Development, comparative structural analysis, and first in vivo evaluation of acellular implanted highly compacted fibrin tubes for arterial bypass grafting.

The generation of small-caliber vascular grafts remains a significant challenge within the field of tissue engineering. In pursuit of this objective, fibrin has emerged as a promising scaffold material. However, its lack of biomechanical strength has limited its utility in the construction of tissue engineered vascular grafts. We have previously reported about the implementation of centrifugal casting molding to generate compacted fibrin tubes with a highly increased biomechanical strength. In this study, we conducted a structural analysis of compacted fibrin tubes using the open-source software Fiji/BoneJ. The primary aim was to validate the hypothesis that the compaction of fibrin leads to a more complex structure characterized by increased crosslinking of fibrin fibers. Structural analysis revealed a strong correlation between fibrin's structure and its biomechanical strength. Moreover, we enhanced fibrin compaction in a subsequent dehydration process, leading to a significant increase of biomechanical strength. Thus, the presented method in combination with an adequate imaging, e.g., micro-CT, has substantial potential as a powerful tool for quality assurance in the development of fibrin-based vascular grafts. To validate this concept, acellular highly compacted fibrin tubes were implanted as substitutes of a segment of the carotid artery in a sheep model (n = 4). After 6 months explanted segments exhibited distinct remodeling, transitioning into newly formed arteries.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

Left Ventricular Wall Reconstruction with Autologous Vascularized Tunica Muscularis of Stomach in a Porcine Pilot Model.

INTRODUCTION: Surgical replacement of dysfunctional cardiac muscle with regenerative tissue is an important option to combat heart failure. But, current available myocardial prostheses like a Dacron or a pericardium patch neither have a regenerative capacity nor do they actively contribute to the heart's pump function. This study aimed to show the feasibility of utilizing a vascularized stomach patch for transmural left ventricular wall reconstruction. METHODS: A left ventricular transmural myocardial defect was reconstructed by performing transdiaphragmatic autologous transplantation of a vascularized stomach segment in six Lewe minipigs. Three further animals received a conventional Dacron patch as a control treatment. The first 3 animals were followed up for 3 months until planned euthanasia, whereas the observation period for the remaining 3 animals was scheduled 6 months following surgery. Functional assessment of the grafts was carried out via cardiac magnetic resonance tomography and angiography. Physiological remodeling was evaluated histologically and immunohistochemically after heart explantation. RESULTS: Five out of six test animals and all control animals survived the complex surgery and completed the follow-up without clinical complications. One animal died intraoperatively due to excessive bleeding. No animal experienced rupture of the stomach graft. Functional integration of the heterotopically transplanted stomach into the surrounding myocardium was observed. Angiography showed development of connections between the gastric graft vasculature and the coronary system of the host cardiac tissue. CONCLUSIONS: The clinical results and the observed physiological integration of gastric grafts into the cardiac structure demonstrate the feasibility of vascularized stomach tissue as myocardial prosthesis. The physiological remodeling indicates a regenerative potential of the graft. Above all, the connection of the gastric vessels with the coronary system constitutes a rationale for the use of vascularized and, therefore, viable stomach tissue for versatile tissue engineering applications.

Serial assessment of early antibody binding to decellularized valved allografts.

