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Cryopreserved pulmonary homograft (CPH) implantation remains the gold standard for reconstruction of the right ventricular outflow tract (RVOT). Harvesting homografts < 24-h post mortem is the international norm, thereby largely excluding cadaveric donors. This study examines the structural integrity and stability of ovine pulmonary homografts harv...
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Homograft availability and durability remain big challenges. Increasing the post-mortem ischaemic harvesting time beyond 24 h increases the potential donor pool. Cryopreservation, routinely used to preserve homografts, damages the extracellular matrix (ECM), contributing to valve degeneration. Decellularization might preserve the ECM, promoting hos...
Citations
... Short ischemic time intend to keep homograft quality and prevent adverse events after implantation, but studies indicate that it would be safe to implant homografts with prolonged ischemic time (Meyns et al. 2005;Kalfa et al. 2011;Axelsson and Malm 2018;Bester et al. 2018). Looking at the homograft structure after prolonged ischemic time, it is shown that cellular viability decreases (Wallace et al. 1992;Niwaya et al. 1995;Gall et al. 1998). ...
... Collagen degeneration signs have previously been shown in vascular diseases such as aortic aneurysm and hypertension, and consist of increased waviness and disrupted fiber orientation, as well as disrupted D-bands with reduced contrast (Jones et al. 2020). So far, studies have shown that mechanical and morphological properties of the extracellular matrix of the homograft vessel walls and cusps seem to keep intact after prolonged cold ischemic time prior to organ harvest (Smit et al. 2015;Bester et al. 2018;Fabian et al. 2022). Forensic studies have shown that tissues with extracellular matrix that is rich in collagen and/or elastin, such as blood vessels, are resistant to both autolytic degeneration and microbiological decomposition and that adequate cooling slow the process even further (Cocariu et al. 2016;Javan et al. 2019). ...
... Current guidelines recommend a maximum of 72 h of total ischemic time. This guideline is based on opinion and experience only, although repeated studies have shown good performance of homografts with prolonged total ischemic times (Meyns et al. 2005;Kalfa et al. 2011;Axelsson and Malm 2018;Bester et al. 2018). In LM, it was difficult to detect any major degeneration of either cells, elastin fibers, or collagen fibers, showing that the overall structural integrity is well preserved at prolonged decontamination time. ...
According to guidelines, total ischemic time for homografts at processing must be kept short to avoid degeneration. Many homografts are discarded due to practical inability to finish all steps from procurement to cryopreservation within the time limit. Although, several studies have shown that homografts with prolonged ischemic time show adequate quality and performance. Twenty aortic and 12 pulmonary homografts were collected and biopsies were retrieved at preparation (day 0) and after 1, 2, 3, 4, 7, 14, 21, 28, and 60 days in antibiotic decontamination at 4 °C. Biopsies were prepared for light microscopy (LM) and transmission electron microscopy (TEM). Assessment generated scores for cells, elastin, and collagen. Relative differences between times were compared with Wilcoxon signed rank test. Bonferroni corrected p value of 0.0056 was considered significant. LM could only reveal decrease in cell count at 60 days in aortic homografts, no other differences was detected. TEM showed affected cell appearance in day 3 and day 4 and beyond for aortic and pulmonary homografts respectively. Elastin appearance was affected at day 60 for aortic and day 21 for pulmonary homografts. Collagen appearance was affected at day 28 for aortic homografts, with no significant differences in pulmonary homografts. Cell degeneration starts early after homograft procurement, but elastic and collagen fibers are more resistant to degeneration. Overall structure integrity as seen in LM was not affected at all, while TEM could reveal small degeneration signs in individual elastic fibers and collagen bundles at 21 and 28 days respectively.
... After preparation, antibiotic decontamination is conducted for a minimum of 24 h and a maximum of 96 h. The allowed extended time spans lead to a larger pool of possible donors, and more time to retrieve and transport the hearts to the tissue bank (Axelsson and Malm 2018;Bester et al. 2018). ...
... Both fibers are important for the mechanical properties of the homograft, providing strength and elasticity to the tissue (Stradins et al. 2004;Burkert et al. 2021). These parameters can be analyzed by calculating the elastic modulus and maximum strength of the tissue (Stradins et al. 2004;Lin et al. 2016;Kubíková et al. 2017;Bester et al. 2018). ...
... This study is the first to evaluate the mechanical properties of the homograft in relation to different decontamination intervals. Similar measurements have been made to evaluate other aspects of the homograft procurement process, such as the impact of different ischemic times and different cryopreservation periods (Bester et al. 2018;Fiala et al. 2019). The main results from this study show that elastic modulus and yield stress had decreased at 7-9 days of decontamination compared to 2-4 days of decontamination. ...
