Biological properties of decellularized porcine heart valve leaflets
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Purpose To investigate the biological properties of decellularized porcine heart valve leaflets. Methods Specimens of porcine aortic valve leaflets were treated with Triton-X100,0.25% sodium deoxycholate and RNase and DNase.Then scanning electron microscopy and hematoxylin-eosin staining were examined.Its biomechanical characteristics were tested and soluble protein content was measured.The decellularized porcine heart valve leaflets were transplanted subcutaneously in rabbit for 12 weeks,the histology was examined. Results The heart valve leaflets were completely removed of the cell components and the construction of the valve was maintained.The biomechanical characteristics of decellularized valve leaflets and the fresh ones were almost similar.Soluble protein content was 365 μg/mL and 1632 μg/mL in decellularized and fresh groups.The immunogenicity of the decellularized valve leaflets were alleviated significantly compared with the fresh ones. Conclusions Decellularized porcine heart valve leaflets can be applied to develop tissue engineering heart valve as scaffold.Keywords:
Decellularization
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To explore the possibility of detergent acellularized porcine heart valve serving as a scaffold for tissue engineering valve.The porcine aortic valves were acellularized by use of trypsin-EDTA. Triton X-100, RNase and DNase treatment. Biomechanical characteristics of fresh valves and acellularized valve were tested; also fresh valves, acellularized valve and valves treated with method of bioprothetic treatment were implanted subcutaneously in rats; frequently seeded with bovine aortic endothelial cells(BAECs), and then cultured for 7 days.The acellularization procedure resulted in complete removal of the cellular components while the construction of matrix was maintained. The matrix could be successfully seeded with in vitro expanded BAECs, which formed a continuous monolayer on the surface. There is no significant difference of PGI2 secretion of BAECs between cells seeded onto the acellular leaflets and that onto the wells of 24-wells plate (P > 0.05).Acellularied porcine aortic valve can be applied as a scaffold to develop tissue engineering heart valve.
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Conventional glutaraldehyde fixation is conducive to calcification of bioprosthetic tissues. The aim of this study was to test calcification resistance of procyanidin-treated decellularized porcine aortic valve in a rat model.We performed cross-linking of the decellularized porcine aortic heart valves by procyanidins and observed morphologic performance and examined the tensile strength and cross-linking index. Then we implanted subcutaneous samples of procyanidin cross-linking decellularized valves, glutaraldehyde cross-linking decellularized valves, and decellularized valves in rats. The retrieved grafts were stained with hematoxylin-eosin and von Kossa and were analyzed with scanning electron microscopy and x-ray energy dispersive spectroscopy (EDS) after 21 and 63 days.After decellularized and cross-linking pretreatment, the procyanidin cross-linked leaflets were soft and stretchable. In addition, the cellular components of the porcine aortic heart valve leaflets were completely removed, and the extracelluar matrix was maintained completely. Examination of tensile strength revealed a significantly higher tissue resistance to tension in procyanidin cross-linked tissue than in other tissues, including the glutaraldehyde group (P< .05), even though the extents of cross-linking of each group were roughly the same at approximately 90%. Histopathologic examination showed that the procyanidin cross-linked valve matrix had no significant calcification, and there were no calcium peaks in the EDS profile of procyanidin cross-linked samples in the 21-day and 63-day rat studies.This study demonstrated that procyanidin cross-linked decellularized heart valves can resist calcification to some extent.
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Glutaraldehyde
Von Kossa stain
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Objective:The purpose of this study is to seek better method of making tissue engineering of heart valves by testing different decellularization procedures for their potential of cell removal and their ability to preserve the matrix. Methods:Specimens of porcine aortic valves were randomly divided into the control groups and the decellularized groups. The porcine aortic valves of decellularized groups were treated with either NaCL and sodium-dodecyl-sulfate(SDS) or trypsin or Triton-X100 with specific concentration. Tissue samples were then processed for Hematoxylin-Eosin and scanning electron microscopy and immunohistochemistric staining for major histocompatibility complex class Ⅰ(MHC-Ⅰ) antigen. Results:NaCL and SDS achieved only incomplete decellularization, the main components of extracellular matrix were reserved completely but the fibrous components become swelling.In contrast, trypsin removed cells completely but caused strong structural alterations.Treatment with Triton-X100 achieved both complete decelluarization and preservation of the matrix structure. The decellularized valves were reserved varied level of immunogenicity. MHC-Ⅰ was decreased remarkably in trypsin and Triton-X100 groups compared with NaCL and SDS group. Conclusion:The decellularization method by Triton-X100 is effective. The decellularized porcine aortic valves reserve matrix structure and have lower immunity which can be used as an ideal valve for developing tissue engineering valve.