Objectives: Decellularized homograft valves (DHV) appear to elicit an immune response despite efficient donor cell removal. Materials and methods: A semiquantitative Dot-Blot analysis for preformed and new recipient antibodies was carried out in 20 patients following DHV implantation on days 0, 1, 7, and 28 using secondary antihuman antibodies. Immune reactions were tested against the implanted DHV as well as against the stored samples of 5 non-implanted decellularized aortic (DAH) and 6 pulmonary homografts (DPH). Results: In this study, 20 patients (3 female and 17 male patients) were prospectively included, with a median age of 18 years and an IQR of 12-30 years. Six patients received DPH and 14 received DAH. The amount of antibody binding, averaged for all patients, decreased on post-operative days 1 and 7 compared to pre-operative values; and on day 28, antibody binding reached close to pre-operative levels (16.8 ± 2.5 on day 0, 3.7 ± 1.9 on day 1, 2.3 ± 2.7 on day 7, and 13.2 ± 3.7 on day 28). In comparison with the results in healthy controls, there was a higher amount of antibody binding to DAH than to DPH. The mean number of arbitrary units was 18.4 ± 3.1 in aortic and 12.9 ± 4.5 in pulmonary DHV (p = 0.140). Male patients exhibited higher antibody binding to aortic DHV than female patients (19.5 ± 2.1 vs. 1.6 ± 6.7). The p-value calculation was limited, as only two female patients received DAH. There was no correlation between the amount of overall antibody binding to DHV with respect to donor age (Kruskal-Wallis test p = 0.550). DHV recipients with a sex mismatch to the donor showed significantly less antibody binding (6.5 ± 1.8 vs. 13.7 ± 1.8; p = 0.003). Our main finding was an increase in antibody binding in younger patients receiving decellularized aortic allografts. This increase was higher in patients with early degeneration signs but was not specific to the individual DHV implanted nor previous DHV implantation. Antibody binding toward explanted DHV was significantly increased in implicating antibody-mediated DHV degeneration. Conclusion: Serial assessment of tissue-specific antibody binding revealed an increase in some patients within 4 weeks after surgery, who subsequently developed early signs of allograft degeneration. Further studies with larger sample sizes are needed to confirm the prognostic relevance of increased antibody activity in addition to targeted research efforts to identify the molecular agents triggering this type of antibody response.

Animal models and animal-free innovations for cardiovascular research: current status and routes to be explored. Consensus document of the ESC Working Group on Myocardial Function and the ESC Working Group on Cellular Biology of the Heart.

Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies, all of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task, in particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and comorbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models do not represent a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on an organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and state-of-the-art animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction, and refinement (3R) as a guiding concept.

Impaired immune response mediated by prostaglandin E2 promotes severe COVID-19 disease.

The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present study finds that risk-factors for severe COVID-19 disease courses, i.e. male sex, older age and sedentary life style are associated with higher prostaglandin E2 (PGE2) serum levels in blood samples from unaffected subjects. In COVID-19 patients, PGE2 blood levels are markedly elevated and correlate positively with disease severity. SARS-CoV-2 induces PGE2 generation and secretion in infected lung epithelial cells by upregulating cyclo-oxygenase (COX)-2 and reducing the PG-degrading enzyme 15-hydroxyprostaglandin-dehydrogenase. Also living human precision cut lung slices (PCLS) infected with SARS-CoV-2 display upregulated COX-2. Regular exercise in aged individuals lowers PGE2 serum levels, which leads to increased Paired-Box-Protein-Pax-5 (PAX5) expression, a master regulator of B-cell survival, proliferation and differentiation also towards long lived memory B-cells, in human pre-B-cell lines. Moreover, PGE2 levels in serum of COVID-19 patients lowers the expression of PAX5 in human pre-B-cell lines. The PGE2 inhibitor Taxifolin reduces SARS-CoV-2-induced PGE2 production. In conclusion, SARS-CoV-2, male sex, old age, and sedentary life style increase PGE2 levels, which may reduce the early anti-viral defense as well as the development of immunity promoting severe disease courses and multiple infections. Regular exercise and Taxifolin treatment may reduce these risks and prevent severe disease courses.

Generation of glycans depleted decellularized porcine pericardium, using digestive enzymatic supplements and enzymatic mixtures for food industry.

