2016 AIChE Annual Meeting

(299a) Decellularization of Porcine Aorta Using Supercritical Carbon Dioxide

Authors

Dominic M. Casali - Presenter, University of South Carolina
Michael A Matthews, University of South Carolina
Currently, there are over 120,000 individuals
in the United States awaiting an organ transplant, but fewer than 30,000
available donors [1].  Fabrication of
artificial tissues and organs via tissue engineering (TE) would alleviate the
current necessity for tissue and organ donors.  However, the need for
functional, biocompatible, and sterile biomaterials for TE scaffolds creates a
significant scientific challenge.  There are two primary methods for producing
TE scaffolds: synthetic scaffolds and naturally-derived scaffolds. 
Naturally-derived scaffolds are produced from the extracellular matrix (ECM) of
decellularized animal tissues [2].  These materials offer several benefits,
including lower risk of implant rejection and a more natural biochemistry and microstructural
environment compared to the native tissue, which promotes post-implant angiogenesis
and constructive remodeling [3].  Currently, natural scaffolds are often
processed with aqueous detergents, which requires long treatment times, can damage
the microstructure, and risks leaving cytotoxic residual material in the matrix
[4].    A novel decellularization technique
using supercritical carbon dioxide (scCO2) offers considerably
faster treatment of natural scaffold materials, on the order of hours instead
of days.  CO2 is non-toxic, non-flammable, and chemically inert, and
has desirable solvent properties and a mild critical temperature (31.1°C), making it viable for use at physiological
temperatures.  scCO2 has been used previously in TE and other
biomedical applications, including polymer foaming of scaffolds [5] and sterilization
of biomaterials [6, 7].  A study on scCO2 decellularization was
first published in 2008 [8], where scCO2 was used to decellularize
porcine aorta, but dehydration of the scaffold during treatment prevented
further progress.  Two years ago, we presented a method to presaturate scCO2
with water that greatly reduces tissue dehydration during treatment with scCO2
[9].  In this work, we proceed to examine the extent of decellularization during
scCO2 treatment, using a variety of additives and thermodynamic
conditions.   Porcine aorta was obtained from a
local butcher and cut into 2 cm x 1 cm rectangles, weighing approximately 200
mg each.  Some tissue samples were treated with scCO2 for 1 hr at 37°C using two pressure conditions: 10.3 MPa (ρCO2 = 0.698 g/mL) and 27.6 MPa (ρCO2 = 0.908 g/mL).  Water and ethanol
were mixed with scCO2 prior to contacting the tissue – water to
prevent dehydration and ethanol to increase the polarity of the CO2. 
Other samples were treated with sodium dodecyl sulfate (SDS) for 48 hr; this
detergent treatment was used as a positive control.  After treatment, DNA was
extracted from the decellularized tissues using Invitrogen DNAzol reagent and
quantified using UV spectrophotometry.  Other samples were stained with
hematoxylin and eosin (H&E) for histological analysis.   In Figure 1, the DNA content found
in native tissue is compared to the remaining DNA after each treatment, which
helps determine the extent of decellularization.  The amount of DNA
significantly decreased in all treatments, though CO2 at the low
pressure condition was much less effective at removing DNA (0.61 μg/mg) than the detergent and high pressure CO2
condition.  Though the high pressure CO2 treatment was unable to
fully decellularize the tissue, with a residual DNA content of 0.14 μg/mg, the ability to remove almost as much DNA in 1 hr
of CO2 as 48 hr with detergent (0.09 μg/mg)
is a promising initial result.  Further development of the method will
potentially lead to complete decellularization.   Figure 1 – DNA Content after
Treatment   Samples were also stained with
H&E and viewed under a light microscope at 4-40x magnification.  In Figure
2, micrographs of untreated, detergent-treated, and high pressure CO2-treated
aortas at 20x magnification are shown.  The detergent treatment removed almost all
cellular material (purple/black), but also caused significant misalignment to
the elastic fibers (pink/red), which suggests likely damage to the
extracellular matrix (ECM).  The CO2 treatment did not disrupt the
elastic fibers, but still left some cellular material intact.   Figure 2 – Aorta Micrographs at 20x
Magnification: (a) Untreated, (b) Detergent, (c) scCO2
  Moving forward, the objective is to
combine the cell removal of the detergent with the ECM preservation of the scCO2
treatment.  Ongoing work includes testing at a liquid CO2
condition (10°C, 27.6 MPa à ρCO2 = 1.012 g/mL),
using a different additive (Ls-54, a non-fluorinated surfactant with known
water and scCO2 solubility [10]), and tensile testing to further assess
how the decellularization process affects the physical properties of the
scaffold.  Completion of this study will elucidate the capabilities of this
novel decellularization method, which offers a significant reduction in
treatment time and potentially less ECM disruption.  

References

1.     The
Need is Real: Data. <http://www.organdonor.gov/about/data.html&gt;

2.    
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3.     Brown BN, Badylak
SF: Extracellular matrix as an inductive scaffold for functional tissue
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4.    
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6.     Qiu
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of Bacterial Spores and Viruses in Biological Material Using Supercritical
Carbon Dioxide With Sterilant.

7.    
Tarafa
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decontamination of biomaterials and tissue scaffolds. J Supercrit Fluids
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8.     Sawada
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supercritical carbon dioxide for acellular artificial tissue. J Chem
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9.    
Casali DM, Matthews MA: Processing Tissue Engineering Matrix
Materials with Supercritical CO2, AIChE Annual Meeting, Atlanta,
GA, November 2014.  Poster and short oral presentation.

10.  Tarafa PJ,
Matthews MA: Phase equilibrium for surfactant Ls-54 in liquid CO2 with water
and solubility estimation using the Peng-Robinson equation of state. Fluid
Phase Equilibria 2010, 298(2):212-218.