(418bt) Processing Tissue Engineering Matrix Materials with Supercritical CO2

This paper addresses a novel process for preparing tissue engineering scaffolds with the aid of supercritical carbon dioxide (CO₂).  Prior research has shown that CO₂ may be useful for sterilizing, removing bioburden, and infusing small molecules, for instance.  Specifically, this study addresses how to preserve the volatile content during treatment with CO₂. 

Background

According to the Department of Health and Human Services, nearly 7,000 people in the United States die each year while waiting for an organ transplant, and this number is likely to continue to grow.  One potential way to address this problem is through the use of artificial organs.  An exciting and relatively novel approach to creating artificial organs is the field of tissue engineering.  Tissue engineering involves seeding cells onto a biocompatible three-dimensional tissue scaffold and then implanting the tissue scaffold into the body. 

There are a number of challenges that have prevented the use of tissue scaffolds in this fashion.  The first challenge is the source of the scaffold.  Two main approaches are possible: producing scaffolds from synthetic biomaterials, or using natural organs or tissues.  Using natural materials is presumed to be advantageous because natural materials should significantly decrease the possibility of bodily rejection and adverse immune response often observed with synthetic biomaterials.  Natural organs or tissues contain foreign cellular material, which must be removed prior to seeding the scaffold.  The process of removing this material is called decellularization.  The objective of any decellularization method is twofold: (1) the removal of all cellular material, and (2) the preservation of the physical and biochemical properties of the extracellular matrix (ECM).  Meeting both of these criteria is of utmost importance for scaffold viability [1].

Decellularization is currently accomplished by contacting xenographic tissue with a combination of chemical detergents and biological agents.  The main challenge regarding decellularization is finding a balance between preserving the properties of the matrix while effectively removing undesired components.  One novel decellularization method involves using a supercritical fluid as a solvent for applying decellularization agents [2].   At temperatures and pressures above the critical point, a substance exists in one homogeneous phase called a supercritical fluid (SCF).  SCFs are potentially useful as solvents because they have desirable transport properties and liquid-like densities that are much greater than those of most gases.  This combination of properties allows them to penetrate through surfaces easily without damaging them.  Upon depressurization, the SCF outgases, leaving no residue in the tissue.

Supercritical carbon dioxide (scCO₂) is particularly promising for biomedical applications. It is nontoxic, nonflammable, and inexpensive.  It has mild critical conditions of 31.1°C and 7.38 MPa, so processing biological materials can take place at or near body temperature (37°C).  Supercritical CO₂ has already been demonstrated other applications, including pasteurization [3], extraction of biological compounds [4], and sterilization, the latter of which our group has published extensively [5-7]. 

There is currently only one report on using supercritical CO₂ in decellularization: Sawada et al. used scCO₂ with an ethanol entrainer to decellularize porcine aortas [2].  Sawada reported 100% removal of DNA and 80-90% removal of phospholipids at relatively mild pressures and temperatures.  However, considerable tissue dehydration caused by SCF extraction was reported, causing embrittlement of the matrix and a subsequent reduction in mechanical properties.  This reported extraction of volatiles during CO₂treatment is not surprising.  In fact, this phenomenon is well-known and is the basis of critical point drying, a process commonly used in tissue engineering and other applications.  In this case, however, it is desired to prevent drying from occurring. 

Hypothesis, Methods, and Results

We hypothesize that the tissue dehydration can be significantly reduced or even eliminated by presaturating scCO₂ with water and other volatiles prior to contacting the tissue.  If the maximum amount of each volatile substance were dissolved in scCO₂to the solubility limit prior to making contact with the tissue, this should prevent volatiles from being extracted.

The objectives of this study were as follows: (1) to construct an apparatus that can presaturate supercritical CO₂ with water and determine the range of CO₂ flow rates at which equilibrium-level presaturation can be attained, (2) to compare the amount of water extracted from model hydrogels and porcine aorta tissue using dry and presaturated CO₂, and (3) to determine if any components of porcine aorta are extracted during CO₂treatment. 

Experiments were conducted with an ECM model poly(acrylic acid-co-acrylamide) potassium salt, a hydrogel) and with porcine aorta.  Both materials were tested by contacting specimens with dry CO₂ and with CO₂ that had been presaturated with water.  The conditions used were a pressure of 2000 psi (13.79 MPa), a temperature of 37°C (and also 50°C for hydrogels only), a depressurization rate of 50 psi/min (0.345 MPa/min).  Treatment ratios of 30 min/0.1 g gel and 60 min/0.25 g tissue were used so that each specimen would be exposed to the same amount of CO₂.

Presaturation maintained almost all of the volatiles in the matrix (about 99% for hydrogels at both temperatures and 97% for tissue), but both materials lost a significant amount of volatiles (50% and 78% weight retention, respectively) when treated with dry CO₂.  These findings confirm the hypothesis and overcome one of the significant barriers to consideration of scCO₂ for decellularization of ECM materials.  They also confirm the presence of volatiles other than water in the porcine aorta tissue and indicate that further experimentation may be necessary to prevent the extraction of those volatiles during CO₂treatment. 

References

1.            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.

2.            Sawada K, Terada D, Yamaoka T, Kitamura S, Fujisato T: Cell removal with supercritical carbon dioxide for acellular artificial tissue. J Chem Technol Biotechnol 2008, 83(6):943-949.

3.            Spilimbergo S, Ciola L: Supercritical CO2 and N2O pasteurisation of peach and kiwi juice. International Journal of Food Science and Technology 2010, 45(8):1619-1625.

4.            Bagheri H, Manap MYB, Solati Z: Response surface methodology applied to supercritical carbon dioxide extraction of Piper nigrum L. essential oil. LWT-Food Sci Technol 2014, 57(1):149-155.

5.            Zhang J, Davis TA, Matthews MA, Drews MJ, LaBerge M, An YHH: Sterilization using high-pressure carbon dioxide. J Supercrit Fluids 2006, 38(3):354-372.

6.            Tarafa PJ, Jimenez A, Zhang JA, Matthews MA: Compressed carbon dioxide (CO2) for decontamination of biomaterials and tissue scaffolds. J Supercrit Fluids 2010, 53(1-3):192-199.

7.            Jimenez A, Zhang J, Matthews MA: Evaluation Of CO2-Based Cold Sterilization of a Model Hydrogel. Biotechnol Bioeng 2008, 101(6):1344-1352.