Ulrike Böer

The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts

Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices were homogenized and extracted by high detergent concentrations westernblot analyses revealed cytosolic and cytosceleton proteins like GAPDH and smooth muscle actin which were depleted to 4.1 ± 1.9% and 13.8 ± 0.55%, resp. Also putative immunogenic MHC I complexes and the alpha-Gal epitop were reduced to only 14.8 ± 1.2% and 15.1 ± 2.05%. Mass spectrometry of matrix extracts identified 306 proteins belonging to cytosol, organelles, nucleus and cell membrane. Moreover, aqueous matrix extracts evoked a pronounced antibody formation when administered in mice and thus display high immunogenic potential. Our data indicate that an established decellularization protocol which results in acellular matrices evaluated by low DNA content reduces but not eliminates cellular components which may contribute to its immunogenic potential in vivo.

The immune response to crosslinked tissue is reduced in decellularized xenogeneic and absent in decellularized allogeneic heart valves

Background The degeneration and failure of xenogeneic heart valves, such as the Matrix P Plus valve (MP-V) consisting of decellularized porcine valves (dec-pV) and equine glutaraldehyde-fixed conduits (ga-eC) have been linked to tissue immunogenicity accompanied by antibody formation. In contrast, decellularized allograft valves (dec-aV) are well-tolerated. Here, we determined tissue-specific antibody levels in patients after implantation of MP-V or dec-aV and related them to valve failure or time period after implantation. Methods and Results Specific antibodies toward whole tissue-homogenates or alphaGal were determined retrospectively by ELISA analyses from patients who received MP-V with an uneventful course of 56.1 ± 5.1 months (n = 15), or with valve failure after 25.3 ± 14.6 months (n = 3), dec-aV for various times from 4 to 46 months (n = 14, uneventful) and from healthy controls (n = 4). All explanted valves were assessed histopathologically. MP-V induced antibodies toward both tissue components with significantly higher levels toward ga-eC than toward dec-pV (68.7 and 26.65 μg/ml IgG). In patients with valve failure, levels were not significantly higher and were related to inflammatory tissue infiltration. Anti-Gal antibodies in MP-V patients were significantly increased in both, the uneventful and the failure group. In contrast, in dec-aV patients only a slight tissue-specific antibody formation was observed after 4 months (6.24 μg/ml) that normalized to control levels after 1 year. Conclusions The strong humoral immune response to glutaraldehyde-fixed tissues is reduced in decellularized xenogeneic valves and almost absent in decellularized allogeneic tissue up to 4.5 years after implantation.

Antibody formation towards porcine tissue in patients implanted with crosslinked heart valves is directed to antigenic tissue proteins and αGal epitopes and is reduced in healthy vegetarian subjects.

Glutaraldehyde‐fixed porcine heart valves (ga‐pV) are one of the most frequently used substitutes for insufficient aortic and pulmonary heart valves which, however, degenerate after 10‐15 years. Yet, xeno‐immunogenicity of ga‐pV in humans including identification of immunogens still needs to be investigated. We here determined the immunogenicity of ga‐pV in patients with respect to antibody formation, identity of immunogens and potential options to reduce antibody levels.

Evaluation of autologous tissue sources for the isolation of endothelial cells and adipose tissue-derived mesenchymal stem cells to pre-vascularize tissue-engineered vascular grafts

Abstract Currently used synthetic vascular grafts bear a high infection risk due to insufficient microvascularization of the graft wall disabling the infiltration of immune cells. Tissue-engineered grafts with a functional pre-vascularization thus would be desirable. However, autologous tissue sources for capillary forming cells need to be evaluated. Here, peripheral blood outgrowth endothelial cells (PB-OEC) from 17 healthy donors and pericyte-like mesenchymal stem cells derived from adipose tissue (ASC) of 17 patients scheduled for visceral surgery were characterized and investigated regarding their ability to form capillary-like networks in plasma-derived fibrin gels. To obtain proliferating PB-OEC with endothelial cell-specific properties (CD31-, VE-cadherin-expression, ac-LDL uptake and three-dimensional (3D)-tube formation in fibrin gels) both enrichment of CD34+ blood cells and young donor age was necessary (7/17, age≤24 years). In contrast, all isolated ASC revealed the expression of surface antigens expressed on pericytes [human neural/glial antigen-2 (hNG2), platelet-derived growth factor receptor β (PDGF-Rβ)] and showed mesodermal differentiation capacity. Moreover, co-culture of PB-OEC and ASC in fibrin gels resulted in highly branched capillary-like networks with significantly increased tube length (2.9-fold, p<0.0001) and number of junctions (8-fold, p<0.0001). In conclusion, successful cell isolation from autologous tissues for pre-vascularization of vascular grafts has been demonstrated although certain limitations for autologous EC require further strategies to enable the use of allogeneic cells.

Dehydration improves biomechanical strength of bioartificial vascular graft material and allows its long-term storage

Abstract 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. Results Thirty-eight fibrin tubes were generated by high-speed rotation-molding from 142±3 mg fibrinogen with an inner diameter of 5.8±0.1 mm and a length of 100 mm. A centrifugal force of nearly 900×g compacted applied fibrin, while fluid was pressed out of the matrix and drained from the mold via holes resulting in a 16-fold compaction of the fibrin matrix. Dehydration was characterized by shrinkage of the tubes to a diameter of 3.2±0.2 mm, while the length remained at 100 mm equivalent to a further two-fold compaction. The biomechanical strength of the dehydrated fibrin tubes significantly increased to values comparable to that of native ovine carotid arteries and maintained during the first 6 months of storage. After 12 months of storage, only five of 10 tubes were intact, and only one showed maintained biomechanical strength. Discussion Compaction of a fibrin matrix in high-speed rotation-moulding and subsequent dehydration enables for the construction of small-caliber fibrin grafts. Over and above, the dehydration process allows their storage and stockpiling as a prerequisite for clinical use.

Low immunogenic endothelial cells maintain morphofunctional properties needed for tissue engineering

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.