Andres Hilfiker - Academia.edu (original) (raw)

Papers by Andres Hilfiker

Cryopreservation and Freeze-Drying Protocols, 2020

Malfunctioning heart valves can cause severe health problems, which if left untreated can lead to... more 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.

European Journal of Cardio-Thoracic Surgery, 2020

OBJECTIVES Decellularized homograft valves (DHVs) have shown promising clinical results, partic... more 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 m...

Cardiovascular diseases are the most frequent cause for morbidity and mortality worldwide. The da... more Cardiovascular diseases are the most frequent cause for morbidity and mortality worldwide. The damage of the heart muscle’s tissue is irreversible since cardiomyocytes do not have the capability to divide, and therefore myocardial regeneration does not take place. A surgical approach for therapy is the substitution of damaged heart tissue by a decellularized and revascularized small intestine mucosa. For the right ventricle and the atria (pressures ≤ 40-60 mmHg) the substitution of the damaged area could already be realized in animal studies. However, for the left ventricle (pressures up to 240 mmHg) the mechanical strength of these myocardial grafts is not sufficient in the early stage after the surgery. In this work first results in the processing of stabilising structures made of magnesium alloys for the cardiovascular surgery by high precision AWIJ are presented. In detail the aspects of cutting strategy, geometrical design, cutting edge roughness and burr generation are discussed.

Cardiovascular disease is one of the main causes of mortality and morbidity worldwide. The "gold ... more Cardiovascular disease is one of the main causes of mortality and morbidity worldwide. The "gold standard" for the replacement/repair of diseased blood vessels is substitution with autologous vessels. However, multiple surgical procedures limit the availability of autologous vessels, whereas synthetic grafts have been reported to demonstrate poor patency rates, especially for small-caliber vascular reconstructions. Decellularization of native vascular or non-vascular tissues for vascular scaffold development has gained significant attention in the past 20 years. A variety of decellularization techniques have been described and employed to achieve effective immunogenic agent removal from the developed vascular scaffold. At the same time, the decellularization must not impair the extracellular matrix (ECM) composition, structure, and mechanical properties of the graft in order to ensure long-term functionality in vivo. The aim of this chapter was to review the various decellularization treatments that have been reported in the literature for the development of decellularized vascular scaffolds.

The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present st... more The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present study finds prostaglandin E2 (PGE2) blood levels elevated in COVID-19 patients with positive correlation 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-lung-precision-slices infected with SARS-CoV-2 display upregulated COX-2. PGE2 in serum of COVID-19 patients lowers the expression of Paired-Box-Protein-Pax-5 (PAX5), a master regulator of B-cell survival, proliferation and differentiation, in both human and mouse pre-B-cells, while the PGE2 inhibitor taxifolin directly reduces SARS-CoV-2-induced PGE2 production and attenuates viral replication. Risk-factors for severe disease courses, i.e. older age, male sex and air pollution are associated with higher PGE2 production and lower PAX5 expression in pr...

Biomedical Spectroscopy and Imaging, 2012

Decellularized heart valve scaffolds can be used for pulmonary heart valve replacements in heart ... more Decellularized heart valve scaffolds can be used for pulmonary heart valve replacements in heart surgery. In order to assess quality parameters of heart valves prior to implantation into patients various techniques can be applied, including ultrastructural evaluation and biomechanical testing. Fourier transform infrared spectroscopy (FTIR) was used here to study protein secondary structure and solvent accessibility in decellularized heart valve matrices. FTIR was used to study proteins in the three main types of structures in decellularized heart valves: leaflet, pulmonary artery wall and heart muscle. The amide-I band region was used to reveal differences in the overall protein secondary structure amongst tissues. Leaflet material contains a relatively high contribution of α-helical structures, whereas artery matrix contains a relatively high content of triple-helix and β-sheet structures. Solvent accessibility of tissue proteins was studied by exposing material to D2O. The exchange of hydrogen of the NH amide-II bond for deuterium originating from D2O was visible as an increase in the area of the amide-II band (1500– 1400 cm−1). The amide-I band also depicted changes in shape and position during incubation in D2O: a low frequency band at around 1625 cm−1 increased as a function of time. Remarkable differences in hydrogen-to-deuterium exchange kinetics and protein solvent accessibility were observed among the three types of heart valve matrices: proteins in artery matrix are highly accessible to solvent, whereas proteins in leaflet and heart muscle structures are relatively inaccessible to solvent and show slow hydrogen/deuterium exchange. The differences in protein solvent accessibility in the different types of matrices likely reflect differences in scaffold porosity.