BACKGROUND: Xenogeneic pericardium has been used largely for various applications in cardiovascular surgery. Nevertheless, xenogeneic pericardial patches fail mainly due to their antigenic components. The xenoantigens identified as playing a major role in recipient immune response are the Galα1-3Gal (α-Gal) epitope, the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), and the porcine SDa antigen, associated with both proteins and lipids. The reduction in glycans from porcine pericardium might hinder or reduce the immunogenicity of xenogeneic scaffolds. METHODS: Decellularized porcine pericardia were further treated at different time points and dilutions with digestive enzymatic supplements and enzymatic mixtures applied for food industry, for the removal of potentially immunogenic carbohydrates. Carbohydrates removal was investigated using up to 8 different lectin stains for the identification of N- and O-glycosylations, as well as glycolipids. Histoarchitectural changes in the ECM were assessed using Elastica van Gieson stain, whereas changes in mechanical properties were investigated via uniaxial tensile test and burst pressure test. RESULTS: Tissues after enzymatic treatments showed a dramatic decrease in lectin stainings in comparison to tissues which were only decellularized. Histological assessment revealed cell-nuclei removal after decellularization. Some of the enzymatic treatments induced elastic lamellae disruption. Tissue strength decreased after enzymatic treatment; however, treated tissues showed values of burst pressure higher than physiological transvalvular pressures. CONCLUSIONS: The application of these enzymatic treatments for tissue deglycosylation is totally novel, low cost, and appears to be very efficient for glycan removal. The immunogenic potential of treated tissues will be further investigated in subsequent studies, in vitro and in vivo.

Fourier transform infrared spectroscopy coupled with machine learning classification for identification of oxidative damage in freeze-dried heart valves.

Freeze-drying can be used to ensure off-the-shelf availability of decellularized heart valves for cardiovascular surgery. In this study, decellularized porcine aortic heart valves were analyzed by nitroblue tetrazolium (NBT) staining and Fourier transform infrared spectroscopy (FTIR) to identify oxidative damage during freeze-drying and subsequent storage as well as after treatment with H2O2 and FeCl3. NBT staining revealed that sucrose at a concentration of at least 40% (w/v) is needed to prevent oxidative damage during freeze-drying. Dried specimens that were stored at 4 °C depict little to no oxidative damage during storage for up to 2 months. FTIR analysis shows that fresh control, freeze-dried and stored heart valve specimens cannot be distinguished from one another, whereas H2O2- and FeCl3-treated samples could be distinguished in some tissue section. A feed forward artificial neural network model could accurately classify H2O2 and FeCl3 treated samples. However, fresh control, freeze-dried and stored samples could not be distinguished from one another, which implies that these groups are very similar in terms of their biomolecular fingerprints. Taken together, we conclude that sucrose can minimize oxidative damage caused by freeze-drying, and that subsequent dried storage has little effects on the overall biochemical composition of heart valve scaffolds.

Residual immune response towards decellularized homografts may be highly individual.

OBJECTIVES: Decellularized homograft valves (DHVs) have shown promising clinical results, particularly in the treatment of congenital heart disease. However, DHV appears to elicit an immune response in a subset of young patients, indicated by early valve degeneration. As the decellularization process is quality controlled for each DHV, we hypothesized that there may be residual immunogenicity within the extracellular matrix of DHV. METHODS: A semi-quantitative dot blot analysis was established to screen for preformed recipient antibodies using secondary anti-human antibodies. Fifteen DHV samples (7 aortic, 8 pulmonary) were solubilized and exposed to serum from 20 healthy controls. RESULTS: The sera from young controls (n = 10, 18-25 years) showed significantly stronger binding of preformed antibodies than sera from older individuals (n = 10, 48-73 years). The difference between the means of arbitrary units was 15.1 ± 6.5 (P = 0.0315). There was high intraindividual variance in the mean amounts of arbitrary units of antibody binding with some healthy controls showing >10 times higher antibody binding towards 2 different DHV. The amount of preformed antibodies bound to DHVs was higher in aortic than in pulmonary DHVs. The mean number of antibody binding (in arbitrary units) was 17.2 ± 4.5 in aortic and 14.5 ± 4.7 in pulmonary DHV (P = 0.27). The amount of preformed antibodies bound to pulmonary DHVs was statistically significantly higher in the sera of healthy males (n = 10) than in the sera of healthy females (n = 10). The mean number of arbitrary units was 17.2 ± 4.2 in male and 11.7 ± 5.3 in female sera (P = 0.036). Antibody binding to aortic DHV was also higher in males, but not significant (18.8 ± 5.0 vs 15.6 ± 4.0). Blood group (ABO) incompatibility between the serum from controls and DHV showed no impact on antibody binding, and there was no age-related impact among DHV donors. CONCLUSIONS: Residual immunogenicity of decellularized homografts appears to exist despite almost complete cell removal. The established dot blot method allows a semi-quantitative assessment of the individual immune response towards extracellular DHV components and potentially the possibility of preoperative homograft matching.