Optimal time spans in homograft procurement are still debatable among tissue banks and needs to be further investigated. Cell viability decreases at longer preparation intervals, but the effect on collagen and elastic fibers has not been investigated to the same extent. These fibers are of importance to the homograft elasticity and strength. The objective of this study was to analyze the mechanical properties of homograft tissue at different time spans in the procurement process. Ten aortic homografts were collected at the Tissue Bank in Lund. Twelve samples were obtained from each homograft, cryopreserved in groups of three after 2-4 days, 7-9 days, 28-30 days, and 60-62 days in antibiotic decontamination. Mechanical testing was performed with uniaxial tensile tests, calculating elastic modulus, yield stress and energy at yield stress. Two randomly selected samples were assessed with light microscopy. Procurement generated a total of 120 samples, with 30 samples in each time group. Elastic modulus and yield stress was significantly higher in samples cryopreserved after 2-4 days (2.7 MPa (2.5-5.0) and 0.78 MPa (0.68-1.0)) compared to 7-9 days (2.2 MPa (2.0-2.6) and 0.53 MPa (0.46-0.69)), p = 0.008 and 0.011 respectively. Light microscopy did not show any difference in collagen and elastin at different time spans. There was a significant decrease in elastic modulus and yield stress after 7 days of decontamination at 4 °C compared to 2-4 days. This could indicate some deterioration of elastin and collagen at longer decontamination intervals. Clinical significance of these findings remains to be clarified.
... The absence of DNA following the decellularization of homograft tissue was also shown with gel electrophoresis (Figure 3). Figure 4C,F,I). The collagen in the cryopreserved group appeared more collapsed compared to fresh untreated tissue [8], and compared to the cryopreserved group, more loosely arranged in the decellularized group and more compact and dense in the decellularized plus EnCap TM -treated group. Figure 4C,F,I). ...
... Figure 4C,F,I). The collagen in the cryopreserved group appeared more collapsed compared to fresh untreated tissue [8], and compared to the cryopreserved group, more loosely arranged in the decellularized group and more compact and dense in the decellularized plus EnCap TM -treated group. Similar results were demonstrated for the wall tissue as for the leaflets, with an endothelial cell layer (white arrow) and uniform donor interstitial cell distribution in the cryopreserved group ( Figure 5A) and no cells present in the decellularized scaffold or decellularized plus EnCap TM -treated group ( Figure 5D,G) on H&E staining. ...
... SEM demonstrated a confluent endothelial cell layer on the leaflet and wall tissue of cryopreserved homografts, even after 48 h of ischaemia ( Figure 6A,B). These endothelial cells presented with prominent nuclei and collapsed extranuclear areas, indicative of non-viable cells [8]. Shenke-Layland and co-workers described the presence of limited collagen-containing structures in the ventricularis of cryopreserved valve leaflets after thawing, with significantly altered and deteriorated collagenous and elastic fiber structures as a result of crystal ice formation in the ECM during cryopreservation [40]. ...
Homografts are routinely stored by cryopreservation; however, donor cells and remnants contribute to immunogenicity. Although decellularization strategies can address immunogenicity, additional fixation might be required to maintain strength. This study investigated the effect of cryopreservation, decellularization, and decellularization with additional glutaraldhyde fixation on the strength and structure of ovine pulmonary homografts harvested 48 h post-mortem. Cells and cellular remnants were present for the cryopreserved group, while the decellularized groups were acellular. The decellularized group had large interfibrillar spaces in the extracellular matrix with uniform collagen distribution, while the additional fixation led to the collagen network becoming dense and compacted. The collagen of the cryopreserved group was collapsed and appeared disrupted and fractured. There were no significant differences in strength and elasticity between the groups. Compared to cryopreservation, decellularization without fixation can be considered an alternative processing technique to maintain a well-organized collagen matrix and tissue strength of homografts.
... For example, naturally occurring and experimental aortic stenosis has been investigated in dogs (Copeland et al., 1974;Kim et al., 1986;Ahlstrom Ast et al., 2008). Sheep are routinely used as a big animal model to investigate calcification of biological aortic valve prosthesis and homografts in vivo (Kheradvar et al., 2017;Theodoridis et al., 2017;Bester et al., 2018). Apparently, calcification occurs very rapidly in sheep compared to humans. ...