Xenotransplantation, 2019

The availability of decellularized human heart valve (dhHV) scaffolds is a general demand in rege... more The availability of decellularized human heart valve (dhHV) scaffolds is a general demand in regenerative heart valve replacement surgery. Particularly, dhHV scaffolds are designed for younger, adolescent patients as they are fully assimilated by the recipient and not restricted in half-life by growth persistence or early calcification 1,2 which has been described for glutaraldehyde and formaldehyde cross-linked bioprosthetics. 3 These are fixed, non-resorbable,

Scientific Reports, 2019

Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering o... more Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering of constructs with clinically relevant size as nourishment of cells is challenged by the diffusion limit. In vitro generation of 3D networks is often achieved under conditions using serum containing cell culture medium and/or animal derived matrices. Here, 3D endothelial cell networks were generated by using human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) employing human collagen I as hydrogel and decellularized porcine small intestinal submucosa as starter matrix. Matrigel/rat tail collagen I hydrogel was used as control. Resulting constructs were cultivated either in serum-free medium or in endothelial growth medium-2 serving as control. Endothelial cell networks were quantified, tested for lumen formation, and interaction of HUVECs and hASCs. Tube diameter was slightly larger in constructs containing human collagen ...

Innovative Surgical Sciences, 2018

Introduction We have recently reported about a novel technique for the generation of bioartificia... more Introduction We have recently reported about a novel technique for the generation of bioartificial vascular grafts based on the use of a compacted fibrin matrix. In this study, we evaluated the effects of a dehydration process on the biomechanical properties of compacted fibrin tubes and whether it allows for their long-term storage. Materials and methods Fibrin was precipitated from fresh frozen plasma by means of cryoprecipitation and simultaneously with a thrombin solution applied in a high-speed rotating casting mold. Subsequent dehydration of the fibrin tubes (29/38) was performed in dry air with a dilator inside the tube to prevent the collapse of the lumen. Dehydrated fibrin tubes were stored for six (n=9) and 12 months (n=10) at room temperature. Comparative analysis was done on initially generated and dehydrated fibrin tubes before and after storage to evaluate the effects of the dehydration process and storage on the biomechanical properties and structure of the tubes. Res...

The International Journal of Artificial Organs, 2019

Background: Autologous pericardium is widely used for the repair of different sized cardiovascula... more 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...

Tissue engineering. Part C, Methods, Dec 1, 2017

Tissue-engineered (TE) grafts based on decellularized grafts have shown very promising results in... more Tissue-engineered (TE) grafts based on decellularized grafts have shown very promising results in preclinical and clinical studies. However, in animal models valves have either been tested in juvenile models or in the clinically less relevant pulmonary valve position. In this study, we tested the grafts in the aortic valve (AV) position of 6-year-old sheep, as geriatric patients in need of an AV substitute due to calcification are the largest patient group benefiting from TE grafts. Decellularized AV (DAV; n = 4) and DAV additionally re-endothelialized with autologous cells (n = 3) were implanted in the AV position of 6-year-old female sheep. Function was investigated at implantation and explantation 12 months later. Regeneration capacity was analyzed by the repopulation degree of the graft with recipient's cells, by the generation of a new endothelial layer and by intracellular staining against pro-collagen type I. DAV and re-endothelialized AV demonstrated excellent function w...

The Thoracic and Cardiovascular Surgeon, 2006

Cells Tissues Organs, 2017

Valvular repair or transplantation, designed to restore the venous valve function of the legs, ha... more Valvular repair or transplantation, designed to restore the venous valve function of the legs, has been proposed as treatment in chronic venous insufficiency. Available grafts or surgeries have provided limited durability so far. Generating venous valve substitutes by means of tissue engineering could be a solution. We generated decellularized jugular ovine vein conduits containing valves (oVVC) after reseeding with ovine endothelial cells differentiated from peripheral blood-derived endothelial cells (oPBEC), cultivated in vitro corresponding to the circulatory situation in the lower leg at rest and under exertion. oVVC were decellularized by detergent treatment. GFP-labeled oPBEC were seeded onto the luminal side of the decellularized oVVC and cultivated under static-rotational conditions for 6 h (group I) and 12 h (group II), respectively. Reseeded matrices of group I were exposed to continuous low flow conditions (“leg at rest”). The tissues of group II were exposed to a gradual...

