Clin Exp Otorhinolaryngol.  2019 Feb;12(1):86-94. 10.21053/ceo.2018.00486.

Decellularization of Trachea With Combined Techniques for Tissue-Engineered Trachea Transplantation

• Batioglu-Karaaltin A
• Ovali E
• Karaaltin
• Yener M
• Yılmaz M
• EyüpoÄŸlu F
• Yılmaz YZ
• Bozkurt ER
• Demir N
• Konuk E
• BozdaÄŸ ES
• YiÄŸit O
• Cansiz H

• KMID: 2437496
• DOI: http://doi.org/10.21053/ceo.2018.00486

Abstract

OBJECTIVES
The purpose of this study is to shorten the decellularization time of trachea by using combination of physical, chemical, and enzymatic techniques.
METHODS
Approximately 3.5-cm-long tracheal segments from 42 New Zealand rabbits (3.5±0.5 kg) were separated into seven groups according to decellularization protocols. After decellularization, cellular regions, matrix and strength and endurance of the scaffold were followed up.
RESULTS
DNA content in all groups was measured under 50 ng/mg and there was no significant difference for the glycosaminoglycan content between group 3 (lyophilization+deoxycholic acid+de-oxyribonuclease method) and control group (P=0.46). None of the decellularized groups was different than the normal trachea in tensile stress values (P>0.05). Glucose consumption and lactic acid levels measured from supernatants of all decellularized groups were close to group with cells only (76 mg/dL and 53 mg/L).
CONCLUSION
Using combination methods may reduce exposure to chemicals, prevent the excessive influence of the matrix, and shorten the decellularization time.

Keyword

Trachea; Scaffold; Freeze Drying; Tissue Engineering; Deoxycholic Acid

MeSH Terms

Deoxycholic Acid
DNA
Freeze Drying
Glucose
Lactic Acid
Rabbits
Tissue Engineering
Trachea*
DNA
Deoxycholic Acid
Glucose
Lactic Acid

Figure

• 

  Fig. 1. Yield load force testing. Graph shows a representative force (N) versus displacement (mm) profile obtained from yield load testing. As outward displacement (tracheal rupture) proceeds, the force increases in linear fashion until tissue failure. The yield load was calculated using a fixed displacement offset (blue line) measured from the point where the load-displacement curve (black line) transitions from the linear response region to enter the plateau region.

• 

  Fig. 2. Sections of decellularized tracheas from groups 1–6. First two rows depict H&E and reticulin staining of epithelial and primary mesenchymal zones (basal membrane, collagen fibers, vesicles, and fibroblasts); third and fourth rows depict H&E and toluidine blue staining of secondary mesenchymal zone (matrix). Magnification, ×200, respectively.

• 

  Fig. 3. Scanning electron microscopy examination of sections from decellularized tracheas of groups 1–6. Group 1: deformed cells on luminal surface, preserved basal membrain (BM), dispersed collagen fibers; preserved matrix, deformed chondrocytes (magnification, ×500). Group 2: no cells on luminal surface; affected BM, compact collagens, deformed isogenic groups, acellular (magnification, ×350 and ×600). Group 3: cellular remnants are observed on luminal surface; BM, collagen integrity, and cartilage matrix are preserved, but chondrocytes are deformed (magnification, ×350 and ×500). Group 4: large deformed cell layer on luminal surface; preserved BM, separation in collagen fibers, deformed isogenic groups and chondrocytes (magnification, ×350). Group 5: luminal surface covered with deformed cells; preserved BM, collagen fibers and matrix, deformed chondrocytes (magnification, ×350). Group 6: no cells on luminal surface; preserved BM, loose collagens; preserved matrix and chondrocytes (magnification, ×1.45 K).

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