Use of supercritical carbon dioxide for decellularization of porcine trachea in an attempt to generate a tissue-engineered tracheal substitute

Abstract

Purpose: Patients with tracheal lesions that exceed half of
the trachea’s total length require a tracheal substitute. Tissue engineering,
using either synthetic materials or decellularized tracheal tissue, opens up
new possibilities for generating tracheal substitutes. Decellularization, a
procedure in which the tissue's immunogenic cellular material is removed while
the extracellular matrix (ECM) is preserved, seems the most promising approach.
The majority of tracheal decellularization methods, however, are reliant on harsh
chemicals and require lengthy wash procedures, resulting in damage to the ECM.
To address these issues, a supercritical carbon dioxide (scCO2)
decellularization approach has been suggested as an alternative solution due to
its ability to both decellularize and sterilize tissues while leaving no toxic
residues and requiring less treatment time. Therefore, the aim of this thesis
was to compare scCO2 treatment with a chemical decellularization method, which
is the current gold standard reported in literature, for creating a
decellularized porcine tracheal scaffold with good cytocompatibility.  Methods: A total of five different protocols
were tested that varied in decellularization and sterilization methods used.
Decellularization efficiency was evaluated in terms of DNA content,
histological appearance, and retention of ECM components. Additionally,
mechanical tensile testing and scanning electron microscopy were used to assess
the effects of the different decellularization protocols. Further,
decellularized scaffolds were recellularized with fibrin-encapsulated porcine
adipose derived stem cells to assess the cytocompatibility of the scaffolds. Results:
The highest reduction in DNA content was observed when samples were subjected
to the detergent-enzymatic protocol, followed by sterilization with gamma
irradiation, and when samples were subjected to scCO2 treatment, followed by
washing with sodium hydroxide. The latter protocol, however, also negatively
impacted the ECM, whereas good preservation of ECM components was seen with the
DEM protocol. DNA and histological analysis showed that treatment with scCO2 in
combination with a hydrogen peroxide (H2O2) washing step was unable to
completely decellularize porcine tracheas. Static surface seeding of the
decellularized scaffolds led to poor cell adherence. Cells encapsulated in
fibrin and seeded on the tracheal scaffolds were able to adhere and survive,
showing the cytocompatibility of the decellularized scaffolds.  Conclusions: Decellularization with scCO2 in
combination with a H2O2 washing step was not successful in completely
decellularizing the porcine tracheas, and possibly requires the use of a
co-solvent or secondary agent for successful decellularization. For
recellularization of decellularized tracheal scaffolds, the use of fibrin as a
cell carrier is an effective and simple seeding method. The findings in this
thesis open up new avenues for potential optimizations in future research.