Journal of Tissue Engineering, 2017

In recent years, circulating progenitors of endothelial cells and smooth muscle cells were identi... more In recent years, circulating progenitors of endothelial cells and smooth muscle cells were identified in the peripheral blood. In our study, we evaluated the utilization of both cell types isolated and differentiated from peripheral porcine blood in terms for their use for tissue engineering purposes. By means of density gradient centrifugation, the monocyte fraction from porcine blood was separated, split, and cultivated with specific culture media with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 for the differentiation of endothelial cells or smooth muscle cells. Obtained cells were characterized at an early stage of cultivation before the first passage and a late stage (fourth passage) on the basis of the expression of the antigens CD31, CD34, CD45, nitric oxide synthase, and the contractile filaments smooth-muscle alpha-actin (sm-alpha-actin) and smoothelin. Functional characterization was done based on the secretion of nitric oxide, the formati...

Tissue Engineering Part A, 2013

Aims: Heart valve tissue engineering aims to create a graft with improved durability compared to ... more Aims: Heart valve tissue engineering aims to create a graft with improved durability compared to routinely used valve substitutes. This study presents the function and morphological changes of a tissue-engineered aortic valve (TEV) compared to the cryopreserved valve (CPV), aortic valve (AV) allografts in an orthotopic position in sheep. Methods and Results: Ovine AV conduits (n = 5) were decellularized with detergents. Autologous endothelial cells (ECs) were seeded onto the valve surface and cultured under physiological conditions using a high pulsatile flow. Grafts were implanted as a root with reimplantation of coronary ostia in sheep. Crystalloid cardioplegia and isogenic blood transfusions from previous sacrificed sheep were used. Only antiplatelet aggregation therapy was used postoperatively. CPVs (n = 4) served as controls. The grafts were investigated for function (echocardiography, magnetic resonance investigation), morpho/histological appearance, graft rejection, and calcification at 3 months. Decellularization led to cell-free scaffolds with preserved extracellular matrices, including the basement membrane. TEVs were covered with ECs expressing typical endothelial markers. Neither dilatation, stenosis, reductions of cusp mobility nor a significant transvalvular gradient, were observed in the TEV group. Explanted valves exhibited normal morphology without signs of inflammation. An endothelial monolayer covered cusps and the valve sinus. In the CPV group, sporadic, macroscopic, calcified degeneration with mild AV insufficiency was noted. Histology revealed signs of rejection and incipient calcification of the tissue. Conclusion: Tissue-engineered AV based on decellularized valve allografts satisfy short-term requirements of the systemic circulation in sheep. Although results of long-term experiments are pending, the lack of degenerative traits thus far, makes these grafts a promising alternative for future aortic heart valve surgery.

Tissue Engineering Part A, 2013

The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for... more The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for the repair of ischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The main drawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus of this study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patch by combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineering an artificial tissue of 11 cm² in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) in SIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were co-isolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize pre-existing vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CC contracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast to undirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology with well-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shaped and random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrode array measurements. A dense network of CD31⁺/eNOS⁺ cells was detected as permeating the whole construct. Superficial supplementation of RHE-A cells to SIS-CC led to the migration of these cells through the CC, resulting in the re-population of pre-existing vessel structures within the decelluarized SIS. By infusion of RHE-A cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well as distribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contact with RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaM represent a promising step toward engineering a functional perfusion system, enabling the engineering of vascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair.

Pharmacology & Therapeutics, 2005

The transcription factor signal transducer and activator of transcription 3 (STAT3) participates ... more The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses, such as proliferation and apoptosis. The constitutive activation of STAT3 promotes tumor growth and angiogenesis and is associated with drug resistance in cancer therapy. In contrast, in the heart, the down-regulation of STAT3 has been associated with end-stage heart failure in patients. Moreover, multiple studies showed that the activation of STAT3 promotes cardiomyocyte survival and hypertrophy, as well as cardiac angiogenesis, in response to various pathophysiologic stimuli, strongly suggesting that STAT3 is beneficial for the heart. Conditional knockout (STAT3-KO) mice harboring a cardiomyocyte-restricted deletion of STAT3 showed enhanced susceptibility to cardiac injury caused by myocardial ischemia, systemic inflammation, or drug toxicity. STAT3-KO mice were also more prone to the pathogenesis of age-related heart failure. Thus, STAT3 is involved in multiple mechanisms required for the protection of the heart from injury and heart failure. These observations should be taken into account in designing novel therapeutic strategies for the prevention of cardiac failure.