Aortic valve stenosis secondary to aortic valve calcification is the most common valve disease in the Western world. Calcification is a result of pathological proliferation and osteogenic differentiation of resident valve interstitial cells. To develop non-surgical treatments, the molecular and cellular mechanisms of pathological calcification must be revealed. In the current overview, we present methods for evaluation of calcification in different ex vivo , in vitro and in vivo situations including imaging in patients. The latter include echocardiography, scanning with computed tomography and magnetic resonance imaging. Particular emphasis is on translational studies of calcific aortic valve stenosis with a special focus on cell culture using human primary cell cultures. Such models are widely used and suitable for screening of drugs against calcification. Animal models are presented, but there is no animal model that faithfully mimics human calcific aortic valve disease. A model of experimentally induced calcification in whole porcine aortic valve leaflets ex vivo is also included. Finally, miscellaneous methods and aspects of aortic valve calcification, such as, for instance, biomarkers are presented.
... In younger patients, however no optimal solution is yet available. The "golden standard" for valve replacement is the use of an allograft, which are today cryopreserved [3]. Disadvantage of these cryopreserved allografts, however, is the immunological response, which might lead to structural valve deterioration [4]. ...
Sir Donald N. Ross established a new era to treat aortic valve disease by introducing the Ross procedure in 1967. The original technique was complex by implanting the pulmonary valve subcoronary into the aortic root with excellent long-term results. The limited part of this specific approach however, was meanly the right ventricular outflow tract (RVOT) reconstruction. Today, the golden standard is the use of a cryopreserved pulmonary allograft, however disadvantageous such as availability and immunogenic response can eventually lead to structural valve deterioration. Tissue engineering could help to create a valve with an excellent durability including remodelling, regeneration and growth potential for RVOT reconstruction. This article reflex a recent literature overview on clinical available solutions of correcting the RVOT by the use of decellularized pulmonary allografts.
... The effects of the PMI (time between death of the cadaver and the harvesting of the tissues), and storage temperatures on the resulting biomechanical properties are hardly investigated to date with varying results [19][20][21] . The PMI in this study revealed no significant correlation with the investigated tensile parameters, which can be interpreted as a biomechanical sign that the tissue is not deteriorating vastly within the here investigated PMI of 144 h under cooled conditions. ...
Background and aims:
The human iliotibial tract (IT) is increasingly used in different types of musculoskeletal models. Previous findings indicate age-dependent changes of the human IT tensile properties, these lack confirmation to date. The relationship of the human IT and anthropometrical parameters, such as body height and weight has not been investigated before.
Materials and methods:
33 fresh human IT samples (age range 4 months to 93 years) were uniaxially tested using digital imaging correlation and the latest advances in 3D-printing to standardize biomechanical soft tissues testing.
Results:
The tensile parameters of the human IT are not age-dependent, except for the maximum strain in males. Height significantly correlated to elastic modulus, tensile strength and maximum strain of the human IT in males. Females just showed a significant correlation between maximum strain and weight, which was contrary to the findings in males.
Discussion and conclusion:
Age-dependency of human IT tensile parameters could not be confirmed in the larger sample size investigated in this study. Due to the strong correlation with the tensile IT parameters in males, we suggest that height should be integrated when the IT is used in simulations, such as finite element analyses of the hip and knee.
Homograft availability and durability remain big challenges. Increasing the post-mortem ischaemic harvesting time beyond 24 h increases the potential donor pool. Cryopreservation, routinely used to preserve homografts, damages the extracellular matrix (ECM), contributing to valve degeneration. Decellularization might preserve the ECM, promoting host-cell infiltration and contributing towards better clinical outcomes. This study compared the performance of cryopreserved versus decellularized pulmonary homografts in the right ventricle outflow tract (RVOT) of a juvenile ovine model. Homografts (n = 10) were harvested from juvenile sheep, subjected to 48 h post-mortem cold ischaemia, cryopreserved or decellularized and implanted in the RVOT of juvenile sheep for 180 days. Valve performance was monitored echocardiographically. Explanted leaflet and wall tissue evaluated histologically, on electron microscopical appearance, mechanical properties and calcium content. In both groups the annulus diameter increased. Cryopreserved homografts developed significant (¾) pulmonary regurgitation, with trivial regurgitation (¼) in the decellularized group. Macroscopically, explanted cryopreserved valve leaflets retracted and thickened while decellularized leaflets remained thin and pliable with good coaptation. Cryopreserved leaflets and walls demonstrated loss of interstitial cells with collapsed collagen, and decellularized scaffolds extensive, uniform ingrowth of host-cells with an intact collagen network. Calcific deposits were shown only in leaflets and walls of cryopreserved explants. Young fibroblasts, with vacuoles and rough endoplasmic reticulum in the cytoplasm, repopulated the leaflets and walls of decellularized scaffolds. Young’s modulus of wall tissue in both groups increased significantly. Cryopreserved valves deteriorate over time due to loss of cellularity and calcification, while decellularized scaffolds demonstrated host-cell repopulation, structural maintenance, tissue remodelling and growth potential.
