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Zouhair, Sabra (2018) Preservation strategies for decellularized cardiovascular scaffolds for off-the-shelf availability. [Ph.D. thesis]

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Abstract (italian or english)

The rapid evolution of heart valve tissue engineering is progressively moving from an in vitro to in vivo setting, pushing human decellularized grafts into preclinical and clinical application. The cost effectiveness and relatively straightforward processing of acellular heart valves would make this concept potentially available for both adult and paediatric patients. This approach relies on the body’s endogenous regenerative capacity. The most appealing results to date have been realized using human acellular biological scaffolds. At present, two crucial limitations pose a significant delay to their application in routine clinical practice: the lack of donor tissues and the limited storage stability of biological scaffolds at 4°C in saline solution. Therefore, decellularized xenogeneic scaffolds, such as pericardium, which is abundantly available and ideally devoid from endogenous cell elements and immunogenic epitopes, could potentially be used for manufacturing cardiovascular substitutes. In order to ensure routine use of cardiovascular scaffolds, off-the-shelf availability requires tissue banking.
The objective of this study was to evaluate the suitability of three different preservation methods for preservation of decellularized bovine and porcine pericardial scaffolds: cryopreservation (as the standard preservation method currently in use), vitrification, and freeze-drying. The implementation of novel preservation technologies for tissue banking of such scaffolds requires careful validation to demonstrate the maintenance of their biological and functional integrity. Bovine and porcine pericardia were decellularized using Triton X-100, sodium cholate and endonucleases. Following decellularization, bovine and porcine samples were subjected to either slow-freezing-rate cryopreservation, vitrification or freeze-drying (n=6 in all cases). Slow-freezing-rate cryopreservation was conducted at ~1°C/min using 10% DMSO as a cryoprotectant. Vitrification was performed usingVS83 (4.65mol/L formamide, 4.65 mol/L DMSO and 3.31 mol/L propylene glycol in EuroCollins solution) and cooling above the vapourphase of liquid nitrogen. Freeze-drying was carried out using a programmable freeze-drier with temperature-controlled shelves, while samples were infiltrated with sucrose for lyoprotection. The impact of these preservation methods on the structural integrity of the scaffolds was assessed using histological staining, scanning electron microscopy (SEM), multiphoton microscopy (TPM) and uniaxial tensile testing. Fourier transform infrared spectroscopy (FTIR) was performed to study the overall protein secondary structure and differential scanning calorimetry (DSC) was used to determine thermal protein denaturation profiles. In addition, cytotoxicity analysis was performed.
Histological staining, SEM and TPM revealed that the extracellular matrix (ECM) integrity was maintained after all preservation treatments compared to the non-preserved control in both species. Inspection of the protein amide-I band (1600–1700 cm−1) in the FTIR spectra showed no statistically significant differences in overall protein secondary structure after preservation and reconstitution. DSC results indicated that the protein denaturation temperature was not significantly affected by any of the preservation protocols. Uniaxial tensile testing demonstrated the preservation of the biomechanical properties of porcine scaffolds, whereas for bovine scaffolds significant differences were observed following cryopreservation treatment.
Furthermore, differently treated scaffolds possess excellent cytocompatibility in vitro. This is of major importance since the preservation of ECM components and their bioactive properties may guarantee endogenous tissue regeneration upon implantation.
The most commonly used preservation method for cardiovascular tissue banking is cryopreservation by slow-rate freezing. It is shown, however, that cryopreservation of bovine pericardial tissues using 10% DMSO and slow-rate freezing results in more rigid tissues compared III to vitrified or freeze-dried tissues, whereas the biomechanical behavior of porcine scaffolds was unaffected by any of the preservation methods. This change in mechanical properties seen in DBP might be caused by damage due to ice crystal formation disturbing the ECM histoarchitecture. However, all preservation technologies were suitable for preserving ECM components with no apparent sign of denaturation of collagen or loss of elastin and sGAGs. Similarly, proteins were found to be stable as no changes were introduced to their structure.
In conclusion, freeze-drying and vitrification represent alternative methods to conventional cryopreservation that demonstrate excellent outcomes regarding preservation of ECM structure and its components. Both cryopreserved and vitrified tissues are usually stored in liquid nitrogen or a mechanical freezer, and include the use of highly toxic cryoprotective agents. Freeze-drying is carried out using non-toxic protective agents and the scaffolds can be stored in operating rooms at room temperature, which gives surgeons the opportunity to choose the ideal graft for the benefit of the patient. Freeze-drying reduces infrastructural costs for storage and shipment and preserves ECM integrity as well as vitrification and even better than conventional cryopreservation.
It is therefore suggested that freeze-drying could replace currently used cryopreservation and vitrification approaches for the preservation of xenogeneic decellularized scaffolds.The rapid evolution of heart valve tissue engineering is progressively moving from an in vitro to in vivo setting, pushing human decellularized grafts into preclinical and clinical application. The cost effectiveness and relatively straightforward processing of acellular heart valves would make this concept potentially available for both adult and paediatric patients. This approach relies on the body’s endogenous regenerative capacity. The most appealing results to date have been realized using human acellular biological scaffolds. At present, two crucial limitations pose a significant delay to their application in routine clinical practice: the lack of donor tissues and the limited storage stability of biological scaffolds at 4°C in saline solution. Therefore, decellularized xenogeneic scaffolds, such as pericardium, which is abundantly available and ideally devoid from endogenous cell elements and immunogenic epitopes, could potentially be used for manufacturing cardiovascular substitutes. In order to ensure routine use of cardiovascular scaffolds, off-the-shelf availability requires tissue banking.
The objective of this study was to evaluate the suitability of three different preservation methods for preservation of decellularized bovine and porcine pericardial scaffolds: cryopreservation (as the standard preservation method currently in use), vitrification, and freeze-drying. The implementation of novel preservation technologies for tissue banking of such scaffolds requires careful validation to demonstrate the maintenance of their biological and functional integrity.
Bovine and porcine pericardia were decellularized using Triton X-100, sodium cholate and endonucleases. Following decellularization, bovine and porcine samples were subjected to either slow-freezingrate cryopreservation, vitrification or freeze-drying (n=6 in all cases).