Immunological and functional features of decellularized xenogeneic heart valves after transplantation into GGTA1-KO pigs.

Decellularization of xenogeneic heart valves might lead to excellent regenerative implants, from which many patients could benefit. However, this material carries various xenogeneic epitopes and thus bears a considerable inherent immunological risk. Here, we investigated the regenerative and immunogenic potential of xenogeneic decellularized heart valve implants using pigs deficient for the galactosyltransferase gene (GGTA1-KO) as novel large animal model. Decellularized aortic and pulmonary heart valves obtained from sheep, wild-type pigs or GGTA1-KO pigs were implanted into GGTA1-KO pigs for 3, or 6 months, respectively. Explants were analyzed histologically, immunhistologically (CD3, CD21 and CD172a) and anti-αGal antibody serum titers were determined by ELISA. Xenogeneic sheep derived implants exhibited a strong immune reaction upon implantation into GGTA1-KO pigs, characterized by massive inflammatory cells infiltrates, presence of foreign body giant cells, a dramatic increase of anti-αGal antibody titers and ultimately destruction of the graft, whereas wild-type porcine grafts induced only a mild reaction in GGTA1-KO pigs. Allogeneic implants, wild-type/wild-type and GGTA1-KO/GGTA1-KO valves did not induce a measurable immune reaction. Thus, GGTA1-KO pigs developed a 'human-like' immune response toward decellularized xenogeneic implants showing that immunogenicity of xenogeneic implants is not sufficiently reduced by decellularization, which detracts from their regenerative potential.

Re-endothelialization of non-detergent decellularized porcine vessels.

Tissue engineering utilizes an interdisciplinary approach to generate constructs for the treatment and repair of diseased organs. Generation of small vessels as vascular grafts or as envisioned central vessel for vascularized constructs is still a challenge. Here, the decellularization of porcine vessels by a non-detergent based protocol was developed and investigated. Perfusion-decellularization with sodium hydroxide solution resulted in removal of cellular material throughout the whole length of the vessel while preserving structural and mechanical integrity. A re-endothelialization of the retrieved matrix with human umbilical vein endothelial cells and cardiac endothelial cells was achieved through rotation-based seeding employing a custom-made bioreactor. A confluent monolayer was detected on the entire luminal surface. Thus, a non-detergent-based decellularization method allowing the re-endothelialization of the luminal surface was developed in this study, thereby paving the way for future implementation of the resulting construct as vascular graft or as central vessel for tissue engineered constructs in need of a perfusion system with readily available anastomosis sites.

Dot blots of solubilized extracellular matrix allow quantification of human antibodies bound to epitopes present in decellularized porcine pulmonary heart valves.

BACKGROUND: The present study reports the development of a sensitive dot blot protocol for determining the level of preformed antibodies against porcine heart valve tissue derived from wild-type (WT) and α-Gal-KO (GGTA1-KO) pigs in human sera. METHODS: The assay uses decellularized and solubilized heart valve tissue; antibody binding found in this dot blot assay could be correlated with antibody titers of preformed anti-α-Gal and anti-Neu5Gc antibodies detected by a sensitive ELISA. RESULTS: The ultimate protocol had an inter-assay variance of 9.5% and an intra-assay variance of 9.2%, showing that the test is reliable and highly reproducible. With the aid of this dot blot assay, we found significant variation with regard to antibody contents among twelve human sera. Binding of preformed antibodies to WT tissue was significantly higher than to GGTA1-KO tissue. CONCLUSIONS: The dot blot assay described herein could be a valuable tool to measure preformed antibody levels in human sera against unknown epitopes on decellularized tissue prior to implantation. Ultimately, this prescreening may allow a matching of the porcine xenograft with the respective human recipients in demand and thus may become an important tool for graft long-term survival similar to current allotransplantation settings.