Mechanisms of Development, 1996

In Drosophila, dosage compensation, i.e. the equalization of levels of X-linked gene products in ... more In Drosophila, dosage compensation, i.e. the equalization of levels of X-linked gene products in the two sexes, is achieved by the hypertranscription of most X-linked genes in males relative to females. The products of at least four genes, collectively termed malespecific lethal (msl) genes, are required for this process and, at least in the case of three of them, mediate this function through an association with the X chromosome in males. We have studied some of the parameters that affect the association of the msl-l gene product and found that its presence is dependent on the wildtype function of the other three genes, leading to the conclusion that these gene products contribute to the formation of a multi-subunit complex. Furthermore, the X-chromosomal association of the msl-l and mle gene products is negatively correlated with the level of function of the master regulatory gene Sxl and can assume either a mosaic or a uniform distribution in the tissues of mutant XX individuals. Surprisingly, we also found that the association of these two msl gene products with the two X chromosomes in females of certain mutant genotypes does not result in the hypertranscription of X-linked genes or in any apparent reduction in viability.

Gastroenterology, 2014

ABSTRACT Background and aims ESD in the esophagus is an expert technique for 'en bloc&amp... more ABSTRACT Background and aims ESD in the esophagus is an expert technique for 'en bloc' resection of mucosal cancers >2cm in size. Secondary stricture formation is the major drawback. Since march 2011 we had been performing animal experiments concerning esophageal ESD and retransplantation of esophageal and gastric mucosal patches in pigs. We report on the first clinical case in man with longterm FU. Clinical case and description of technology 72 y old male p; early esophageal SCC (17.5-25cm, cervical esophagus to hypopharynx; Paris IIa; >75% circumferential; EUS UT1a, m, N0). Discussion in tumor board/with patient. On April 13, 2011 as compassionate use at first tubular esophageal ESD 17-27cm aborally followed by a 9×4-6 cm gastric ESD in the antrum (video). Attachment of 3 gastric mucosal stripes to the denuded esophageal muscular layer by hemoclips, secured by a non-covered SEMS to allow luminal and vascular nutrition of the specimen. Sphincter area of 1.5cm spared. Specimen: low horny early SCC (pT1a, G2, L-, V-, R0; invasion to lp <150mc; low risk). Stent removal at day 20, multiple islets of gastric mucosa with stepwise circular spread out over 5-6cm within the next 6 month and successful prevention of stenosis. In contrast, the 1-1.5cm non-transplant covered segment in the sphincter area showed a tendency to stricture formation requiring repetitive dilations. Conclusions The so far unique case of successful endoscopic gastro-esophageal mucosal transplantation for stricture prevention after wide-spread ESD in the esophagus opens a new perspective for systematic research in this field.

European Journal of Cardio-Thoracic Surgery, 2010

Objectives: A persistent problem in generating a functional myocardial patch is maintaining contr... more Objectives: A persistent problem in generating a functional myocardial patch is maintaining contractions in a thicker construct. Thus far, we have successfully created contracting constructs with a defined directionality by seeding neonatal rat cardiomyocytes (CMs) on decellularised porcine small-intestinal submucosa (SIS). Here, we report our efforts in generating a thicker contracting construct by combining CM cell sheets with CM-seeded SIS. Methods: Porcine SIS was decellularised, opened along the longitudinal axis, fixed in a metal frame (45 mm  25 mm) and seeded onto the submucosal side with neonatal rat CMs at a density of 1.8  10 5 cells cm À2. CM sheets were prepared using temperatureresponsive dishes by seeding CMs at a density of 4.0  10 5 cells cm À2. Three days after CM seeding, one-or three-layered CMs sheet(s) were stacked onto seeded SIS. Construct contraction was observed for an additional 10 days followed by histological analysis. Results: Stacked CM sheets contracted spontaneously and synchronously with seeded SIS after adherence. A large portion of analysed constructs showed a defined contraction direction, parallel to the longitudinal axis (seeded SIS: 83%, seeded SIS + 1 sheet: 70%, seeded SIS + 3 layered sheets: 71%). This finding was in agreement to the histological finding of aligned CMs parallel to the longitudinal axis. The thickness of seeded SIS with and without three-layered sheets was approximately 800 mm and 500 mm, respectively. Conclusions: By combining layered CM sheets with CM-seeded SIS, a three-dimensional myocardial patch with contraction in a defined direction was successfully generated. This may represent an intermediate step to a multiple layered, vascularised contractile myocardial graft.

