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.

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.

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.

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.

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.

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.

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.

Simultaneous monitoring of different vitrification solution components permeating into tissues.

Cryopreservation can be used for long-term preservation of tissues and organs. It relies on using complex mixtures of cryoprotective agents (CPAs) to reduce the damaging effects of freezing, but care should be taken to avoid toxic effects of CPAs themselves. In order to rationally design cryopreservation strategies for tissues, it is important to precisely determine permeation kinetics of the protectants that are used to ensure maximum permeation, while minimizing the exposure time and toxicity effects. This is particularly challenging with protectant solutions consisting of multiple components each with different physical properties and diffusing at a different rate. In this study, we show that an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) setup can be used to simultaneously monitor diffusion of multiple components in a mixture into tissues in real time. Diffusion studies were done with decellularized heart valves using a sucrose-DMSO mixture as well as vitrification solution VS83. To assess diffusion kinetics of different solutes in mixtures, the increase in specific infrared absorbance bands was monitored during diffusion through the tissue. Solute specific wavenumber ranges were selected, and the calculated area was assumed to be proportional to the CPA concentration in the tissue. A diffusion equation based on Fick's second law of diffusion fitted the experimental data quite well, and clear differences in permeation rates were observed among the different mixture components dependent on molecular size and physical properties.

Tissue Engineering of Vein Valves Based on Decellularized Natural Matrices.

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 gradually increasing flow ("leg under effort"). After 5 days, the grafts of group I revealed a uniform luminal endothelial cell coverage of the examined areas of the venous walls and adjacent venous valve leaflets. In group II, the cell coverage on luminal areas of the venous wall parts was found to be nearly complete. The endothelial cell coverage of adjacent venous valve leaflets was revealed to be less dense and confluent. Endothelial cells cultured on acellular vein tissues of both groups were distinctly orientated uniformly in the flow direction, clearly creating a stable and flow-orientated layer. Thus, an endothelium could successfully be reestablished on the luminal surface of a decellularized venous valve by seeding peripheral blood endothelial cells and culturing under different conditions.

Decellularized GGTA1-KO pig heart valves do not bind preformed human xenoantibodies.

Pre-clinical and clinical data have unequivocally demonstrated the usefulness of decellularized heart valve (HV) matrices implanted for HV replacement therapy. However, human donor valves applicable for decellularization are in short supply, which prompts the search for suitable alternatives, such as porcine grafts. Since decellularization might be insufficient to remove all xenoantigens, we analysed the interaction of human preformed antibodies with decellularized porcine HV in vitro to assess potential immune reactions upon implantation. Detergent-decellularized pulmonary HV from German Landrace wild-type (wt) or α1,3-galactosyltransferase knockout (GGTA1-KO) pigs were investigated by inhibition ELISA and GSL I-B4 staining to localize and quantify matrix-bound αGal epitopes, which represent the most prominent xenoantigen. Additionally, preformed human xenoantibodies were affinity purified by perfusing porcine kidneys. Binding of purified human antibodies to decellularized HV was investigated by inhibition ELISA. Furthermore, binding of human plasma proteins to decellularized matrices was determined by western blot. Decellularized human pulmonary artery served as controls. Decellularization of wt HV led to a reduction of αGal epitopes by 70 %. Residual epitopes were associated with the subendothelial extracellular matrix. As expected, no αGal epitopes were found on decellularized GGTA1-KO matrix. The strongest binding of preformed human anti-pig antibodies was found on wt matrices, whereas GGTA1-KO matrices bound similar or even fewer xenoantibodies than human controls. These results demonstrate the suitability of GGTA1-KO pigs as donors for decellularized heart valves for human patients. Besides the presence of αGal antibodies on decellularized heart valves, no further preformed xenoantibodies against porcine matrix were detected in tested human sera.

Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules.

Decellularized tissues can be used as matrix implants. The aims of this study were to investigate protein stability and solvent accessibility in decellularized pulmonary heart valve tissues. Protein denaturation profiles of tissues were studied by differential scanning calorimetry. Protein solvent accessibility of tissue exposed to D2O, and diffusion kinetics of various protective molecules were studied by Fourier transform infrared spectroscopy. Little changes were observed in the protein denaturation temperature during storage, at either 5 or 40°C. Glycerol was found to stabilize proteins; it increased the protein denaturation temperature. The stabilizing effect of glycerol disappeared after washing the sample with saline solution. Hydrogen-to-deuterium exchange rates of protein amide groups were fastest in leaflet tissue, followed by artery and muscle tissue. Diffusion of glycerol was found to be fastest in muscle tissue, followed by artery and leaflet tissue. Diffusion coefficients were derived and used to estimate the time needed to reach saturation. Fixation of tissue with glutaraldehyde had little effects on exchange and diffusion rates. Diffusion rates decreased with increasing molecular size. Proteins in decellularized heart valve tissue are stable during storage. Glycerol increases protein stability in a reversible manner. Solvent accessibility studies of protein amide groups provide an additional tool to study proteins in tissues. Diffusion coefficients can be derived to simulate diffusion kinetics of protective molecules in tissues. This study provides novel tools to evaluate protein stability and solvent accessibility in tissues, which can be used to develop biopreservation strategies.

