Artur Lichtenberg

Clinical Application of Tissue Engineered Human Heart Valves Using Autologous Progenitor Cells

Background— Tissue engineering (TE) of heart valves reseeded with autologous cells has been successfully performed in vitro. Here, we report our first clinical implantation of pulmonary heart valves (PV) engineered with autologous endothelial progenitor cells (EPCs) and the results of 3.5 years of follow-up. Methods and Results— Human PV allografts were decellularized (Trypsin/EDTA) and resulting scaffolds reseeded with peripheral mononuclear cells isolated from human blood. Positive stain for von Willebrand factor, CD31, and Flk-1 was observed in monolayers of cells cultivated and differentiated on the luminal surface of the scaffolds in a dynamic bioreactor system for up to 21 days, indicating endothelial nature. PV reseeded with autologous cells were implanted into 2 pediatric patients (age 13 and 11) with congenital PV failure. Postoperatively, a mild pulmonary regurgitation was documented in both children. Based on regular echocardiographic investigations, hemodynamic parameters and cardiac morphology changed in 3.5 years as follows: increase of the PV annulus diameter (18 to 22.5 mm and 22 to 26 mm, respectively), decrease of valve regurgitation (trivial/mild and trivial, respectively), decrease (16 to 9 mm Hg) or a increase (8 to 9.5 mm Hg) of mean transvalvular gradient, remained 26 mm or decreased (32 to 28 mm) right-ventricular end-diastolic diameter. The body surface area increased (1.07 to 1.42 m2 and 1.07 to 1.46 m2, respectively). No signs of valve degeneration were observed in both patients. Conclusions— TE of human heart valves using autologous EPC is a feasible and safe method for pulmonary valve replacement. TE valves have the potential to remodel and grow accordingly to the somatic growth of the child.

Use of Fresh Decellularized Allografts for Pulmonary Valve Replacement May Reduce the Reoperation Rate in Children and Young Adults Early Report

Background— Degeneration of xenografts or homografts is a major cause for reoperation in young patients after pulmonary valve replacement. We present the early results of fresh decellularized pulmonary homografts (DPH) implantation compared with glutaraldehyde-fixed bovine jugular vein (BJV) and cryopreserved homografts (CH). Methods and Results— Thirty-eight patients with DPH in pulmonary position were consecutively evaluated during the follow-up (up to 5 years) including medical examination, echocardiography, and MRI. These patients were matched according to age and pathology and compared with BJV (n=38) and CH (n=38) recipients. In contrast to BJV and CH groups, echocardiography revealed no increase of transvalvular gradient, cusp thickening, or aneurysmatic dilatation in DPH patients. Over time, DPH valve annulus diameters converge toward normal z-values. Five-year freedom from explantation was 100% for DPH and 86±8% and 88±7% for BJV and CH conduits, respectively. Additionally, MRI investigations in 17 DPH patients with follow-up time >2 years were compared with MRI data of 20 BJV recipients. Both patient groups (DPH and BJV) were at comparable ages (mean, 12.7±6.1 versus 13.0±3.0 years) and have comparable follow-up time (3.7±1.0 versus 2.7±0.9 years). In DPH patients, the mean transvalvular gradient was significantly (P=0.001) lower (11 mm Hg) compared with the BJV group (23.2 mm Hg). Regurgitation fraction was 14±3% and 4±5% in DPH and BJV groups, respectively. In 3 DPH recipients, moderate regurgitation was documented after surgery and remained unchanged in follow-up. Conclusions— In contrast to conventional homografts and xenografts, decellularized fresh allograft valves showed improved freedom from explantation, provided low gradients in follow-up, and exhibited adaptive growth.

Alterations in Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling in patients with end-stage dilated cardiomyopathy.

