Hug Aubin

A novel customizable modular bioreactor system for whole-heart cultivation under controlled 3D biomechanical stimulation.

In the last decade, cardiovascular tissue engineering has made great progress developing new strategies for regenerative medicine applications. However, while tissue engineered heart valves are already entering the clinical routine, tissue engineered myocardial substitutes are still restrained to experimental approaches. In contrast to the heart valves, tissue engineered myocardium cannot be repopulated in vivo because of its biological complexity, requiring elaborate cultivation conditions ex vivo. Although new promising approaches—like the whole-heart decellularization concept—have entered the myocardial tissue engineering field, bioreactor technology needed for the generation of functional myocardial tissue still lags behind in the sense of user-friendly, flexible and low cost systems. Here, we present a novel customizable modular bioreactor system that can be used for whole-heart cultivation. Out of a commercially obtainable original equipment manufacturer platform we constructed a modular bioreactor system specifically aimed at the cultivation of decellularized whole-hearts through perfusion and controlled 3D biomechanical stimulation with a simple but highly flexible operation platform based on LabVIEW®. The modular setup not only allows a wide range of variance regarding medium conditioning under controlled 3D myocardial stretching but can also easily be upgraded for e.g. electrophysiological monitoring or stimulation, allowing for a tailor-made low-cost myocardial bioreactor system.

Decellularized whole heart for bioartificial heart.

Whole-organ decellularization has opened the gates to the creation of 3D extracellular matrix (ECM) templates that mimic natures design to a degree that-as for today-is not reproducible with any synthetic materials. Here, we describe a whole-heart decellularization approach through software-controlled automated coronary perfusion with standard decellularization detergents, enabling us to create native ECM-derived 3D templates that preserve the basic anatomy, vascular network, and critical ECM characteristics of the native heart. Such a cardiac ECM platform directly derived from nature itself might help us to better understand and reproduce cardiac biology and may even lay the grounds for the construction of a bioartificial heart in the future.

Bio-active coating of decellularized vascular grafts with a temperature-sensitive VEGF-conjugated hydrogel accelerates autologous endothelialization in vivo.

No ideal small‐diameter vascular graft for widespread clinical application has yet been developed and current approaches still suffer from graft failure because of thrombosis or degeneration. Decellularized vascular grafts are a promising strategy as they preserve native vessel architecture while eliminating cell‐based antigens and allow for autologous recellularization. In the present study, a functional in vivo rodent aortic transplantation model was used in order to evaluate the benefit of bioactive coating of decellularized vascular grafts with vascular endothelial growth factor (VEGF) conjugated to a temperature‐sensitive aliphatic polyester hydrogel (HG). Luminal HG‐VEGF coating persistence up to 4 weeks was confirmed in vivo by rhodamine‐labelling. Doppler‐sonography showed that the grafts were functional for up to 8 weeks in vivo. Histological and immunohistochemical analysis of the explanted grafts after 4 weeks and 8 weeks in vivo demonstrated significantly increased endothelium formation in the HG‐VEGF group compared with the control group (luminal surface covered with single‐layered endothelium, 4 weeks: 64.8 ± 7.6% vs. 40.4 ± 8.3%, p = 0.025) as well as enhanced media recellularization (absolute cell count, 8 weeks: 22.1 ± 13.0 vs. 3.2 ± 3.6, p = 0.0039). However, HG‐VEGF coating also led to increased neo‐intimal hyperplasia, resulting in a significantly increased intima‐to‐media ratio in the perianastomotic regions (intima‐to‐media ratio, 8 weeks: 1.61 ± 0.17 vs. 0.93 ± 0.09, p = 0.008; HG‐VEGF vs. control). The findings indicate that HG‐VEGF coating has potential for the development of engineered small‐diameter artificial grafts, although further research is needed to prevent neo‐intimal hyperplasia. Copyright

Rheology of perfusates and fluid dynamical effects during whole organ decellularization: a perspective to individualize decellularization protocols for single organs.