Slow-freezing-rate cryopreservation was conducted at ~1°C/min using 10% DMSO as a cryoprotectant. Vitrification was performed using VS83 (4.65mol/L formamide, 4.65 mol/L DMSO and 3.31 mol/L propylene glycol in EuroCollins solution) and cooling above the vapour phase of liquid nitrogen. Freeze-drying was carried out using a programmable freeze-drier with temperature-controlled shelves, while samples were infiltrated with sucrose for lyoprotection. The impact of these preservation methods on the structural integrity of the scaffolds was assessed using histological staining, scanning electron microscopy (SEM), multiphoton microscopy (TPM) and uniaxial tensile testing. Fourier transform infrared spectroscopy (FTIR) was performed to study the overall protein secondary structure and differential scanning calorimetry (DSC) was used to determine thermal protein denaturation profiles. In addition, cytotoxicity analysis was performed.
Histological staining, SEM and TPM revealed that the extracellular matrix (ECM) integrity was maintained after all preservation treatments compared to the non-preserved control in both species. Inspection of the protein amide-I band (1600–1700 cm−1) in the FTIR spectra showed no statistically significant differences in overall protein secondary structure after preservation and reconstitution. DSC results indicated that the protein denaturation temperature was not significantly affected by any of the preservation protocols. Uniaxial tensile testing demonstrated the preservation of the biomechanical properties of porcine scaffolds, whereas for bovine scaffolds significant differences were observed following cryopreservation treatment.
Furthermore, differently treated scaffolds possess excellent cytocompatibility in vitro. This is of major importance since the preservation of ECM components and their bioactive properties may guarantee endogenous tissue regeneration upon implantation. The most commonly used preservation method for cardiovascular tissue banking is cryopreservation by slow-rate freezing. It is shown, however, that cryopreservation of bovine pericardial tissues using 10% DMSO and slow-rate freezing results in more rigid tissues compared to vitrified or freeze-dried tissues, whereas the biomechanical behavior of porcine scaffolds was unaffected by any of the preservation methods. This change in mechanical properties seen in DBP might be caused by damage due to ice crystal formation disturbing the ECM histoarchitecture. However, all preservation technologies were suitable for preserving ECM components with no apparent sign of denaturation of collagen or loss of elastin and sGAGs. Similarly, proteins were found to be stable as no changes were introduced to their structure.
In conclusion, freeze-drying and vitrification represent alternative methods to conventional cryopreservation that demonstrate excellent outcomes regarding preservation of ECM structure and its components. Both cryopreserved and vitrified tissues are usually stored in liquid nitrogen or a mechanical freezer, and include the use of highly toxic cryoprotective agents. Freeze-drying is carried out using non-toxic protective agents and the scaffolds can be stored in operating rooms at room temperature, which gives surgeons the opportunity to choose the ideal graft for the benefit of the patient. Freeze-drying reduces infrastructural costs for storage and shipment and preserves ECM integrity as well as vitrification and even better than conventional cryopreservation.
It is therefore suggested that freeze-drying could replace currently used cryopreservation and vitrification approaches for the preservation of xenogeneic decellularized scaffolds.


EPrint type:Ph.D. thesis
Tutor: Gerosa, Gino
Supervisor:Wolkers, Wim and Iop, Laura and Aguiari, Paola and Korossis, Sotirios
Ph.D. course:Ciclo 31 > Corsi 31 > MEDICINA SPECIALISTICA TRASLAZIONALE "G.B. MORGAGNI"
Data di deposito della tesi:06 December 2018
Anno di Pubblicazione:30 October 2018
Key Words:Pericardium, Cryopreservation, Vitrification, Freeze-drying, Decellularization
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Cardiologiche, Toraciche e Vascolari
Codice ID:11594
Depositato il:05 Nov 2019 17:11
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