Background—Experimental studies indicate that interleukin-6 (IL-6)–related cytokines, signaling via the shared receptor gp130, Janus kinases (JAKs), and signal transducers and activators of transcription (STATs), provide a critical cardiomyocyte survival pathway in vivo. Little is known about the activation of this signaling pathway in the myocardia of patients with end-stage dilated cardiomyopathy (DCM). Methods and Results—We performed a comprehensive expression and activation analysis of IL-6–related cytokines, receptors, signal transducers, and signal transduction inhibitors in left ventricular (LV) myocardia from patients with DCM (n=10) and non-failing (NF) donor hearts (n=5). Differential expression (DCM versus NF) was observed by immunoblotting at each level of the signaling cascade, including receptor ligands (IL-6: −59%, P <0.01; leukemia inhibitory factor [LIF]: +54%, P <0.05), receptor subunits (LIF receptor: −16%, P <0.05), signaling molecules (the Janus kinase TYK2: −48%, P <0.01; STAT3: −47%, P <0.01), and suppressors of cytokine signaling (SOCS1: +97%, P <0.05; SOCS3: −49%, P <0.01). Tyrosine-phosphorylation status of gp130 was increased (+60%, P <0.05), whereas tyrosine-phosphorylation status of JAK2 was reduced in DCM (−72%, P <0.01). Moreover, as shown by immunohistochemistry, the number of STAT3-positive cardiomyocytes was reduced in DCM (−42%, P <0.01). Conclusion—Signaling via gp130 and JAK-STAT is profoundly altered in DCM. Importantly, tyrosine-phosphorylation of JAK2 is reduced in the face of increased gp130 phosphorylation, indicating impaired downstream activation of this critical pathway in DCM.

Preclinical Testing of Tissue-Engineered Heart Valves Re-Endothelialized Under Simulated Physiological Conditions

Background— The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months. Methods and Results— PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-α-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-α-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types. Conclusion— In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.

Myocardial tissue engineering: the extracellular matrix

More than a decade after the first reports on successful three-dimensional cardiac cell culture for experimental and potential therapeutic application, the interest and experimental efforts in the field of myocardial tissue engineering continues to grow. The hope that tissue cultures may one day act as graft substitute for malfunctioning myocardium continues to drive current scientific activity. Against this background interest seem to have progressively shifted towards the aim of engineering single tissue components. Accordingly, elements of the extracellular matrix (ECM) have gained increasing attention as potentially crucial mediators in developing and maintaining the characteristics of three-dimensional cardiac cell cultures. The ECM is now no longer regarded as merely a scaffold for developing tissue, a concept that is widely acknowledged in modern tissue engineering. The understanding of the role of precursor and stem cells has highlighted new complicated aspects of cell proliferation and differentiation and ECM proves to play an important role in providing essential signals to influence major intracellular pathways such as proliferation, differentiation and cell metabolism. Furthermore, progress in biochemical engineering has provided the perspective of application of synthetic ECM-linked molecules with bioactive potential. With the advent and continuous refinement of cell removal techniques, a new class of native acellular ECM has emerged with some striking advantages. The presently available ECM materials aim to closely resemble the in vivo microenvironment by acting as an active component of the developing tissue construct. It is therefore not surprising that most of the focus in myocardial tissue engineering has been on cell-matrix interaction, for both naturally derived and synthetic ECM. This article provides a review of established models of myocardial tissue engineering with respect to the employed ECM materials including current frontiers in material development.

Achilles Tendon and Paratendon Microcirculation in Midportion and Insertional Tendinopathy in Athletes