Freeze-Drying of Decellularized Heart Valves for Off-the-Shelf Availability.

Malfunctioning heart valves can cause severe health problems, which if left untreated can lead to death. One of the treatment options is to replace a diseased heart valve with a decellularized valve construct prepared from human or animal material. Decellularized tissue scaffolds closely resemble properties of native tissue, while lacking immunogenic factors of cellular components. After transplantation, circulating stem and progenitor cells of the patient adhere to the scaffold resulting in in vivo tissue regeneration of the valve. Decellularized heart valve scaffold implants need to be stored to be readily available whenever needed, which can be done by freeze-drying. The advantage of freeze-drying is that it does not require bulky and energy-consuming freezing equipment for storage and allows easy transport. This chapter outlines the entire process from decellularization to freeze-drying to obtain dry decellularized heart valves, which after a simple rehydration step, can be used as implants. The protocol is described for porcine heart valves, but procedures can easily be adapted for material obtained from other species.

Characterization of Tissue Engineered Endothelial Cell Networks in Composite Collagen-Agarose Hydrogels.

Scaffolds constitute an important element in vascularized tissues and are therefore investigated for providing the desired mechanical stability and enabling vasculogenesis and angiogenesis. In this study, supplementation of hydrogels containing either MatrigelTM and rat tail collagen I (MatrigelTM/rCOL) or human collagen (hCOL) with SeaPlaqueTM agarose were analyzed with regard to construct thickness and formation and characteristics of endothelial cell (EC) networks compared to constructs without agarose. Additionally, the effect of increased rCOL content in MatrigelTM/rCOL constructs was studied. An increase of rCOL content from 1 mg/mL to 3 mg/mL resulted in an increase of construct thickness by approximately 160%. The high rCOL content, however, impaired the formation of an EC network. The supplementation of MatrigelTM/rCOL with agarose increased the thickness of the hydrogel construct by approximately 100% while supporting the formation of a stable EC network. The use of hCOL/agarose composite hydrogels led to a slight increase in the thickness of the 3D hydrogel construct and supported the formation of a multi-layered EC network compared to control constructs. Our findings suggest that agarose/collagen-based composite hydrogels are promising candidates for tissue engineering of vascularized constructs as cell viability is maintained and the formation of a stable and multi-layered EC network is supported.

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.

Effects of six month personalized endurance training on work ability in middle-aged sedentary women: a secondary analysis of a randomized controlled trial.

BACKGROUND: To test the effects of guided endurance training on work ability in middle-aged female hospital workers of various occupations. METHODS: We randomized 265 healthy, sedentary, middle-aged women (45-65 years) to an endurance training group (EG 210 min/week) or a wait-list control group (CG). At baseline and at 6-month follow-up, we assessed work ability (Work Ability Index [WAI]), physical activity (Freiburger activity questionnaire) and peak oxygen uptake (VO2peak) by cardiopulmonary exercise testing. To examine the influence of baseline work ability, participants were divided into poor-moderate (WAI 1, 7-36 points, n = 83), good (WAI 2, 37-43 points, n = 136) and excellent (WAI 3, 44-49 points, n = 46) WAI subgroups. RESULTS: Cardiorespiratory fitness improved significantly after 6 months in the EG but not in the CG. The WAI total score increased significantly in the EG (38.3 ± 5.0 to 39.8 ± 4.9 points) but not in the CG (39.4 ± 4.7 to 39.3 ± 4.9 points), with a significant difference between groups (p < 0.01). In the EG, only the poor-moderate subgroup (WAI 1, 33.0 ± 2.9 to 36.6 ± 4.8 points, p < 0.05) increased the WAI total score, with this increase being significantly higher compared to the good (WAI 2, 40.2 ± 2.1 to, 40.4 ± 3.7 points) and excellent (WAI 3, 45.6 ± 1.5 to 45.7 ± 1.8 points) subgroup. CONCLUSIONS: A 6-month guided exercise training intervention significantly increases cardiorespiratory fitness with concomitant improvements in work ability in middle-aged previously sedentary hospital employees. Women with low baseline work ability seem to particularly benefit from the intervention, which implies that similar interventions may be particularly beneficial for this group of individuals. TRIAL REGISTRATION: German Clinical Trails Register Identifier: DRKS00005159. Registered 25 September 2013.

