Unit:
Τομέας ΧειρουργικήςLibrary of the School of Health Sciences
Author:
Dimou Zacharoula-Georgia
Dissertation committee:
Περικλής Τόμος, Αναπλ. Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Γρηγόριος Κουράκλης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Νικόλαος Νικητέας, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Κωνσταντίνος Κόντζογλου, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Δημήτριος Δημητρούλης, Αναπλ. Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ευστάθιος Αντωνίου, Αναπλ. Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Γεράσιμος Τσουρούφλης, Επίκ. Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Original Title:
Παραγωγή ικριώματος τραχείας για την αντιμετώπιση σοβαρών παθήσεων της αναπνευστικής οδού
Translated title:
Development of trachea scaffold for the treatment of serious diseases of the respiratory tract
Summary:
Regenerative medicine is an evolving domain that combines tissue engineering and cell therapies and is now considered that may open up new prospects for a promising future in medical science. The aim of this thesis is to produce tracheal scaffold in order to treat serious diseases of the respiratory system without the use of bioreactor. The acquired or iatrogenic injury of the airways and particularly of trachea is relatively common and is considered to be deadly for the patient. Therefore, the reconstruction/replacement of portions of the trachea due to injury, damage or congenital anomaly and the need to strengthen tissue regeneration are still a wide field of research. To carry out the present study, rat tracheas were resected and decellularized with protocols that have already been used before. The absence of genetic material and the maintenance of the tissue morphology were then tested. The test was performed by evaluation of decellularisation through histological, quantitative and qualitative control of scaffolds. Trachea tissues were stained with Hematoxylin-Eosin, Toloudine Blue, Masson's Trichrome, Elastic Van Gieson and 4', 6-diamidino-2-phenylindole (DAPI). Furthermore, DNA, glycosaminoglycans and hydroxyproline were quantitated in both native and decellularised tracheas. Also, a qualitative evaluation of tissues was performed using scanning electron microscopy. These tests confirmed the removal of the nuclear and cellular elements of the tissue and the maintenance of the extracellular matrix of scaffolds. Ultimate Tensile Strength (UTS) was applied to cartilage rings and strips of native and decellularized tissue. Compared to the former, the latter displayed significantly increased failure strain, but decreased failure stress and peak elastic modulus in the longitudinal direction. There were no differences between native and decellularized tracheas in the circumferential direction. In native tracheas, greater parameter values were found longitudinally than circumferentially, with all directional differences attaining high statistical significance. Unlike native tracheas, there were no directional differences in failure stress in decellularized tracheas, while failure strain was significantly higher longitudinally and peak elastic modulus was significantly higher circumferentially. Biomechanical testing revealed certain differences in failure parameters between native and decellularized tracheas, suggesting that the latter were significantly weaker and softer, but more extensible than the former, albeit only longitudinally. Subsequently, in vitro cytotoxic test of scaffolds was performed using human mesenchymal cells (hMSCs). Successful cell growth was observed around the decellularised scaffold suggesting the removal of most toxic biological and chemical elements that would inhibit cell proliferation and growth.
Finally, following the above tests, in vivo biocompatibility and recellularization test of the decellularised scaffolds was performed in rats (n = 8) for 1 month. The decellularised tracheas were implanted in the rat back, while biopsies were taken to assess tracheal recellularisation and the risk of immune response. Following euthanasia of rats, histological and phenotypic testing was performed on implanted tissues. Control for the expression of CD3, CD4, CD11-b immunological markers and S100 marker was performed as well as hematoxylin-eosin staining for confirmatory check. No expression of CD4 cells was observed in decellularised tissue, whereas the presence of CD3 and CD11b was very low. Therefore, decellularised trachea implants showed a low immune response, which was also confirmed by histological staining of hematoxylin and eosin (H&E). S100 expression and presence of fibroblast-like cells was also observed, a characteristic of tissue regeneration and recellularization. Cartilage tissue exhibits elements of regeneration indicating that the body can function as a bioreactor in order to restore tissue structure and function. The above indicates initiation of tissue regeneration and the low immunogenicity of the decellularised scaffold. The aim of this thesis was to create a cellular, tissue-engineered airway through the process decellularisation and recellularisation, without the use of a bioreactor, that could have the structure and function of native tissue without the risk of an immune response. The purpose of this study was to evaluate the decellularised scaffold and study whether the organism can function as a bioreactor in order to restore the structure and function of the tissue so that in future there will be successful functional solutions for serious clinical disorders of the respiratory tract.
Main subject category:
Health Sciences
Other subject categories:
Respiratory tract diseases
Keywords:
Trachea, Scaffold, Decellularisation, Transplantation, Diseases of respiratory system, Bioreactor, Recellularisation, Regenerative medicine, Tissue-engineering
Number of references:
200