Cryopreservation and Freeze-Drying Protocols, 2020

Malfunctioning heart valves can cause severe health problems, which if left untreated can lead to... more 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.

European Journal of Cardio-Thoracic Surgery, 2020

OBJECTIVES Decellularized homograft valves (DHVs) have shown promising clinical results, partic... more 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 m...

Cardiovascular diseases are the most frequent cause for morbidity and mortality worldwide. The da... more Cardiovascular diseases are the most frequent cause for morbidity and mortality worldwide. The damage of the heart muscle’s tissue is irreversible since cardiomyocytes do not have the capability to divide, and therefore myocardial regeneration does not take place. A surgical approach for therapy is the substitution of damaged heart tissue by a decellularized and revascularized small intestine mucosa. For the right ventricle and the atria (pressures ≤ 40-60 mmHg) the substitution of the damaged area could already be realized in animal studies. However, for the left ventricle (pressures up to 240 mmHg) the mechanical strength of these myocardial grafts is not sufficient in the early stage after the surgery. In this work first results in the processing of stabilising structures made of magnesium alloys for the cardiovascular surgery by high precision AWIJ are presented. In detail the aspects of cutting strategy, geometrical design, cutting edge roughness and burr generation are discussed.

Cardiovascular disease is one of the main causes of mortality and morbidity worldwide. The "gold ... more Cardiovascular disease is one of the main causes of mortality and morbidity worldwide. The "gold standard" for the replacement/repair of diseased blood vessels is substitution with autologous vessels. However, multiple surgical procedures limit the availability of autologous vessels, whereas synthetic grafts have been reported to demonstrate poor patency rates, especially for small-caliber vascular reconstructions. Decellularization of native vascular or non-vascular tissues for vascular scaffold development has gained significant attention in the past 20 years. A variety of decellularization techniques have been described and employed to achieve effective immunogenic agent removal from the developed vascular scaffold. At the same time, the decellularization must not impair the extracellular matrix (ECM) composition, structure, and mechanical properties of the graft in order to ensure long-term functionality in vivo. The aim of this chapter was to review the various decellularization treatments that have been reported in the literature for the development of decellularized vascular scaffolds.

The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present st... more The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present study finds prostaglandin E2 (PGE2) blood levels elevated in COVID-19 patients with positive correlation 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-lung-precision-slices infected with SARS-CoV-2 display upregulated COX-2. PGE2 in serum of COVID-19 patients lowers the expression of Paired-Box-Protein-Pax-5 (PAX5), a master regulator of B-cell survival, proliferation and differentiation, in both human and mouse pre-B-cells, while the PGE2 inhibitor taxifolin directly reduces SARS-CoV-2-induced PGE2 production and attenuates viral replication. Risk-factors for severe disease courses, i.e. older age, male sex and air pollution are associated with higher PGE2 production and lower PAX5 expression in pr...

Biomedical Spectroscopy and Imaging, 2012

Decellularized heart valve scaffolds can be used for pulmonary heart valve replacements in heart ... more Decellularized heart valve scaffolds can be used for pulmonary heart valve replacements in heart surgery. In order to assess quality parameters of heart valves prior to implantation into patients various techniques can be applied, including ultrastructural evaluation and biomechanical testing. Fourier transform infrared spectroscopy (FTIR) was used here to study protein secondary structure and solvent accessibility in decellularized heart valve matrices. FTIR was used to study proteins in the three main types of structures in decellularized heart valves: leaflet, pulmonary artery wall and heart muscle. The amide-I band region was used to reveal differences in the overall protein secondary structure amongst tissues. Leaflet material contains a relatively high contribution of α-helical structures, whereas artery matrix contains a relatively high content of triple-helix and β-sheet structures. Solvent accessibility of tissue proteins was studied by exposing material to D2O. The exchange of hydrogen of the NH amide-II bond for deuterium originating from D2O was visible as an increase in the area of the amide-II band (1500– 1400 cm−1). The amide-I band also depicted changes in shape and position during incubation in D2O: a low frequency band at around 1625 cm−1 increased as a function of time. Remarkable differences in hydrogen-to-deuterium exchange kinetics and protein solvent accessibility were observed among the three types of heart valve matrices: proteins in artery matrix are highly accessible to solvent, whereas proteins in leaflet and heart muscle structures are relatively inaccessible to solvent and show slow hydrogen/deuterium exchange. The differences in protein solvent accessibility in the different types of matrices likely reflect differences in scaffold porosity.

