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

Sterilisation methods for decellularised scaffolds

The lack of adequate sterilisation procedures represents one of the main hindrance for clinical adoption of cardiovascular substitutes composed of xenogeneic decellularised material. Effective methods are required to be tailored and optimized for the utilization on tissues derived from animal sources and should not impair the functional properties of the original scaffolds. The purpose of this study is to establish two protocols for sterilisation of pericardial decellularised scaffolds: Sterilisation Protocol 1, based on antibiotic/antimycotic (AA) + peracetic acid (PAA) treatments; and Sterilisation Protocol 2, based on g-irradiation. Assessment of the sterility effectiveness of the proposed methods required the design of a novel proper rigorous protocol. The sterilisation assessment methodology consists in the verification of sterility and subsequent quantification/qualification of the bioburden. A preliminary controlled contamination (CC) with Gram+ and Gram- bacteria was performed on all scaffolds to assess the effectiveness of the developed sterilisation protocols. Sterility test was performed according to the European Pharmacopoeia guidelines by inoculation of a small amount of sample or its rinsing solution in bacteria media. Turbid medium is indicative of contaminated samples while clear medium indicates that the sample is sterile. Identification of contaminants was assessed by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry, while microorganism quantification was performed by Most Probable Number (MPN) method. This method was applied to evaluate both sterilisation methods in study, however it can also be tailored to other sterilisation methodologies or scaffolds. The scaffold subjected to sterilisation are pericardia form porcine and bovine origin, decellularised according to an established protocol called TRICOL. The Sterilisation Protocol 1 consists in the incubation of tissues with a cocktail of AA comprising vancomycin, gentamicin, cefoxitin and amphotericin B during 24 h at 37 °C followed by treatment with PAA at 0.05% and 0.1% (v/v) for 3h at 27 °C. The second protocol developed (Sterilisation Protocol 2) consists in the application of five dosages of g-irradiation: 1.5, 5, 10, 15 and 25 kGy. Sterility assessment was carried out through the sterilisation assessment protocol developed. The effects of the sterilisation on the pericardial scaffolds were evaluated by histology, biochemical and biomechanical tests, scanning electron microscopy (SEM) analysis and cell seeding experiments with human bone marrow mesenchymal stem cells (hBM-MSC) for evaluation of cytotoxicity and cell proliferation. The outcomes of the Sterilisation Protocol 1 showed that AA+PAA treatment provides sterile tissues in both concentrations tested. Histological, biomechanical and biochemical evaluation demonstrated that AA+PAA at both concentrations does not lead to substantial modifications of the decellularised pericardial scaffolds in terms of collagen fibres architecture, ECM protein content and appearance. However, the higher concentration of PAA has been shown to induce damages on porcine pericardial scaffolds ECM, as collagen network fragmentation. In addition, biomechanical evaluation demonstrates the preservation of mechanical properties of bovine scaffolds, whereas for porcine differences were observed after PAA treatments. Cell seeding experiments disclosed the non-cytotoxic effect of treatments in both pericardial scaffolds, as a sustained cell proliferation was observed in all tissues during time. Furthermore, histological assessment of cell seeded sterilised scaffolds showed the presence of a uniform monolayer of cells on scaffold surface after 14 days. The Sterilisation Protocol 2, based on g-irradiation treatment, was proven effective in the complete elimination of all the contaminants from both porcine and bovine pericardial scaffolds, even in the lower dosage applied. In addition, the biological indicator for this treatment (spore form of Bacilus Pumilus), employed to simulate the worst-case scenario, was removed after all the dosages employed in the test. Furthermore, after histological, biochemical and SEM analyses a general well preserved ECM was observed. In summary, in this PhD project a sterilisation assessment protocol has been designed. This protocol has been designed for the verification of the effectiveness of two different sterilisation methods for decellularised animal biomaterials. In addition, the two sterilisation protocols (Sterilisation Protocol 1, comprising AA + 0.05% PAA and Sterilisation Protocol 2, dosage 1.5kGy) were both adequate in the effective sterilisation of pericardial decellularised tissues without affecting ECM structure and functionality. In addition, the sterilisation methodologies developed were optimised for the tissues in study but can be putatively applied on any decellularised animal scaffold. Furthermore, they can be easily exploited in research laboratories and in tissue banks, since they do not require expensive and complex equipment to be implemented.La mancanza di adeguate procedure di sterilizzazione rappresenta uno dei principali ostacoli per l'adozione nell’ambito clinico di sostituti cardiovascolari composti da materiale xenogenico decellularizzato. Tra i requisiti fondamentali dei metodi di sterilizzazione vi è quello di essere specifico e ottimizzato per l'utilizzo su tessuti animali ed