Date of Award

2017

Document Type

Open Access Dissertation

Department

Chemical Engineering

Sub-Department

College of Engineering and Computing

First Advisor

Michael A. Matthews

Abstract

The number of people requiring an organ transplant in the United States has increased considerably over the past 25 years, but the number of organ donations has stagnated; over 8,000 people now die annually while awaiting a transplant or become too sick to receive one. Tissue engineering (TE), the design and production of artificial tissues and organs in vitro, has been proposed to alleviate this problem. Though synthetic polymers offer tunable mechanical and biochemical properties, natural biomaterials have recently garnered attention in TE for their high degree of biocompatibility and ability to direct cell proliferation and constructive tissue remodeling. Yet scaffold processing remains challenging and a need for novel treatment and fabrication methods still exists.

One underexplored method for creating TE scaffolds is treatment with supercritical fluids (SCFs). SCFs are appealing for treating biomaterials because of their desirable solvent properties; liquid-like densities and gas-like viscosities allow supercritical fluids to wet and penetrate matrices easily without damaging surface tension effects. Supercritical carbon dioxide (scCO2) is of particular interest. scCO2 is a non-toxic, non-flammable substance that is relatively inert and can be used to process biomaterials at physiologic temperatures and mild pressures. scCO2 treatment avoids organic solvents, does not leave cytotoxic residue, and has already been utilized in similar biomedical applications, including sterilization, pasteurization, biomolecule extraction, and removal of endotoxins, bioburden, and allergenic proteins.

Supercritical CO2 has been used in foaming of synthetic polymer scaffolds, but it is almost completely unexplored in treatment of natural biomaterials for TE. In this dissertation, the potential of scCO2 in natural biomaterial TE is extensively explored. Two commonly-studied natural TE scaffold biomaterials were examined: a single-component biomaterial, type I collagen, and a multi-component biomaterial, extracellular matrix (ECM) obtained by decellularization of porcine aorta. Both biomaterials were studied at the fundamental and applied level.

First, the chemical compatibility of collagen and liquid and scCO2 was assessed. Compatibility was determined based on changes in four biochemical properties: thermal stability, molecular weight, secondary structure, and overall appearance. For scCO2, no significant differences were observed, indicating chemical compatibility. Liquid CO2 treatment caused significant denaturing, though it was hypothesized that the apparent incompatibility may be a result of treatment conditions rather than total incompatibility.

After chemical compatibility between collagen and scCO2 was established, scCO2 was applied to crosslinked collagen films to extract residual glutaraldehyde after crosslinking. After 1 hr of scCO2/ethanol treatment, over 95% of residual glutaraldehyde was removed, reducing the concentration below 1 ppm. Differential scanning calorimetry analysis showed a high degree of crosslinking and a denaturation temperature of about 63°C both before and after scCO2 treatment. Tensile testing did reveal a significant increase in both stiffness and tensile strength caused by scCO2 treatment, likely resulting from dehydration caused by the ethanol additive. However, this dehydration is preventable and less disruptive than heat-based removal of residual glutaraldehyde.

Decellularized ECM is also commonly used as a TE scaffolds. Current decellularization methods often utilize chemical detergents, which are residually cytotoxic and can damage ECM composition and ultrastructure. scCO2 has been proposed as a decellularizing agent, but added ethanol severely dehydrates the matrix. The second half of this dissertation explores how scCO2 can decellularize a tissue without dehydrating it. To prevent dehydration, a novel presaturation method was developed where scCO2 and water are thoroughly mixed before treatment. Presaturation with water led to mass retention of over 99% in a model hydrogel and over 97% in porcine aorta during scCO2 treatment, compared to only 46% and 78%, respectively, when dry (pure) scCO2 was used, proving that dehydration during scCO2 treatment is easily prevented.

Finally, scCO2 was used to decellularize porcine aorta. Contrary to a previous report, scCO2 alone was unable to achieve complete cell removal, even with a polar additive. However, when an SDS pretreatment step was used, the same scCO2 treatment completely decellularized porcine aorta as indicated by histology and DNA quantitation. Presaturation of scCO2 with water maintained the hydration state of the matrix, better maintaining the mechanical properties of the native tissue.

This dissertation confirms the potential of supercritical CO2 as a processing method for naturally-derived biomaterial scaffolds. Further work can be performed to determine the efficacy of CO2 on different scaffold compositions and morphologies as well as decellularization of other tissue types. More complex treatments may also be possible, such as simultaneous sterilization and decellularization. These studies provide insight into the mechanisms and applications of scCO2 in TE and offer a springboard for impactful discoveries in the future.

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