A Comprehensive Comparison of Bovine and Porcine Decellularized Pericardia: New Insights for Surgical Applications

Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e. bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species' scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species' scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use

Preservation strategies for decellularized cardiovascular scaffolds for off-the-shelf availability

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

Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability

Decellularized biological scaffolds hold great promise in cardiovascular surgery. In order to ensure off-the-shelf availability, routine use of decellularized scaffolds requires tissue banking. In this study, the suitability of cryopreservation, vitrification and freeze-drying for the preservation of decellularized bovine pericardial (DBP) scaffolds was evaluated. Cryopreservation was conducted using 10% DMSO and slow-rate freezing. Vitrification was performed using vitrification solution (VS83) and rapid cooling. Freeze-drying was done using a programmable freeze-dryer and sucrose as lyoprotectant. The impact of the preservation methods on the DBP extracellular matrix structure, integrity and composition was assessed using histology, biomechanical testing, spectroscopic and thermal analysis, and biochemistry. In addition, the cytocompatibility of the preserved scaffolds was also assessed. All preservation methods were found to be suitable to preserve the extracellular matrix structure and its components, with no apparent signs of collagen deterioration or denaturation, or loss of elastin and glycosaminoglycans. Biomechanical testing, however, showed that the cryopreserved DBP displayed a loss of extensibility compared to vitrified or freeze-dried scaffolds, which both displayed similar biomechanical behavior compared to non-preserved control scaffolds. In conclusion, cryopreservation altered the biomechanical behavior of the DBP scaffolds, which might lead to graft dysfunction in vivo. In contrast to cryopreservation and vitrification, freeze-drying is performed with non-toxic protective agents and does not require storage at ultra-low temperatures, thus allowing for a cost-effective and easy storage and transport. Due to these advantages, freeze-drying is a preferable method for the preservation of decellularized pericardium

Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability

© 2018 Decellularized biological scaffolds hold great promise in cardiovascular surgery. In order to ensure off-the-shelf availability, routine use of decellularized scaffolds requires tissue banking. In this study, the suitability of cryopreservation, vitrification and freeze-drying for the preservation of decellularized bovine pericardial (DBP) scaffolds was evaluated. Cryopreservation was conducted using 10% DMSO and slow-rate freezing. Vitrification was performed using vitrification solution (VS83) and rapid cooling. Freeze-drying was done using a programmable freeze-dryer and sucrose as lyoprotectant. The impact of the preservation methods on the DBP extracellular matrix structure, integrity and composition was assessed using histology, biomechanical testing, spectroscopic and thermal analysis, and biochemistry. In addition, the cytocompatibility of the preserved scaffolds was also assessed. All preservation methods were found to be suitable to preserve the extracellular matrix structure and its components, with no apparent signs of collagen deterioration or denaturation, or loss of elastin and glycosaminoglycans. Biomechanical testing, however, showed that the cryopreserved DBP displayed a loss of extensibility compared to vitrified or freeze-dried scaffolds, which both displayed similar biomechanical behavior compared to non-preserved control scaffolds. In conclusion, cryopreservation altered the biomechanical behavior of the DBP scaffolds, which might lead to graft dysfunction in vivo. In contrast to cryopreservation and vitrification, freeze-drying is performed with non-toxic protective agents and does not require storage at ultra-low temperatures, thus allowing for a cost-effective and easy storage and transport. Due to these advantages, freeze-drying is a preferable method for the preservation of decellularized pericardium. Statement of Significance: Clinical use of DBP scaffolds for surgical reconstructions or substitutions requires development of a preservation technology that does not alter scaffold properties during long-term storage. Conclusive investigation on adverse impacts of the preservation methods on DBP matrix integrity is still missing. This work is aiming to close this gap by studying three potential preservation technologies, cryopreservation, vitrification and freeze-drying, in order to achieve the off-the-shelf availability of DBP patches for clinical application. Furthermore, it provides novel insights for dry-preservation of decellularized xenogeneic scaffolds that can be used in the routine clinical cardiovascular practice, allowing the surgeon the opportunity to choose an ideal implant matching with the needs of each patient

Supplementary information files for A comprehensive comparison of bovine and porcine decellularized pericardia: new insights for surgical applications

Supplementary files for article A comprehensive comparison of bovine and porcine decellularized pericardia: new insights for surgical applications. Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e., bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species’ scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species’ scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use.</div

A comprehensive comparison of bovine and porcine decellularized pericardia: new insights for surgical applications

Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e., bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species’ scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species’ scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use