Background Neovascularisation can be detected qualitatively by Power Doppler in Achilles tendinopathy. Quantitative data regarding tendon microcirculation have not been established and may be substantial. Purpose To assess the microcirculation of the Achilles tendon and the paratendon in healthy volunteers as well as in athletes with either midportion or insertional tendinopathy. Study Design Cohort study; Level of evidence, 2. Methods In 66 physically active volunteers, parameters of Achilles tendon and paratendon microcirculation, such as tissue oxygen saturation, relative postcapillary venous filling pressures, and microcirculatory blood flow, were determined at rest at 2-mm and 8-mm tissue depths. Forty-one patients never had Achilles pain (25 men, 27 ± 8 years), 14 patients had insertional pain (7 men, 29 ± 8 years), and 11 patients had midportion tendinopathy (7 men, 38 ± 13 years, not significant). Results Achilles tendon diameter 2 cm and 6 cm proximal to the insertion was increased in symptomatic tendons. Compared with the uninvolved opposite tendon, deep microcirculatory blood flow was significantly elevated at insertional (160 ± 79 vs 132 ± 42, P<. 05) as well as in midportion tendinopathy (150 ± 74 vs 119 ± 34, P<. 05). The microcirculation in the uninvolved opposite tendon and the normal athlete controls were not significantly different from each other (132 ± 42 insertional asymptomatic vs 119 ± 34 mid-portion vs 120 ± 48 healthy tendon). Insertional paratendon deep microcirculatory flow was elevated in all groups, whereas tissue oxygen saturation and relative postcapillary venous filling pressures were not significantly different. Conclusion Microcirculatory blood flow is significantly elevated at the point of pain in insertional and midportion tendinopathy. Postcapillary venous filling pressures are increased at both the midportion Achilles tendon and the midportion paratendon, whereas tissue oxygen saturation is not different among the studied groups. We found no evidence of an abnormal microcirculation of the asymptomatic limb in Achilles tendinopathy.

Implantation and follow-up of totally subcutaneous versus conventional implantable cardioverter-defibrillators: A multicenter case-control study

BACKGROUND The approval of an entirely subcutaneous implantable-cardioverter defibrillator (ICD) system (S-ICD) has raised attention about this promising technology. It was developed to overcome lead failure and infection problems of conventional transvenous ICD systems. Nevertheless, lead migration of the initial design and inappropriate shock rates have raised concerns regarding its reliability and safety. OBJECTIVE The purpose of this study was to report the largest multicenter series to date of patients with the new device in comparison with a matched conventional transvenous ICD collective with focus on perioperative complications, conversion of induced ventricular fibrillation (VF), and short-term follow-up. METHODS/RESULTS Sixty-nine patients (50 male and 19 female; mean age 45.7 ± 15.7 years) received an S-ICD in three German centers and were randomly assigned to 69 sex- and age-matched conventional ICD patients. The indication was primary prevention in 41 patients (59.4%) without difference between groups (34 control patients; P = .268). The predominant underlying heart disease was ischemic cardiomyopathy in 11 (15.9%), dilated cardiomyopathy in 25 (36.2%), and hypertrophic cardiomyopathy in 10 (14.5%) in the S-ICD group. Mean implantation time was 70.8 ± 27.9 minutes (P = .398). Conversion rates of induced VF were 89.5% for 65 J (15-J safety margin) and 95.5% including reversed shock polarity (15-J safety margin) in the study group. Termination of induced VF was successful in 90.8% (10-J safety margin, device dependent) of the control patients (P = .815). Procedural complications were similar between the 2 groups. Mean follow-up was 217 ± 138 days. During follow-up, 3 patients with S-ICD were appropriately treated for ventricular arrhythmias. Three inappropriate episodes (5.2%) occurred in 3 S-ICD patients due to T-wave oversensing, whereas atrial fibrillation with rapid conduction was the predominant reason for inappropriate therapy in conventional devices (P = .745). CONCLUSION The novel S-ICD system can be implanted safely with similar perioperative adverse events compared with standard transvenous devices. Our case-control study demonstrates a 10.4% failure of conversion of induced VF with the S-ICD set to standard polarity and 15-J safety margin and comparable inappropriate shock rates during short-term follow-up.

Orthotopic replacement of the aortic valve with decellularized allograft in a sheep model

Tissue engineered (TE) allografts have been successfully applied in pulmonary circulation. The behavior of TE valves based on decellularized scaffolds in systemic circulation remains unexplored. We investigated the function, histological changes, potential of in-vivo re-endothelialization of decellularized aortic valve allografts in orthotopic position in sheep. Ovine aortic valve conduits (n=12) were decellularized with detergents and implanted as an aortic root in lambs (35-45kg). For controls, fresh native ovine aortic valve conduits (n=6) were implanted. The valves were explanted at 3 and 9 months. In the experimental group, the valves exhibited trivial regurgitation and normal morphology with no signs of graft dilatation, degeneration or rejection. In some animals (n=2), we documented minimal calcification in the area of arterial anastomosis and in one, microthrombi formation on the leaflet surface. The luminal sides of the grafts were partially covered with an endothelial cell monolayer, neovasculogenesis was observed at the adventitial side. The valves in the control group appeared thickened, shrunken with marked calcification/degeneration signs, and advanced valve insufficiency. Detergent decellularized aortic valve allografts satisfy the higher requirements of the systemic circulation in sheep. As valve conduits become repopulated by endothelial and interstitial cells, they may re-gain the potential for growth.

