Biocompatibility and immunogenicity of decellularised allogeneic aorta in the orthotopic rat model

Background and aim of the study: Peripheral arterial disease causes blood vessel dysfunction that requires surgical intervention. Current surgical interventions employ synthetic or allogeneic vascular grafts, which offer biocompatible materials solutions that are not able to regenerate or grow with the patient. Decellularised scaffolds have gained significant momentum in the past few years, since they have the potential to regenerate in the patient. The aim of this study was to investigate the effects of modified decellularisation protocol on the biocompatibility and immunogenicity of allogeneic rat abdominal aorta in an orthotopic rat model. Methods: Native syngeneic Wistar (W) and allogeneic Dark Agouti (DA) aortas, together with decellularised allogeneic DA aortas, were assessed histologically, immunohistochemically and biomechanically. The immunogenicity of the untreated and decellularized syngeneic and allogeneic grafts was assessed in W rats, implanted orthotopically. Following implantation for 6 weeks, the grafts were explanted and assessed for the presence of T cells and macrophages by immunohistochemistry, and for their biomechanical integrity and histoarchitecture. Results: No obvious histoarchitectural differences were observed between the native W and DA aortas, with both presenting similar three-layered structures. Histological analysis of decellularized DA aortas did not reveal any remaining cells. Explanted native DA allografts showed media necrosis, partial elastic fibre degradation and adventitia thickening, as well as infiltration by lymphocytes (CD3+, CD4+) and macrophages (CD68+) in the adventitia. The explanted decellularized DA allografts indicated reduced immune injury compared to the explanted native DA allografts. The explanted native W syngeneic grafts showed a mild immune response, with an intact media and no lymphocyte infiltration. The explanted native DA allografts showed significantly lower collagen phase slope than the decellularized DA allografts prior implantation, and significantly higher thickness than the explanted decellularized DA allografts. Conclusions: The results indicated that the modified decellularization protocol did not affect significantly the mechanical and histological properties of the native DA rat aorta. Overall, the immune response was improved by decellularization. Native DA allografts induced an adverse immune response in W rats, whereas syngeneic W grafts showed good tissue integration

Development of a dual-component infection-resistant arterial replacement for small-caliber reconstructions: A proof-of-concept study

Introduction: Synthetic vascular grafts perform poorly in small-caliber (<6mm) anastomoses, due to intimal hyperplasia and thrombosis, whereas homografts are associated with limited availability and immunogenicity, and bioprostheses are prone to aneurysmal degeneration and calcification. Infection is another important limitation with vascular grafting. This study developed a dual-component graft for small-caliber reconstructions, comprising a decellularized tibial artery scaffold and an antibiotic-releasing, electrospun polycaprolactone (PCL)/polyethylene glycol (PEG) blend sleeve.Methods: The study investigated the effect of nucleases, as part of the decellularization technique, and two sterilization methods (peracetic acid and γ-irradiation), on the scaffold’s biological and biomechanical integrity. It also investigated the effect of different PCL/PEG ratios on the antimicrobial, biological and biomechanical properties of the sleeves. Tibial arteries were decellularized using Triton X-100 and sodium-dodecyl-sulfate.Results: The scaffolds retained the general native histoarchitecture and biomechanics but were depleted of glycosaminoglycans. Sterilization with peracetic acid depleted collagen IV and produced ultrastructural changes in the collagen and elastic fibers. The two PCL/PEG ratios used (150:50 and 100:50) demonstrated differences in the structural, biomechanical and antimicrobial properties of the sleeves. Differences in the antimicrobial activity were also found between sleeves fabricated with antibiotics supplemented in the electrospinning solution, and sleeves soaked in antibiotics.Discussion: The study demonstrated the feasibility of fabricating a dual-component small-caliber graft, comprising a scaffold with sufficient biological and biomechanical functionality, and an electrospun PCL/PEG sleeve with tailored biomechanics and antibiotic release