Xenotransplantation, 2019

The availability of decellularized human heart valve (dhHV) scaffolds is a general demand in rege... more The availability of decellularized human heart valve (dhHV) scaffolds is a general demand in regenerative heart valve replacement surgery. Particularly, dhHV scaffolds are designed for younger, adolescent patients as they are fully assimilated by the recipient and not restricted in half-life by growth persistence or early calcification 1,2 which has been described for glutaraldehyde and formaldehyde cross-linked bioprosthetics. 3 These are fixed, non-resorbable,

Scientific Reports, 2019

Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering o... more Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering of constructs with clinically relevant size as nourishment of cells is challenged by the diffusion limit. In vitro generation of 3D networks is often achieved under conditions using serum containing cell culture medium and/or animal derived matrices. Here, 3D endothelial cell networks were generated by using human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) employing human collagen I as hydrogel and decellularized porcine small intestinal submucosa as starter matrix. Matrigel/rat tail collagen I hydrogel was used as control. Resulting constructs were cultivated either in serum-free medium or in endothelial growth medium-2 serving as control. Endothelial cell networks were quantified, tested for lumen formation, and interaction of HUVECs and hASCs. Tube diameter was slightly larger in constructs containing human collagen ...

Innovative Surgical Sciences, 2018

Introduction We have recently reported about a novel technique for the generation of bioartificia... more Introduction We have recently reported about a novel technique for the generation of bioartificial vascular grafts based on the use of a compacted fibrin matrix. In this study, we evaluated the effects of a dehydration process on the biomechanical properties of compacted fibrin tubes and whether it allows for their long-term storage. Materials and methods Fibrin was precipitated from fresh frozen plasma by means of cryoprecipitation and simultaneously with a thrombin solution applied in a high-speed rotating casting mold. Subsequent dehydration of the fibrin tubes (29/38) was performed in dry air with a dilator inside the tube to prevent the collapse of the lumen. Dehydrated fibrin tubes were stored for six (n=9) and 12 months (n=10) at room temperature. Comparative analysis was done on initially generated and dehydrated fibrin tubes before and after storage to evaluate the effects of the dehydration process and storage on the biomechanical properties and structure of the tubes. Res...

The International Journal of Artificial Organs, 2019

Background: Autologous pericardium is widely used for the repair of different sized cardiovascula... more 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...

Tissue engineering. Part C, Methods, Dec 1, 2017

Tissue-engineered (TE) grafts based on decellularized grafts have shown very promising results in... more Tissue-engineered (TE) grafts based on decellularized grafts have shown very promising results in preclinical and clinical studies. However, in animal models valves have either been tested in juvenile models or in the clinically less relevant pulmonary valve position. In this study, we tested the grafts in the aortic valve (AV) position of 6-year-old sheep, as geriatric patients in need of an AV substitute due to calcification are the largest patient group benefiting from TE grafts. Decellularized AV (DAV; n = 4) and DAV additionally re-endothelialized with autologous cells (n = 3) were implanted in the AV position of 6-year-old female sheep. Function was investigated at implantation and explantation 12 months later. Regeneration capacity was analyzed by the repopulation degree of the graft with recipient's cells, by the generation of a new endothelial layer and by intracellular staining against pro-collagen type I. DAV and re-endothelialized AV demonstrated excellent function w...

The Thoracic and Cardiovascular Surgeon, 2006

Cells Tissues Organs, 2017

Valvular repair or transplantation, designed to restore the venous valve function of the legs, ha... more Valvular repair or transplantation, designed to restore the venous valve function of the legs, has been proposed as treatment in chronic venous insufficiency. Available grafts or surgeries have provided limited durability so far. Generating venous valve substitutes by means of tissue engineering could be a solution. We generated decellularized jugular ovine vein conduits containing valves (oVVC) after reseeding with ovine endothelial cells differentiated from peripheral blood-derived endothelial cells (oPBEC), cultivated in vitro corresponding to the circulatory situation in the lower leg at rest and under exertion. oVVC were decellularized by detergent treatment. GFP-labeled oPBEC were seeded onto the luminal side of the decellularized oVVC and cultivated under static-rotational conditions for 6 h (group I) and 12 h (group II), respectively. Reseeded matrices of group I were exposed to continuous low flow conditions (“leg at rest”). The tissues of group II were exposed to a gradual...