The approach of whole organ decellularization is rapidly becoming more widespread within the tissue engineering community. Today it is well known that the effects of decellularization protocols may vary with the particular type of treated tissue. However, there are no methods known to individualize decellularization protocols while automatically ensuring a standard level of quality to minimize adverse effects on the resulting extracellular matrix. Here we follow this idea by introducing two novel components into the current practice. First, a non-invasive method for online monitoring of resulting fluid dynamical characteristics of the coronary system is demonstrated for application during the perfusion decellularization of whole hearts. Second, the observation of the underlying rheological characteristics of the perfusates is employed to detect ongoing progress and maturation of the decellularization process. Measured data were contrasted to the respective release of specific cellular components. We demonstrate rheological measurements to be capable of detecting cellular debris along with a discriminative capture of DNA and protein ratios. We demonstrate that this perfusate biomass is well correlated to the biomass loss in the extracellular matrix produced by decellularization. The appearance of biomass components in the perfusates could specifically reflect the appearance of fluid dynamical characteristics that we monitored during the decellularization process. As rheological measuring of perfusate samples can be done within minutes, without any time-consuming preparation steps, we predict this to be a promising novel analytic strategy to control decellularization protocols, in time, by the actual conditions of the processed organ.

Characterization of the Epicardial Adipose Tissue in Decellularized Human-Scaled Whole Hearts: Implications for the Whole-Heart Tissue Engineering

Whole-organ engineering is an innovative field of regenerative medicine with growing translational perspectives. Recent reports suggest the feasibility of decellularization and repopulation of entire human size hearts. However, little is known about the susceptibility of epicardial adipose tissue (EAT) to decellularization. In this study, human size hearts of ovine donors were subjected to perfusion-based decellularization using detergent solutions. Upon basic histological evaluation and total DNA measurement myocardial regions prove largely decellularized while EAT demonstrated cellular remnants, further confirmed by transmission electron microscopy. Western blot analysis showed a significant reduction in lipid-associated and cardiac proteins. However, gas chromatography revealed unchanged proportional composition of fatty acids in EAT of decellularized whole hearts. Finally, cell culture medium conditioned with EAT from decellularized whole hearts had a significant deleterious effect on cardiac fibroblasts. These data suggest that perfusion decellularization of human size whole hearts provides inconsistent efficacy regarding donor material removal from myocardial regions as opposed to EAT.

Whole-Heart Construct Cultivation Under 3D Mechanical Stimulation of the Left Ventricle

Today the concept of Whole-Heart Tissue Engineering represents one of the most promising approaches to the challenge of synthesizing functional myocardial tissue. At the current state of scientific and technological knowledge it is a principal task to transfer findings of several existing and widely investigated models to the process of whole-organ tissue engineering. Hereby, we present the first bioreactor system that allows the integrated 3D biomechanical stimulation of a whole-heart construct while allowing for simultaneous controlled perfusion of the coronary system.

Additional right-sided upper “Half-Mini-Thoracotomy” for aortocoronary bypass grafting during minimally invasive multivessel revascularization

BackgroundAlthough minimally invasive coronary artery bypass grafting (MICS-CABG) has been shown to result in excellent clinical outcomes overall adoption rates still remain low. Traditional strategies for minimally invasive multivessel revascularization - usually performed through single-thoracotomy – have to deal with restricted grafting possibilities and possible increased susceptibility of arterial grafts to competitive flow, restraining their applicability to very specific indications or hybrid approaches and on top, are prone to conversion to full-sternotomy in case of left internal thoracic artery (LITA) insufficiency.MethodsHere, we present a novel alternative to the traditional MICS-CABG approaches by adding a right-sided upper “half-mini-thoracotomy”, which allows for aortocoronary bypass grafting in standard “off-pump” manner and adoption of similar revascularization principles as with conventional CABG during minimally invasive multivessel revascularization, though reducing restrictions inherent to current MICS-CABG strategies.ResultsSo far, feasibility and safety of this new approach has been successfully shown in 7 consecutive patients requiring surgical revascularization with no procedure-specific complications and graft configuration as well as intraoperative flow assessment comparable to those of similar patients operated via standard full-sternotomy off-pump coronary artery bypass (OPCAB) surgery.ConclusionsFurther evaluation warranted, this technique might have the potential to develop into an additional approach for minimally invasive multivessel revascularization, especially in cases where competitive flow to arterial grafts is feared, while also serving as a bailout-strategy for traditional approaches in case of LITA insufficiency.