Sternal microcirculation after skeletonized versus pedicled harvesting of the internal thoracic artery: a randomized study.

OBJECTIVE In human patients the influence of skeletonized internal thoracic artery harvesting on the sternal microcirculation in the perioperative phase has not been well investigated. METHODS Twenty-four consecutive male patients who were scheduled for isolated coronary artery bypass grafting were prospectively randomized into 2 groups. The left internal thoracic artery was harvested by using the skeletonized technique in group 1, and it was harvested with a pedicle in group 2. Superficial (2 mm) and deep (8 mm) tissue oxygen saturation and blood flow were measured presternally and retrosternally in the upper, middle, and lower sternal parts with a novel laser Doppler flowmetric and remission spectroscopic system (Oxygen-to-See; LEA Medizintechnik, Giessen, Germany). RESULTS Presternal tissue oxygen saturation deteriorated at the upper and middle sternum, and presternal blood flow deteriorated at all measurement points after internal thoracic artery harvesting in both groups. Skeletonization had no advantage in maintaining presternal microcirculation. Retrosternal microcirculation also deteriorated at all measurement points after internal thoracic artery harvesting in both groups. However, the deterioration of the retrosternal microcirculation was significantly less in group 1 at the middle and lower sternum; values of oxygen saturation to the baseline were 86% +/- 3.8% versus 60% +/- 4.3% (P = .001) at 2-mm depth and 82% +/- 4.2% versus 61% +/- 6.1% (P = .009) at 8-mm depth at the middle sternum and 95% +/- 3.2% versus 78% +/- 1.3% (P = .001) at 2-mm depth and 94% +/- 2.2% versus 78% +/- 4.6% (P = .004) at 8-mm depth at the lower sternum in groups 1 and 2, respectively. CONCLUSION The damage of the tissue microcirculation in the middle and lower retrosternal area is significantly less after internal thoracic artery skeletonization compared with that after the pedicled internal thoracic artery harvesting technique.

Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating

Decellularization is a promising option to diminish immune and inflammatory response against donor grafts. In order to accelerate the autologous in vivo recellularization of aortic conduits for an enhanced biocompatibility, we tested fibronectin surface coating in a standardized rat implantation model. Detergent-decellularized rat aortic conduits (n = 36) were surface-coated with covalently Alexa488-labeled fibronectin (50 μg/ml, 24 h) and implanted into the systemic circulation of Wistar rats for up to 8 weeks (group FN; n = 18). Uncoated implants served as controls (group C; n = 18). Fibronectin-bound fluorescence on both surfaces of the aortic conduits was persistent for at least 8 weeks. Cellular repopulation was examined by histology and immunofluorescence (n = 24). Luminal endothelialization was significantly accelerated in group FN (p = 0.006 after 8 weeks), however, local myofibroblast hyperplasia with significantly increased ratio of intima-to-media thickness occurred (p = 0.0002 after 8 weeks). Originating from the adventitial surface, alpha-smooth muscle actin and desmin positive cell invasion into the media of fibronectin-coated conduits was significantly increased as compared to group C (p < 0.0001). In these medial areas, in situ zymography revealed enhanced matrix metalloproteinase activity. In both groups, inflammatory cell markers (CD3 and CD68) and signs of thrombosis proved negative. With regard to several markers of cell adhesion, inflammation and calcification, quantitative real-time PCR (n = 12) revealed no significant inter-group differences. Fibronectin surface coating of decellularized cardiovascular implants proved feasible and persistent for at least 8 weeks in the systemic circulation. Biofunctional protein coating accelerated the autologous in vivo endothelialization and induced a significantly increased medial recellularization. Therefore, this strategy may contribute to the improvement of current clinically applied bioprostheses.