Journal of Tissue Engineering, 2017

In recent years, circulating progenitors of endothelial cells and smooth muscle cells were identi... more In recent years, circulating progenitors of endothelial cells and smooth muscle cells were identified in the peripheral blood. In our study, we evaluated the utilization of both cell types isolated and differentiated from peripheral porcine blood in terms for their use for tissue engineering purposes. By means of density gradient centrifugation, the monocyte fraction from porcine blood was separated, split, and cultivated with specific culture media with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 for the differentiation of endothelial cells or smooth muscle cells. Obtained cells were characterized at an early stage of cultivation before the first passage and a late stage (fourth passage) on the basis of the expression of the antigens CD31, CD34, CD45, nitric oxide synthase, and the contractile filaments smooth-muscle alpha-actin (sm-alpha-actin) and smoothelin. Functional characterization was done based on the secretion of nitric oxide, the formati...

Tissue Engineering Part A, 2013

Aims: Heart valve tissue engineering aims to create a graft with improved durability compared to ... more Aims: Heart valve tissue engineering aims to create a graft with improved durability compared to routinely used valve substitutes. This study presents the function and morphological changes of a tissue-engineered aortic valve (TEV) compared to the cryopreserved valve (CPV), aortic valve (AV) allografts in an orthotopic position in sheep. Methods and Results: Ovine AV conduits (n = 5) were decellularized with detergents. Autologous endothelial cells (ECs) were seeded onto the valve surface and cultured under physiological conditions using a high pulsatile flow. Grafts were implanted as a root with reimplantation of coronary ostia in sheep. Crystalloid cardioplegia and isogenic blood transfusions from previous sacrificed sheep were used. Only antiplatelet aggregation therapy was used postoperatively. CPVs (n = 4) served as controls. The grafts were investigated for function (echocardiography, magnetic resonance investigation), morpho/histological appearance, graft rejection, and calcification at 3 months. Decellularization led to cell-free scaffolds with preserved extracellular matrices, including the basement membrane. TEVs were covered with ECs expressing typical endothelial markers. Neither dilatation, stenosis, reductions of cusp mobility nor a significant transvalvular gradient, were observed in the TEV group. Explanted valves exhibited normal morphology without signs of inflammation. An endothelial monolayer covered cusps and the valve sinus. In the CPV group, sporadic, macroscopic, calcified degeneration with mild AV insufficiency was noted. Histology revealed signs of rejection and incipient calcification of the tissue. Conclusion: Tissue-engineered AV based on decellularized valve allografts satisfy short-term requirements of the systemic circulation in sheep. Although results of long-term experiments are pending, the lack of degenerative traits thus far, makes these grafts a promising alternative for future aortic heart valve surgery.

Tissue Engineering Part A, 2013

The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for... more The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for the repair of ischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The main drawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus of this study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patch by combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineering an artificial tissue of 11 cm² in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) in SIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were co-isolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize pre-existing vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CC contracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast to undirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology with well-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shaped and random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrode array measurements. A dense network of CD31⁺/eNOS⁺ cells was detected as permeating the whole construct. Superficial supplementation of RHE-A cells to SIS-CC led to the migration of these cells through the CC, resulting in the re-population of pre-existing vessel structures within the decelluarized SIS. By infusion of RHE-A cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well as distribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contact with RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaM represent a promising step toward engineering a functional perfusion system, enabling the engineering of vascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair.

Pharmacology & Therapeutics, 2005

The transcription factor signal transducer and activator of transcription 3 (STAT3) participates ... more The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses, such as proliferation and apoptosis. The constitutive activation of STAT3 promotes tumor growth and angiogenesis and is associated with drug resistance in cancer therapy. In contrast, in the heart, the down-regulation of STAT3 has been associated with end-stage heart failure in patients. Moreover, multiple studies showed that the activation of STAT3 promotes cardiomyocyte survival and hypertrophy, as well as cardiac angiogenesis, in response to various pathophysiologic stimuli, strongly suggesting that STAT3 is beneficial for the heart. Conditional knockout (STAT3-KO) mice harboring a cardiomyocyte-restricted deletion of STAT3 showed enhanced susceptibility to cardiac injury caused by myocardial ischemia, systemic inflammation, or drug toxicity. STAT3-KO mice were also more prone to the pathogenesis of age-related heart failure. Thus, STAT3 is involved in multiple mechanisms required for the protection of the heart from injury and heart failure. These observations should be taken into account in designing novel therapeutic strategies for the prevention of cardiac failure.