Guided functional re-engineering of the mitral valve leaflet

Valvular heart disease is a major cause of mortality worldwide. Mitral valve regurgitation represents the second major valvular disorder in the western world. Current strategies for mitral valve reconstruction are imperfect. The aim of this study was to investigate the tissue engineering of mitral valve leaflets for mitral valve leaflets reconstruction. The approach taken was to utilise decellularised porcine pericardium seeded with porcine mesenchymal stem cells (pMSCs) and to mechanically condition the cell seeded constructs using biaxial strain in a bespoke bioreactor. The biomechanical and biological properties of porcine mitral valve leaflets and native and decellularised porcine pericardium were studied for comparative purposes. The porcine pericardium was decellularised using a propriety method based upon low concentration sodium dodecyl sulphate (SDS) and proteinase inhibitors. Histological characterisation showed the four and three layered structure of leaflets and fresh/decellularised pericardium, respectively. Histological analysis of decellularised pericardium did not reveal any remaining cells. Moreover, the histoarchitecture of collagen and elastic fibres appeared to be well preserved. Biochemical analysis showed that the mitral valve leaflets and the fresh pericardium had hydroxyproline and glycosaminoglycan (GAG) contents similar to those reported in the literature. Decellularised pericardium had higher hydroxyproline content than that of fresh pericardium but lower GAG content. The levels of deoxyribonucleic acid (DNA) in fresh and decellularised pericardium were determined and this showed that there was a 99% reduction in the DNA content following decellularisation. In vitro biocompatibility studies showed that the decellularised pericardium was not cytotoxic to porcine skin fibroblasts and pMSCs. Biomechanical properties were determined using low strain rate uniaxial loading to failure. Both fresh and decellularised pericardium demonstrated rather isotropic behaviours, possessing similar mechanical properties along the two orthogonal directions studied. Anterior leaflet specimens cut circumferentially were stiffer than those cut radially and the posterior leaflets. This anisotropic mechanical behaviour of the anterior leaflet was related to the main orientation of the collagen fibres along the circumferential direction. Conversely, the posterior leaflets were more isotropic. The optimum seeding density for culturing decellularised pericardial samples was 1×105 cells·cm-2 for pMSCs and the ideal time for culturing prior to loading the reseeded scaffold in the bioreactor stations was 3 days. With regards to fibroblasts and porcine smooth muscle cells (pSMCs), the optimum seeding density was 2×105 cells·cm-2 at 1 week culture for fibroblasts, whereas pSMCs had the tendency of forming agglomerates on the surface of tissue rather than penetrating throughout the thickness of the scaffold, thus, they were not considered ideal for remodelling the histoarchitecture of tissue during dynamic experiments. The actuator of the bioreactor was calibrated and the general set up of the system was completed. Suitable conditions to apply during static culture in the bioreactor, in terms of oxygen and pH control, in order to maintain the ECM integrity and cell viability in the bioreactor, were initially determined using fresh pericardium, however this proved problematic due to the variability in the tissue that was available. One day static experiment performed with the bioreactor by using decellularised pericardium reseeded with pMSCs showed that there was no difference in terms of viability when keeping the tissue statically in the tissue holders and in the bioreactor stations. Thus, tissue holders could be used as positive static controls during dynamic experiments. One day dynamic experiments (10% strain) performed with the bioreactor by using decellularised pericardium reseeded with pMSCs showed that cells were viable both when kept dynamically in the bioreactor and statically in the tissue holders. Cells started aligning along specific directions when kept dynamically in the bioreactor. Overall porcine mitral valve leaflets and porcine fresh/decellularised pericardium shared similar histoarchitectures, but had different biochemical composition and biomechanics. Decellularised pericardium was shown to be an optimum material for cell repopulation, delivering the necessary biological and biomechanical cues to seeded cells. The bioreactor was optimised for both static and dynamic culture and is now ready for further investigation at longer time points. This research provided the basics into the optimal strategy for mechanostimulation of cell-seeded pericardial scaffolds in vitro in order to generate heart-valve like tissue

Investigation of the suitability of decellularized porcine pericardium in mitral valve reconstruction