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Sodium dodecyl sulfate
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Objective:To study the histology and the biomechanical properties of porcine aortic valves decellularized by different methods. Methods: The porcine aortic valves were decellularized by 4 methods. Group 1:1% Triton X-100 + en-donuclease; Group III ,0.01% Trypsin + endonuclease; Group IV : 0. 05% Trypsin; Group V ,1% Triton X-100 + 0. 01% Trypsin + endonuclease. Group I was control group. Both fresh valves and decellularized valves were implanted subcuta-neously in rats. Biomechanical properties of fresh valves and decellularized valves were tested. Results: The decellular matrices were all intact except those in Group Ⅳ , and the maximal resistance to mechanical force of matrices in Group IN was obviously reduced compared to that of fresh valves (F0. 05). The inflammation reactions of Group Ⅳ and V were weaker than those of Group Ⅰ and Ⅱ . Conclusion: Using 1% Triton X-100 + 0. 01% Trypsin+endonuclease for decellularization can completely decellularize the porcine aortic valves, keep the biomechanical properties and reduce the immunogenicity; the scaffold made by this decellularization method may be ideal for constructing tissue-engineered heart valve.
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Histology
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Tissue-engineered heart valves offer the potential to deliver a heart valve replacement that will develop with the young patient. The present authors' approach is to use decellularized aortic heart valves reseeded in vitro or in vivo with the patient's own cells. It has been reported that treatment of porcine aortic valve leaflets with 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer produced complete leaflet acellularity without affecting tissue strength. The present study aim was to investigate the effect of an additional treatment incorporating 1.25% (w/v) trypsin and 0.1% (w/v) SDS on the biomechanics and hydrodynamics of the aortic root. This treatment has been shown to produce decellularization of both the aorta and valve leaflets.Fresh porcine aortic roots were treated to reduce the thickness of their aortic wall, and incubated in hypotonic buffer for 24 h. The leaflets were masked with agarose gel, and the aorta was treated with 1.25% (w/v) trypsin for 4 h at 37 degrees C. The trypsin and agarose were removed and the roots incubated with 0.1% (w/v) SDS in hypotonic buffer for 24 h. Fresh and treated circumferential and axial aortic specimens were subjected to uniaxial tensile testing, while intact porcine aortic roots were subjected to dilation and pulsatile flow testing.Decellularized aortic wall specimens demonstrated significantly decreased elastin phase slope and increased transition strain compared to the fresh control. However, the treatment did not impair tissue strength. Decellularized intact roots presented complete leaflet competence under systemic pressures, increased dilation and effective orifice areas, reduced pressure gradients, physiological leaflet kinematics and reduced leaflet deformation.The excellent leaflet kinematics and hydrodynamic performance of the decellularized roots, coupled with the excellent biomechanical characteristics of their aortic wall, form a promising platform for the creation of an acellular valve scaffold with adequate mechanical strength and functionality to accommodate dynamic cell repopulation in vitro or in vivo. This approach can be used for both allogeneic and xenogeneic tissue matrices.
Decellularization
Pulsatile flow
Agarose
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Each year, more than 800,000 vascular and cardiac surgeries are performed therefore, there is a great need for suitable material for bioprosthetic operations. Porcine pericardium is a double-walled sac that covers the heart and can be used in vascular and cardiac thoracic surgery.The aim of the present study was to evaluate the decellularization process and biomechanical properties in porcine pericardial tissue after the decellularization treatment.A detergent based protocol was used for the decellularization of porcine pericardium. Histological analysis and contact cytotoxicity assay were performed. Additionally, biomechanical testing and in vivo biocompatibility by implantation into Wistar Rats were performed.The histological analysis showed the preservation of the extracellular matrix, without any observable cellular remnants. No toxic effects were noticed when contact cytotoxicity assay performed. The decellularized tissues, after implantation in Wistar Rats, remained for up to 12 weeks without being rejected. Finally, the biomechanical testing showed no significant differences between native and decellularized tissues.In this study, the decellularization of the porcine pericardium produced a non toxic scaffold, free of any cellular remnants, thus serving as an alternative material for tissue engineering applications including heart valve and vascular patch development.