Mechanisms of Development, 1996

In Drosophila, dosage compensation, i.e. the equalization of levels of X-linked gene products in ... more In Drosophila, dosage compensation, i.e. the equalization of levels of X-linked gene products in the two sexes, is achieved by the hypertranscription of most X-linked genes in males relative to females. The products of at least four genes, collectively termed malespecific lethal (msl) genes, are required for this process and, at least in the case of three of them, mediate this function through an association with the X chromosome in males. We have studied some of the parameters that affect the association of the msl-l gene product and found that its presence is dependent on the wildtype function of the other three genes, leading to the conclusion that these gene products contribute to the formation of a multi-subunit complex. Furthermore, the X-chromosomal association of the msl-l and mle gene products is negatively correlated with the level of function of the master regulatory gene Sxl and can assume either a mosaic or a uniform distribution in the tissues of mutant XX individuals. Surprisingly, we also found that the association of these two msl gene products with the two X chromosomes in females of certain mutant genotypes does not result in the hypertranscription of X-linked genes or in any apparent reduction in viability.

Gastroenterology, 2014

ABSTRACT Background and aims ESD in the esophagus is an expert technique for 'en bloc&amp... more ABSTRACT Background and aims ESD in the esophagus is an expert technique for 'en bloc' resection of mucosal cancers >2cm in size. Secondary stricture formation is the major drawback. Since march 2011 we had been performing animal experiments concerning esophageal ESD and retransplantation of esophageal and gastric mucosal patches in pigs. We report on the first clinical case in man with longterm FU. Clinical case and description of technology 72 y old male p; early esophageal SCC (17.5-25cm, cervical esophagus to hypopharynx; Paris IIa; >75% circumferential; EUS UT1a, m, N0). Discussion in tumor board/with patient. On April 13, 2011 as compassionate use at first tubular esophageal ESD 17-27cm aborally followed by a 9×4-6 cm gastric ESD in the antrum (video). Attachment of 3 gastric mucosal stripes to the denuded esophageal muscular layer by hemoclips, secured by a non-covered SEMS to allow luminal and vascular nutrition of the specimen. Sphincter area of 1.5cm spared. Specimen: low horny early SCC (pT1a, G2, L-, V-, R0; invasion to lp <150mc; low risk). Stent removal at day 20, multiple islets of gastric mucosa with stepwise circular spread out over 5-6cm within the next 6 month and successful prevention of stenosis. In contrast, the 1-1.5cm non-transplant covered segment in the sphincter area showed a tendency to stricture formation requiring repetitive dilations. Conclusions The so far unique case of successful endoscopic gastro-esophageal mucosal transplantation for stricture prevention after wide-spread ESD in the esophagus opens a new perspective for systematic research in this field.

European Journal of Cardio-Thoracic Surgery, 2010

Objectives: A persistent problem in generating a functional myocardial patch is maintaining contr... more Objectives: A persistent problem in generating a functional myocardial patch is maintaining contractions in a thicker construct. Thus far, we have successfully created contracting constructs with a defined directionality by seeding neonatal rat cardiomyocytes (CMs) on decellularised porcine small-intestinal submucosa (SIS). Here, we report our efforts in generating a thicker contracting construct by combining CM cell sheets with CM-seeded SIS. Methods: Porcine SIS was decellularised, opened along the longitudinal axis, fixed in a metal frame (45 mm  25 mm) and seeded onto the submucosal side with neonatal rat CMs at a density of 1.8  10 5 cells cm À2. CM sheets were prepared using temperatureresponsive dishes by seeding CMs at a density of 4.0  10 5 cells cm À2. Three days after CM seeding, one-or three-layered CMs sheet(s) were stacked onto seeded SIS. Construct contraction was observed for an additional 10 days followed by histological analysis. Results: Stacked CM sheets contracted spontaneously and synchronously with seeded SIS after adherence. A large portion of analysed constructs showed a defined contraction direction, parallel to the longitudinal axis (seeded SIS: 83%, seeded SIS + 1 sheet: 70%, seeded SIS + 3 layered sheets: 71%). This finding was in agreement to the histological finding of aligned CMs parallel to the longitudinal axis. The thickness of seeded SIS with and without three-layered sheets was approximately 800 mm and 500 mm, respectively. Conclusions: By combining layered CM sheets with CM-seeded SIS, a three-dimensional myocardial patch with contraction in a defined direction was successfully generated. This may represent an intermediate step to a multiple layered, vascularised contractile myocardial graft.