Four-year experience of providing mobile extracorporeal life support to out-of-center patients within a suprainstitutional network—Outcome of 160 consecutively treated patients

AIM Mobile extracorporeal life support (ECLS) may soon be on the verge to become a fundamental part of emergency medicine. Here, we report on our four-year experience of providing advanced mechanical circulatory support for out-of-center patients within the Düsseldorf ECLS Network (DELSN). METHODS This retrospective cohort study analyses the outcome of 160 patients with refractory circulatory failure consecutively treated with mobile veno-arterial extracorporeal membrane oxygenation (vaECMO) between July 2011 and October 2015 within the DELSN. RESULTS Out of the 160 patients (56±16years, vaECMO initiation under CPR 68%), 59 patients (36%) survived to primary discharge, with 50 patients (31%) still alive after a median follow-up of 1.74 years. Time-discrete mortality was highest during the first 24h. There was no difference between survivors and non-survivors regarding age, etiology of circulatory failure, presence of CPR during implantation or distance to implantation site. Incidence of kidney injury requiring dialysis (61% vs. 24%, p<0.0001), shock liver (27% vs. 12%, p=0.031) and visceral ischemia (19% vs. 3%, p=0.013) were the only complications increased in non-survivors. Subgroup analysis showed no significant outcome difference for ECPR vs. non-ECPR patients. Outcome was significantly impaired with initial neuron-specific enolase ≥45.4μg/L (AUC 0.75, p<0.0001) and lactate ≥5.5mmol/L (AUC 0.70, p<0.0001). Program-year-dependent in-center mortality showed an increasing trend, while program-year-dependent follow-up mortality decreased over time. CONCLUSIONS This study illustrates that regional mobile ECLS rescue therapy can be provided with encouraging outcomes, although patient selection criteria and early outcome parameters reflecting on therapy success or futility still need to be refined.

Assessment of decellularization of heart bioimplants using a Raman spectroscopy method

Abstract. We report the results of experimental studies on cardiac implants using a Raman spectroscopy method (RS). Raman spectra characteristics of leaves and walls of cardiac implants were obtained; the implants were manufactured by protocols of detergent-enzymatic technique (DET) and biological, detergent-free (BIO) decellularization, using detergents (group DET) or a detergent-free, nonproteolytic, actin-disassembling regimen (BIO). There were input optical coefficients that allowed us to carry out evaluation of the protocols of DET and BIO decellularization on the basis of the concentrations of glycosaminoglycans, proteins, amides, and DNA. It was shown that during DET and BIO decellularization, composition aberrations of proteins and lipids do not occur and the integrity of the collagenous structures is preserved. It was found that during the DET decellularization, preservation of glycosaminoglycans is better than during BIO decellularization.

The impact of left ventricular stretching in model cultivations with neonatal cardiomyocytes in a whole-heart bioreactor.

Here, we investigate the impact of integrated three‐dimensional (3D) left ventricular (LV) stretching on myocardial maturation in a whole‐heart bioreactor setting. Therefore, decellularized rat hearts were selectively repopulated with rodent neonatal cardiomyocytes (5 · 106 cells per heart) and cultured over 5 days. Continuous medium perfusion was maintained through the coronary artery system in a customized whole‐heart bioreactor system with or without integrated biomechanical stimulation of LV. 3D repopulation effectiveness and cellular vitality were evaluated by repetitive metabolic WST‐1 assays and 3D confocal microscopy analysis through fluorescent staining, also assessing cellular organization. Moreover, specific myocardial vitality was verified by detecting spontaneous electrophysiological activity using a multielectrode assay. Western blot analysis of cardiac myosin heavychain (MHC) and quantitative RT‐PCR for Connexin 43 was used to analyze cardiomyocyte maturation. Decellularized whole‐heart constructs repopulated with neonatal cardiomyocytes (repopWHC) showed vital 3D cell populations throughout the repopulation sites within the LV with a significant increase in metabolic activity (326 ± 113% for stimulated constructs vs. 162 ± 32% for non‐stimulated controls after 96 h of continuous cultivation as compared to their state 24 h after injection, directly prior to bioreactor cultivation). Further, bioreactor cultivation under integrated mechanical LV stimulation not only led to a higher degree of cellular organization and an increased MHC content, but also to a significant increase of Cx43 gene expression resulting in a regain of 60 ± 19% of native neonatal hearts expression level in contrast to 20 ± 9% for non‐stimulated controls (P = 0.03). Therefore, our study suggests that the integration of LV stretching into whole‐heart bioreactor cultivation may enhance cardiac maturation not only by promoting cellular organization but also through adaptive protein and gene expression with particular implications for the formation of the conductive apparatus. Further, this study emphasizes the importance of suitable bioprocessing strategies within sophisticated bioreactor systems as tools for customized stimulation and cultivation of tissue engineered tissues and organs. Biotechnol. Bioeng. 2017;114: 1107–1117.