No evidence for αGal epitope transfer from media containing FCS onto human endothelial cells in culture.

BACKGROUND: Current clinical applications of cell therapies and tissue engineered (TE) constructs aim to generate non-immunogenic cells in the best-case scenario of autologous origin. As the cells are cultured, it is theoretically possible that immunoreactive molecules present in xenogenic cell culture media components, such as fetal calf serum (FCS), are transmitted in the culturing process. This problem has propelled the search for xeno-free culture media; however, in vitro culturing of many cell types, especially TE constructs which consist of several cell types, still relies to a great extent on FCS. In this study, we investigated the degree to which xenoantigens are transmitted to human endothelial cells (EC) cultured in medium containing FCS. METHODS: Human EC were isolated from pulmonary artery fragments and atrial appendage tissue samples by enzymatic digestion followed by magnetic-activated cell separation (MACS) utilizing CD31 antibodies. The cells were cultured in EGM-2 medium containing 10% FCS for several passages. Griffonia Simplicifolia Lectin I - Isolectin B4 (GSL I-B4) was used to detect cell surface-bound αGal epitopes either microscopically or flow cytometrically. Antibody binding to cells exposed to human sera prepared from healthy blood donors was investigated to detect surface-located xenoantigens. An antibody-dependent cytotoxicity assay was conducted with heat-inactivated human serum supplemented with rabbit complement and analyzed by flow cytometry after staining for living and dead cells (LIVE/DEAD assay kit). In all experiments, cells cultured in EGM-2 supplemented with 10% human serum (HS) served as controls. RESULTS: Human EC were isolated and cultured successfully for ≥6 passages. GSL I-B4 staining showed no difference between human EC cultured in FCS and in HS. In contrast to porcine EC which showed strong staining with GSL I-B4 and binding of preformed human serum antibodies, human EC cultured in FCS media did not bind human antibodies from high titer anti-αGal and anti-Neu5GC antibody serum. Along these lines, the antibody-dependent cytotoxicity assay showed that human EC cultures independent of FCS or HS usage were not affected, whereas about 40% of porcine EC did not survive. CONCLUSION: Despite culturing cells in an environment containing xenoantigens, we were unable to demonstrate the translocation of xenogenic epitopes onto the surface of human EC or find an increased sensitivity in preformed human xenoantibody-dependent complement activity. Therefore, our results suggest that the use of human cells for TE or cell therapy grown in cell culture systems complemented with FCS does not necessarily lead to an acute rejection reaction upon implantation.

Successful re-endothelialization of a perfusable biological vascularized matrix (BioVaM) for the generation of 3D artificial cardiac tissue.

Generating cellularized 3D constructs with clinical relevant dimensions is challenged by nutrition supply. This is of utmost importance for cardiac tissue engineering, since cardiomyocytes are extremely sensitive to malnutrition and hypoxia in vitro and after implantation. To develop a perfusable myocardial patch, we have focused on seeding a decellularized biological vascularized matrix (BioVaM) with endothelial cells. BioVaM is produced by decellularization of porcine small intestinal segments with preserved arterial and venous pedicles, which can be connected to a perfusion system in vitro or the host vasculature in vivo. The BioVaM vessel bed was re-seeded with porcine primary endothelial cells (pCEC). Seeding efficiency was influenced by detergent composition used for decellularization (sodium dodecyl sulfate (SDS) and/or Triton X-100) and the medium composition used for re-seeding. After decellularization, residual SDS was detected in the matrix affecting the survival of pCEC which showed a low tolerance to SDS and Triton X-100. Sensitivity to detergents was attenuated by supplementation of the medium with bovine serum albumin (BSA) or fetal calf serum (FCS). Pre-conditioning of the BioVaM with 20% FCS was not sufficient to attain pCEC survival in the vascular bed. However, re-cellularization was achieved by prolonged FCS supplementation during cultivation, resulting in a perfusable, re-endothelialized matrix of 11 cm2 in size. This achievement represents a promising step towards engineering of perfusable, 3D cardiac constructs with clinically relevant dimensions.

Moderate and severe periodontitis are independent risk factors associated with low cardiorespiratory fitness in sedentary non-smoking men aged between 45 and 65 years.

AIM: To investigate the association between periodontal disease severity and cardiorespiratory fitness (CRF) in a cross-sectional study of sedentary men. MATERIALS & METHODS: Seventy-two healthy men (45-65 years) who did not join any sport activity and had a preferentially sitting working position were recruited. Periodontal status was recorded and CRF was measured by peak oxygen uptake (VO2 peak ) during exercise testing on a cycle ergometer. Physical activity was assessed by a validated questionnaire and data were transformed to metabolic equivalent of task scores. Univariate and multivariate regression analyses were performed to investigate associations. RESULTS: Differences between VO2 peak levels in subjects with no or mild, moderate or severe periodontitis were statistically significant (p = 0.026). Individuals with low VO2 peak values showed high BMI scores, high concentrations of high-sensitive C-reactive protein, low levels of high-density lipoprotein-cholesterol, and used more glucocorticoids compared to individuals with high VO2 peak levels. Multivariate regression analysis showed that high age (p = 0.090), high BMI scores (p < 0.001), low levels of physical activity (p = 0.031) and moderate (p = 0.087), respectively, severe periodontitis (p = 0.033) were significantly associated with low VO2 peak levels. CONCLUSIONS: This study demonstrated that moderate and severe periodontitis were independently associated with low levels of CRF in sedentary men aged between 45 and 65 years.