essere privo di eventuali effetti nocivi sulle proprietà funzionali del tessuto di origine. Lo scopo di questo studio è di sviluppare due protocolli per la sterilizzazione di scaffold pericardici decellularizzati: il Protocollo di Sterilizzazione 1, basato su un cocktail di antibiotici/antimicotici (AA) e sul trattamento con acido peracetico (PAA); e il Protocollo di Sterilizzazione 2, basato sull’utilizzo di raggi γ. La valutazione dell'efficacia dei metodi di sterilizzazione proposti ha reso necessaria la progettazione di un rigoroso protocollo di analisi. Quest’ultimo consiste nella verifica della sterilità e nella successiva quantificazione/qualificazione della carica batterica residua. Per valutare l'efficacia di tali protocolli, è stata eseguita una contaminazione controllata (CC) preliminare dei tessuti con batteri Gram+ e Gram-. In seguito, il test di sterilità è stato realizzato, in base alle linee guida della Farmacopea Europea, tramite l’inoculazione specifici di una piccola quantità di campione o della sua soluzione di lavaggio in mezzi di coltura batterici. Un mezzo di coltura torbido è stato considerato indicativo di campioni contaminati, mentre un mezzo trasparente indicava la sterilità del campione. L'identificazione di contaminanti è stata eseguita mediante spettrometria di massa MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight), mentre la quantificazione dei microorganismi è stata effettuata con il metodo del Most Probable Number (MPN). Questo metodo è stato applicato per valutare entrambi i metodi di sterilizzazione in studio, ma può considerarsi adattato anche ad altre metodologie di sterilizzazione e su altri tessuti. I biomateriali su cui i metodi di sterilizzazione sono stati testati sono pericardi di origine suina e bovina, decellularizzati secondo un protocollo consolidato denominato TRICOL. Il Protocollo di Sterilizzazione 1 consiste nell’incubazione di tessuti con un cocktail di AA comprendenti vancomicina, gentamicina, cefoxitina e anfotericina B per 24 ore a 37 °C, seguita da un trattamento con PAA al 0,05% e lo 0,1% (v/v) per 3h a 27 °C. Il secondo protocollo sviluppato (Protocollo di Sterilizzazione 2) consiste nell'applicazione di raggi γ, testata in cinque diversi dosaggi: 1.5, 5, 10, 15 e 25 kGy. L’efficacia di sterilizzazione è stata testata tramite il protocollo di analisi dell’efficacia di sterilizzazione sviluppato in questo progetto. Gli effetti della sterilizzazione sugli scaffold pericardici sono stati valutati tramite istologia, test biochimici e biomeccanici, microscopia elettronica a scansione (SEM) ed esperimenti di semina con cellule staminali mesenchimali umane da midollo osseo (hBM-MSC), allo scopo di valutarne la citotossicità e la proliferazione cellulare. I risultati del protocollo di sterilizzazione 1 hanno mostrato che il trattamento con AA + PAA è in grado di fornire tessuti sterili ad entrambe le concentrazioni testate. Le valutazioni istologica, biomeccanica e biochimica hanno dimostrato che il metodo AA + PAA, ad entrambe le concentrazioni di acido peracetico, non porta a sostanziali modifiche degli scaffold pericardici decellularizzati per quanto riguarda l’architettura delle fibre di collagene, il contenuto e la disposizione di proteine nella ECM. Tuttavia, PAA alla maggiore concentrazione è stato dimostrato indurre danni sul tessuto pericardico porcino ed, in particolare, la frammentazione della rete di collagene. Inoltre, la valutazione biomeccanica ha confermato la conservazione delle proprietà meccaniche degli scaffold bovini, al contrario dei tessuti di origine porcina in cui sono state osservate differenze dopo trattamento con PAA. Gli esperimenti di semina cellulare hanno rivelato la non citotossicità dei trattamenti su entrambi gli scaffold pericardici, confermata dalla mantenuta proliferazione cellulare osservata su tutti i tessuti nel tempo. Inoltre, la valutazione istologica dei campioni di pericardio sterilizzato e seminato ha mostrato la presenza di un monolayer cellulare uniforme sulla superficie tissutale dopo 14 giorni di coltura. Il Protocollo di Sterilizzazione 2, basato sul trattamento con raggi γ, si è dimostrato efficace nella eliminazione completa di tutti i contaminanti da pericardio sia porcino che bovino, anche a seguito dell’applicazione del dosaggio inferiore. Inoltre, l'indicatore biologico per questo trattamento (Bacillus pumilus), impiegato per simulare lo scenario peggiore, è stato rimosso dopo il trattamento con tutti i dosaggi testati. Infine, dopo analisi istologiche, biochimiche e SEM è stata osservata una ECM generalmente ben conservata. In sintesi, in questo progetto di dottorato è stato sviluppato un protocollo di valutazione dell’efficacia della sterilizzazione. Questo protocollo è stato progettato per verificare l’efficienza dei due diversi metodi di sterilizzazione di scaffold animali decellularizzati. Inoltre, i due protocolli di sterilizzazione (Protocollo di Sterilizzazione 1, comprendente AA + 0,05% PAA e Protocollo di Sterilizzazione 2, a dosaggio 1.5kGy) si sono dimostrati entrambi adeguati nella sterilizzazione dei tessuti senza alterare la struttura della ECM o la sua funzionalità. Le metodologie di sterilizzazione sviluppate sono state ottimizzate per i tessuti in studio, ma possono essere potenzialmente applicate su qualsiasi scaffold animale acellulare. Inoltre, questi metodi possono essere facilmente impiegati in laboratori di ricerca e in banche di tessuti, in quanto non richiedono attrezzature dispendiose e complesse da utilizzare