International audienceBACKGROUND AND AIM OF THE STUDY: Autologous and glutaraldehyde-treated xenogeneic and homogeneic pericardium has been used extensively in mitral valve repair, but there are a number of limitations associated with its use. These include calcification, limited durability and lack of in vivo regeneration with glutaraldehyde-treated xenografts, as well as the sacrifice of the patient's own pericardium in the case of repair with autologous pericardium. The study aim was to investigate the suitability of decellularized porcine pericardium for heterotopic repair of the mitral valve leaflets, and its potential to regenerate through endogenous cell repopulation in vivo, or in vitro cell seeding prior to implantation. METHODS: Fresh porcine anterior and posterior mitral valve leaflets, together with fresh and decellularized porcine pericardium, were tested histologically, biochemically and biomechanically to investigate potential similarities and differences between the different types of tissue. Subsequently, the decellularized pericardial scaffolds were tested both in terms of biocompatibility, using contact and extract cytotoxicity assays, and in terms of regenerative capacity through porcine mesenchymal stem cell (pMSC) seeding. RESULTS: Histological examination of fresh pericardium and leaflets showed the typical trilaminar and quadlaminar structures of the two tissues, respectively. No cell remnants were observed in the decellularized pericardium, whereas the histoarchitecture of the collagen, elastin and glycosaminoglycan (GAG) matrix appeared well preserved. Significant differences were found in the GAG and hydroxyproline contents and the biomechanics between the leaflet and the pericardial groups. No indication of cytotoxicity was observed with the decellularized pericardial scaffolds. The optimum cell seeding density of pMSCs was 1 x 10(5) cells per cm2, which represented the lowest density at which the cells were capable of repopulating the scaffold by migrating through its full thickness. CONCLUSION: Porcine mitral valve leaflets and porcine fresh/decellularized pericardium shared similar histoarchitectures, but had different biochemical compositions and biomechanics. Decellularized pericardium was shown to be an optimum material for cell repopulation, delivering the necessary biological and biomechanical cues to seeded or migrating cells, and representing a plausible scaffold option for the regeneration of the mitral leaflets in vitro or in vivo, respectively

Regional biomechanical and histological characterization of the mitral valve apparatus: Implications for mitral repair strategies

The aim of this study was to investigate the regional and directional differences in the biomechanics and histoarchitecture of the porcine mitral valve (MV) apparatus, with a view to tailoring tissue-engineered constructs for MV repair. The anterior leaflet displayed the largest directional anisotropy with significantly higher strength in the circumferential direction compared to the posterior leaflet. The histological results indicated that this was due to the circumferential alignment of the collagen fibers. The posterior leaflet demonstrated no significant directional anisotropy in the mechanical properties, and there was no significant directionality of the collagen fibers in the main body of the leaflet. The thinner commissural chordae were found to be significantly stiffer and less extensible than the strut chordae. Histological staining demonstrated a tighter knit of the collagen fibers in the commissural chordae than the strut chordae. By elucidating the inhomogeneity of the histoarchitecture and biomechanics of the MV apparatus, the results from this study will aid the regional differentiation of MV repair strategies, with tailored mitral-component-specific biomaterials or tissue-engineered constructs

Investigation of the biomechanical integrity of decellularized rat abdominal aorta

Objectives. The loss or damage of an organ or tissue is one of the most common and devastating problems in healthcare today. Tissue engineering applies the principles of engineering and biology toward the development of functional biological replacements that are able to maintain, improve, or restore the function of pathological tissues. The aim of the overall project is to study an already existing method for the decellularization of homograft vascular grafts for use in vascular surgery. Materials and Methods. The biomechanical integrity of native and decellularized rat aortas was assessed under uniaxial tension tests. For this purpose, 36 male rats (12 Wistar and 24 Dark Agouti [DA]) were used to excise their abdominal aortas. Twelve of the aortas were tested fresh (Wistar and DA rats), within 24 hours from euthanasia, and the rest were decellularized using a modified protocol (DA rats only). Fresh and decellularized samples (n ¼ 12) were subjected to uniaxial tensile loading to failure, and the recorded stress-strain behaviour of each specimen was assessed in terms of 6 biomechanical parameters. Results. No statistically significant differences were found in any of the biomechanical parameters studied between the decellularized DA rat aorta group and both the native DA and Wistar rat aorta groups (P > .05). Also, no significant difference was shown between the native DA and native Wistar rat aorta groups. Conclusions. The results from this study have shown that the decellularization protocol did not affect the mechanical properties of the native rat aorta. In addition to this, both native Wistar and native/decellularized DA rat aorta groups shared similar mechanical properties