Decellularization combined with enzymatic removal of N-linked glycans and residual DNA reduces inflammatory response and improves performance of porcine xenogeneic pulmonary heart valves in an ovine in vivo model.

BACKGROUND: Limited availability of decellularized allogeneic heart valve substitutes restricts the clinical application thereof. Decellularized xenogeneic valves might constitute an attractive alternative; however, increased immunological hurdles have to be overcome. This study aims for the in vivo effect in sheep of decellularized porcine pulmonary heart valves (dpPHV) enzymatically treated for N-glycan and DNA removal. METHODS: dpPHV generated by nine different decelluarization methods were characterized in respect of DNA, hydroxyproline, GAGs, and SDS content. Orthotopic implantation in sheep for six months of five groups of dpPHV (n = 3 each; 3 different decellularization protocols w/o PNGase F and DNase I treatment) allowed the analysis of function and immunological reaction in the ovine host. Allogenic doPHV implantations (n = 3) from a previous study served as control. RESULTS: Among the decellularization procedures, Triton X-100 & SDS as well as trypsin & Triton X-100 resulted in highly efficient removal of cellular components, while the extracellular matrix remained intact. In vivo, the functional performance of dpPHV was comparable to that of allogeneic controls. Removal of N-linked glycans and DNA by enzymatic PNGase F and DNase I treatment had positive effects on the clinical performance of Triton X-100 & SDS dpPHV, whereas this treatment of trypsin & Triton X-100 dpPHV induced the lowest degree of inflammation of all tested xenogeneic implants. CONCLUSION: Functional xenogeneic heart valve substitutes with a low immunologic load can be produced by decellularization combined with enzymatic removal of DNA and partial deglycosylation of dpPHV.

Decellularized pig pulmonary heart valves-Depletion of nucleic acids measured by proviral PERV pol.

BACKGROUND: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients. However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. METHODS: The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences. Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times. DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out. RESULTS: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 107 to 5 × 103  copies/mg in nuclease treated tissues. CONCLUSIONS: Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background.

Assessment of cytocompatibility and mechanical properties of detergent-decellularized ovine pericardial tissue.

BACKGROUND: Autologous pericardium is widely used for the repair of different sized cardiovascular defects. However, its use is limited especially in redo cardiac surgery. We developed an engineered tissue based on decellularized pericardium reseeded with blood-derived endothelial cells. MATERIALS AND METHODS: Decellularization of ovine pericardium was performed using detergent treatment. Ovine outgrowth blood-derived and green fluorescent protein-labeled endothelial cells were used to reseed the decellularized ovine pericardium on the mesothelial side. The cell adhesion was assessed using fluorescent microscopy up to 15 days of in vitro cultivation. The mechanical properties of the pericardium were evaluated using suturability, burst pressure, and suture retention strength tests. RESULTS: After decellularization the pericardial sheets appeared cell-free and repopulation using ovine blood-derived endothelial cells was successful by forming a robust monolayer. Detergent treatment did not affect the extracellular matrix. The thickness of decellularized tissue was similar to native ovine pericardium (285.3 ± 28.2 µm, respective 276.9 ± 23.8 µm, p = 0.48). Decellularized patch showed similar suturability comparable to the native ovine pericardium. Resulted burst pressure was not significantly different (native/decellularized: 312.5 ± 13.6/304.2 ± 16, p = 0.35). The suture retention strength of native pericardium was 638.33 ± 90.2 gr and comparable to decellularized tissue (622.2 ± 89.9 gr, p = 0.76). No differences were observed concerning elongation of native and decellularized pericardium (8.33 ± 1.5 and 8.5 ± 0.84 mm, respectively; p = 0.82). CONCLUSION: Mesothelial surface of decellularized ovine pericardium is suitable for reseeding with ovine blood-derived endothelial cells. The mechanical properties of detergent-treated pericardium were comparable to native tissue.