

Document Type

Open Access Dissertation

Department

Biomedical Science

First Advisor

Wayne Carver

Abstract

In 2017, over 3.5 million peripheral vascular surgeries were performed worldwide with over 400,000 vascular repair or replacement surgeries being performed in the United States each year alone. As the number of vascular repair surgeries, including both coronary and peripheral bypass grafting procedures, continues to increase each year, these statistics indicate an urgent need for more effective and readily available replacement materials. Regenerative medicine and tissue engineering (TE) approaches, including the design, fabrication, and validation of suitable biomaterials in vitro that direct the repair and regeneration of damaged tissues, have been proposed to alleviate this problem. While advanced biomaterials have made significant headway in the engineering of complex replacement tissue scaffolds, several hurdles remain before this technology reaches its full potential. In the application of tissue-engineered blood vessels as a platform for coronary and peripheral artery bypass grafting procedures these hurdles include increased thrombogenicity, poor recellularization, and mechanical mismatch, ultimately leading to decreased patency of the vessel. In addressing these hurdles, decellularized blood vessels have gained substantial popularity as a platform for TE vascular grafts; however, this remains challenging, as there is a pressing need to optimize decellularization protocols for small-diameter blood vessels to produce vascular scaffolds with properties mirroring that of native tissue.

One method for producing effectively decellularized TE scaffolds is chemical decellularization, which has been successfully achieved through the use of the anionic detergents sodium dodecyl sulfate (SDS) and sodium deoxycholate (SDC). These homogeneously disrupting surfactants solubilize cell membranes and can be very effective decellularization agents; however, exposure at high concentrations or for long durations can damage the extracellular matrix and can be cytotoxic. We initially investigated the differential response of varying 0%−6% SDS and SDC anionic detergent concentrations after 24 and 72 h in the presence of DNase using biochemical, histological, and biaxial mechanical analyses to optimize the decellularization process for xenogeneic vascular tissue sources, specifically the porcine internal thoracic artery (ITA). Detergent concentrations greater than 1% were successful at removing cytoplasmic and cell surface proteins, but not DNA content after 24 h. A progressive increase in porosity and decrease in glycosaminoglycan (GAG) content was observed with increasing detergent concentration. Prolonged treatment significantly improved decellularization by reducing DNA content to trace amounts after 72 h, but also reduced laminin content and influenced the vessel’s mechanical behavior. Collectively, DNase with 1% detergent for 72 h provided an effective and efficient decellularization strategy to be employed in the preparation of porcine ITAs as bypass graft scaffolding materials. Yet this was at the cost of altered mechanical properties and laminin retention.

Supercritical carbon dioxide (scCO2) has received attention as an alternative strategy for tissue decellularization to potentially alleviate the problems associated with the traditional chemical and physical decellularization methods; however, this strategy has been evaluated in limited tissue types and methods are far from optimized with this approach. We turned our focus to developing a novel optimized scCO2 protocol that would lead to a more time-efficient decellularization method and would yield a small- diameter vascular scaffold with enhanced structural, biomechanical, and biochemical properties compared to the decellularized scaffold products generated with chemical methods alone. The experimental conditions utilized in this study, 0.5% SDS for 2 and 24 h, were established to evaluate the effects of scCO2, DNase, and SDS alone or in combination on porcine ITA decellularization. Both the 2 h and 24 h SDS/scCO2 hybrid treatments removed sufficient DNA content, which allowed us to selectively continue our experiments using the 2 h SDS/scCO2 hybrid treatment as to prevent the tissue from being exposed to detergent for longer than is necessary to achieve adequate decellularization. 2 h of 0.5% SDS treatment plus scCO2 was successful at removing cytoplasmic and cell surface proteins and DNA content. There was not a significant difference in sGAG concentration across any of the conditions analyzed, yet an increase in the concentration was observed in the samples treated with detergent alone and with detergent plus scCO2. However, tissue porosity appeared to increase between detergent alone and detergent plus scCO2-treated samples, but this was not directly quantified. Collectively, these findings suggest that very low detergent concentrations coupled with scCO2 treatment can be used in future decellularization strategies, but at the cost of some laminin retention. However, further study is required to determine the possibilities and limitations of this method, including bioactivity studies, testing scCO2-generated scaffolds as replacement material in animal models, and assessing biochemical modification of the scaffolds.

This dissertation confirms the use of a hybrid protocol including detergent plus scCO2 as: (1) a method of decellularization that can be optimized for the decellularization of porcine ITAs and, (2) a more-efficient decellularization method that yields a small-diameter vascular scaffold with similar structural and biochemical properties compared to those of the enzyme-detergent methods also presented in this work. The use of scCO2 for decellularization can be expanded to additional tissues once the SDS/scCO2 hybrid method is better understood in porcine ITAs. Additionally, it is possible that scCO2 could be used to simultaneously decellularize and sterilize tissues in the future. These studies provide insight into the effects of anionic detergent concentration and incubation time on porcine ITA decellularization and demonstrate an optimized decellularization procedure using a scCO2-based hybrid treatment. These are significant contributions to the field of tissue engineering, especially involving the potential development of more appropriate replacement materials for both coronary and peripheral bypass grafting procedures.

Rights

© 2022, Julia Elizabeth Hohn

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