Towards the development of osteochondral allografts with reduced immunogenicity

Nowadays, repair and replacement of hyaline articular cartilage still challenges orthopedic surgery. Using a graft of decellularized articular cartilage as a structural scaffold is considered as a promising therapy. So far, successful cell removal has only been possible for small samples with destruction of the macrostructure or loss of biomechanics. Our aim was to develop a mild, enzyme-free chemical decellularization procedure while preserving the biomechanical properties of cartilage. Porcine osteochondral cylinders (diameter: 12 mm; height: 10 mm) were divided into four groups: Native plugs (NA), decellularized plugs treated with PBS, Triton-X-100 and SDS (DC), and plugs additionally treated with freeze-thaw-cycles of - 20 ◦C, - 80 ◦C or shock freezing in nitrogen (N2) before decellularization. In a nondecalcified HE stain the decellularization efficiency (cell removal, cell size, depth of decellularization) was calculated. For biomechanics the elastic and compression modulus, transition and failure strain as well as transition and failure stress were evaluated. The - 20 ◦C, - 80 ◦C, and N2 groups showed a complete decellularization of the superficial and middle zone. In the deep zone cells could not be removed in any experimental group. The biomechanical analysis showed only a reduced elastic modulus in all decellularized samples. No significant differences were found for the other biomechanical parameters.</p

Toward acellular xenogeneic heart valve prostheses: histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices

The use of decellularized xenogeneic heart valves might offer a solution to overcome the issue of human valve shortage. The aim of this study was to revise decellularization protocols in combination with enzymatic deglycosylation, in order to reduce the immunogenicity of porcine pulmonary heart valves, in means of cells, carbohydrates, and, primarily, Galα1-3Gal (α-Gal) epitope removal. In particular, the valves were decellularized with sodium dodecylsulfate/sodium deoxycholate (SDS/SD), Triton X-100 + SDS (Tx + SDS), or Trypsin + Triton X-100 (Tryp + Tx) followed by enzymatic digestion with PNGaseF, Endoglycosidase H, or O-glycosidase combined with Neuraminidase. Results showed that decellularization alone reduced carbohydrate structures only to a limited extent, and it did not result in an α-Gal free scaffold. Nevertheless, decellularization with Tryp + Tx represented the most effective decellularization protocol in means of carbohydrates reduction. Overall, carbohydrates and α-Gal removal could strongly be improved by applying PNGaseF, in particular in combination with Tryp + Tx treatment, contrary to Endoglycosidase H and O-glycosidase treatments. Furthermore, decellularization with PNGaseF did not affect biomechanical stability, in comparison with decellularization alone, as shown by burst pressure and uniaxial tensile tests. In conclusion, valves decellularized with Tryp + Tx and PNGaseF resulted in prostheses with potentially reduced immunogenicity and maintained mechanical stability

Development and characterization of a porcine mitral valve scaffold for tissue engineering

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/ v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colonyforming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IVon the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics

Development of a dual-component infection-resistant arterial replacement for small-caliber reconstructions: A proof-of-concept study

Introduction: Synthetic vascular grafts perform poorly in small-caliber ( Methods: The study investigated the effect of nucleases, as part of the decellularization technique, and two sterilization methods (peracetic acid and γ-irradiation), on the scaffold’s biological and biomechanical integrity. It also investigated the effect of different PCL/PEG ratios on the antimicrobial, biological and biomechanical properties of the sleeves. Tibial arteries were decellularized using Triton X-100 and sodium-dodecyl-sulfate. Results: The scaffolds retained the general native histoarchitecture and biomechanics but were depleted of glycosaminoglycans. Sterilization with peracetic acid depleted collagen IV and produced ultrastructural changes in the collagen and elastic fibers. The two PCL/PEG ratios used (150:50 and 100:50) demonstrated differences in the structural, biomechanical and antimicrobial properties of the sleeves. Differences in the antimicrobial activity were also found between sleeves fabricated with antibiotics supplemented in the electrospinning solution, and sleeves soaked in antibiotics. Discussion: The study demonstrated the feasibility of fabricating a dual-component small-caliber graft, comprising a scaffold with sufficient biological and biomechanical functionality, and an electrospun PCL/PEG sleeve with tailored biomechanics and antibiotic release.</p