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Biocompatibility
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Objective To obtain the decellularized scaffold for tissue engineering heart valvefrom porcine heart valves.Methods Trypsin and EDTA were used to decellularize fresh porcine aorticvalves. The morphological integrity of the acellular scaffold was investigated by means of light andscanning electron microscopy. The shrinkage temperature, tension and strain at fracture were comparedbetween fresh porcine aortic valves and acellular matrix.Results Light and electron microscopyconfirmed that all the cellular constituents were removed without ultrastructural damage to fibrouscomponents. Compared with the fresh sample, there were no significant difference [P0.05] at theevaluation of Shrinkage temperature, tension at fracture and strain at fracture.Conclusion Porcineaortic valves can be almost completely acellularized by trypsin/EDTA procedure without impairing themechanical property of the tissue. This kind of acellular tissue could possibly be used to develop tissueengineering for heart valve substitute.
Decellularization
Shrinkage
Strain (injury)
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Objective:To compare the histological, immunological, and biomechanical characteristics of decellularized porcine aortic valve scaffold created by 3 different decellularization protocols and to search for a more suitable technique for creating acellular tissue-engineered cardiac valve conduit. Methods: Porcine aortic valve leaflets and whole aortic roots were decellularized by 3 different protocols. Decellularization procedure in group Ⅰ involved treatment with 0.01% trypsin, 1% Triton, and nuclease for 24 h; that in group Ⅱ involved treatment with 0.01% trypsin (8 h),1% DCA, and nuclease for 24 h; and that in group Ⅲ involved treatment with 1% DCA and nuclease for 32 h. All the treatments were conducted during continuous shaking at 37℃. Porcine aortic valve leaflets and whole aortic roots treated with PBS were taken as control.The decellularization efficiencies of each protocol were assessed by H-E staining, scanning electron microscopy, and transmission electron microscopy. The biomechanical features of the acellular valve matrices were examined by stress-strain tests and tensile strength tests.The immunogenicity and inflammatory responses of the decellularized matrices, valve leaflets, and aortic wall were investigated by subcutaneous implantation of them in rats. Results: The native cells in porcine aortic valve leaflets and aortic roots were completely removed in groupⅡ, which was superior to groupⅠand Ⅲ. The values of elasticity modulus and ultimate tensile strength (UTS) of group Ⅱ were greater than those in group Ⅰ( MPa vs MPa and MPa vs MPa, respectively; P0.05). The extension ratios at 1.5 MPa and at rupture in group Ⅱwere less than those in group Ⅰ( vs and vs ; P0.05), but the extension ratio at rupture was similar to that of fresh porcine aortic valves ( vs (). Histological analysis showed only slight inflammatory responses in groupⅡand the host cells grew into the matrix, rebuilding the acellular matrices gradually. Conclusion: Decellularization using 8-hour pretreatment with 0.01% trypsin, followed by 24 hours incubation with 1% DCA plus nuclease is effective and convenient; it not only removes the cells but also decreases the immunogenicity of the aortic valve matrices, making the product an excellent material for tissue-engineered cardiac valve conduit.
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Endothelial cells harbor many antigenic determinants that may be targets for the immune system. The aim of this study was to determine the immunologic effects of decellularization, using 3 different methods, on xenograft rejection.In a sterile plate containing phosphate-buffered saline, fresh sheep aortic heart valves were decellularized using 3 different enzymatic methods: with 900 μg/mL of collagenase at 40°C (method A), with 450 μg/mL of collagenase at 4°C (method B), and with 900 μg/mL of collagenase at 4°C (method C). Intact and decellularized valves were implanted subdermally into inbred male albino rabbits and extracted after 21 days (extra valve pieces were also extracted after 60 days, as control samples, for assessing chronic rejection). Valves were histologically analyzed for inflammatory cell infiltration. Subendothelial structure integrity was determined using surface electron microscope.No inflammatory cell infiltration was seen around the decellularized valve with method A, and no subendothelial structure change was observed by surface electron microscope. Infiltration of immune cells involved in rejection was not seen around valves decellularized with method B, although the subendothelial structure was relatively preserved and valve stiffness was increased. With method C, we observed a foreign body-type reaction around the intact valve and the decellularized valve.Method A is considered the optimal method of decellularization in our study, as this method significantly reduced the immune response to xenograft tissue, while maintaining subendothelial tissue.
Decellularization
Infiltration (HVAC)
Foreign-body giant cell
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