Fabrication of heart tubes from iPSC derived cardiomyocytes and human fibrinogen by rotating mold technology.

Due to its structural and functional complexity the heart imposes immense physical, physiological and electromechanical challenges on the engineering of a biological replacement. Therefore, to come closer to clinical translation, the development of a simpler biological assist device is requested. Here, we demonstrate the fabrication of tubular cardiac constructs with substantial dimensions of 6 cm in length and 11 mm in diameter by combining human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and human foreskin fibroblast (hFFs) in human fibrin employing a rotating mold technology. By centrifugal forces employed in the process a cell-dense layer was generated enabling a timely functional coupling of iPSC-CMs demonstrated by a transgenic calcium sensor, rhythmic tissue contractions, and responsiveness to electrical pacing. Adjusting the degree of remodeling as a function of hFF-content and inhibition of fibrinolysis resulted in stable tissue integrity for up to 5 weeks. The rotating mold device developed in frame of this work enabled the production of tubes with clinically relevant dimensions of up to 10 cm in length and 22 mm in diameter which-in combination with advanced bioreactor technology for controlled production of functional iPSC-derivatives-paves the way towards the clinical translation of a biological cardiac assist device.

Reduction in Xenogeneic Epitopes on Porcine Endothelial Cells by Periodate Oxidation.

BACKGROUND: Patterns of humoral immune responses represent a major hurdle in terms of pig-to-human xenotransplantation approaches. The best-known xenogeneic glycan antigens present in pigs are the αGal (Galili antigen) and the non-human sialic acid Neu5Gc. As there are further differences between porcine and human cellular surface glycosylation, a much broader range of glycan epitopes with xeno-reactive relevance can be anticipated. Therefore, we set out to chemically modify porcine cellular surface glycans in a global approach by applying sodium periodate (NaIO4) oxidation. METHODS: Porcine endothelial cells were exposed to oxidation with 1 to 5 mM NaIO4 for different time periods at 37 °C or 4 °C and under static or dynamic conditions. The impact on cellular survival was determined by applying live/dead assays. Oxidation of αGal-epitopes was assessed by fluorescence microscopy-based quantification of isolectin-B4 (IL-B4) staining. Overall immunogenicity of porcine cells was determined by human serum antibody binding. RESULTS: Treatment of porcine endothelial cells and tissues with NaIO4 led to reduced binding of the αGal-specific IL-B4 and/or human serum antibodies. NaIO4 was revealed to be cytotoxic when performed at elevated temperatures and for a prolonged time. However, by applying 2 mM NaIO4 for 60 min at 4 °C, a high extent of cellular viability and a relevant reduction in detectable αGal epitope were observed. No differences were detected irrespectively on whether the cells were oxidized under static or flow conditions. CONCLUSIONS: Glycan epitopes on living cells can be oxidized with NaIO4 while maintaining their viability. Accordingly, this strategy holds promise to prevent immune reactions mediated by preformed anti-glycan antibodies.

Isolation and chondrogenic differentiation of porcine perichondrial progenitor cells for the purpose of cartilage tissue engineering.

In vivo, cartilage has a limited regenerative capacity. Clinical replacement strategies require a suitable cell source to provide a stable chondrocyte phenotype without hypertrophic cartilage development, while being broadly available, and harboring a high proliferative potential. Thus, the aim of this study was to analyze the proliferation and chondrogenic differentiation capacity of porcine perichondrial progenitor cells (PPC) isolated from auricular (ePPC) and tracheal cartilage (tPPC) as an alternative cell source to mesenchymal stem cells (MSC). The proliferative potential of these cell types was analyzed by means of doubling times. Cell pellets were cultured in chondrogenic differentiation medium for 4 weeks. Potential chondrogenic differentiation was investigated by histology and immunohistology in addition to gene expression analysis of the cartilage markers collagen II, aggrecan, cartilage oligomeric matrix protein (COMP), the precartilage marker collagen I, and the hypertrophic cartilage marker collagen X. PPC showed a proliferative behavior comparable to that of MSC. Chondrogenic stimulation resulted in a higher expression of collagen II, aggrecan, and COMP in ePPC as compared to tPPC and MSC, whereas the expression of collagen I was comparable in all cell types independently of differentiation stimulation. Collagen type X, however, could not be detected. The production of cartilage-like extracellular matrix components in PPC pellets was confirmed by histological and immunohistological stains. Elastin, a component of auricular cartilage, however, was not detected in ePPC-derived pellets. Thus, PPC present a promising cell source for tissue engineering of cartilage. Furthermore, ePPC may be more convenient than tPPC due to their higher chondrogenic potential and better accessibility.

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.

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.