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

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 (ρ_CO2 = 0.698 g/mL) and 27.6 MPa (ρ_CO2 = 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 ρ_CO2 = 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>

2.    
Keane
TJ, Swinehart IT, Badylak SF: Methods of tissue decellularization used for
preparation of biologic scaffolds and in vivo relevance. Methods 2015,
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3.     Brown BN, Badylak
SF: Extracellular matrix as an inductive scaffold for functional tissue
reconstruction. Translational Research 2014, 163(4):268-285.

4.    
Keane
TJ, Londono R, Turner NJ, Badylak SF: Consequences of ineffective
decellularization of biologic scaffolds on the host response. Biomaterials
2012, 33(6):1771-1781.

5.    
Bhamidipati
M, Scurto AM, Detamore MS: The Future of Carbon Dioxide for Polymer
Processing in Tissue Engineering. Tissue Eng Part B-Rev 2013, 19(3):221-232.

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
2010, 53(1-3):192-199.

8.     Sawada
K, Terada D, Yamaoka T, Kitamura S, Fujisato T: Cell removal with
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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
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