A sterilization method for decellularized xenogeneic cardiovascular scaffolds

Decellularized xenogeneic scaffolds have shown promise to be employed as compatible and functional cardiovascular biomaterials. However, one of the main barriers to their clinical exploitation is the lack of appropriate sterilization procedures. This study investigated the efficiency of a two-step sterilization method, antibiotics/antimycotic (AA) cocktail and peracetic acid (PAA), on porcine and bovine decellularized pericardium. In order to assess the efficiency of the method, a sterilization assessment protocol was specifically designed, comprising: i) controlled contamination with a known amount of bacteria; ii) sterility test; iii) identification of contaminants through MALDI-TOF (Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight) mass spectrometry and iv) quantification by the Most Probable Number (MPN) method. This sterilization assessment protocol proved to be a successful tool to monitor and optimize the proposed sterilization method. The treatment with AA + PAA method provided sterile scaffolds while preserving the structural integrity and biocompatibility of the decellularized porcine and bovine tissues. However, surface properties and cellular adhesion resulted slightly impaired on porcine pericardium. This work developed a sterilization method suitable for decellularized pericardial scaffolds that could be adopted for in vivo tissue engineering. Together with the proposed sterilization assessment protocol, this decontamination method will foster the clinical translation of decellularized xenogeneic substitutes

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

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