Jörn Hülsmann

Transplantation material bovine pericardium: biomechanical and immunogenic characteristics after decellularization vs. glutaraldehyde-fixing

Hülsmann J, Grün K, El Amouri S, Barth M, Hornung K, Holzfuß C, Lichtenberg A, Akhyari P. Transplantation material bovine pericardium: biomechanical and immunogenic characteristics after decellularization vs. glutaraldehyde‐fixing. Xenotransplantation 2012; 19: 286–297.

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.

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.

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.

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.

Electrophysiological Stimulation of Whole Heart Constructs in an 8-Pole Electrical Field: Electrophysiological Stimulation of Whole Heart

Today 2D and 3D electrophysiological stimulation represents a well established concept to enhance myocardial development and maturation in tissue-engineered constructs. However, electrical field stimulation has never been adapted to complex whole heart constructs (WHC). This study demonstrates the impact of three-dimensional electrophysiological stimulation of tissue-engineered WHC in a custom made eight-pole electrical field stimulation system by short model cultivations with neonatal rat cardiomyocytes (CM). Therefore, WHC were generated by repopulation of decellularized rat hearts with neonatal CM and subjected to perfusion based cultivation with or without additional biophysicalstimulation for 96 h. Spontaneous electrophysiological (EP) activity of the processed WHC was analyzed by qualitative evaluation of multielectrode assay (MEA) signal sequences, descriptive comparative spike sorting, and direct contrasting assessment in simple numerical quantities complemented by impulse response tests after phasing out spontaneous EP activity. As strong reduction of voltage signals by the decellularized extracellular matrix (ECM) component of WHC was observed, the active principle was determined and used to estimate the spectrum of source signals to recorded values by calculative elimination. Western blotting of key myocardial markers was employed to substantiate the functional EP evaluation by classical biochemical analysis. We observed stable spontaneous EP activity showing clear R and S, but predominantly rS patterns, for both stimulated WHC and non-stimulated controls. By the impact of stimulation, mean voltage amplitudes and beating frequencies could be significantly increased. The active principle of signal reduction in decellularized ECM could be shown to follow a nonlinear damping function with remarkable accuracy, illustrating that recorded signals of moderate voltage amplitudes can also represent far-field measurements of strong signals that are emitted in distant depths of the ECM while small amplitudes are limited to actually represent also rather weak source-signals. After phasing out spontaneous activity, both stimulated WHC and non-stimulated controls could be excited again to emit immediate impulse responses. The observed beneficial impact of 8-pole field stimulation on functional EP activity could finally be validated on the biochemical level by showing increased ratios for myosin heavy chain, cardiac tropnin T, desmin, and connexin 43 for stimulated WHC by Western blot analysis. In conclusion, we found that although electrophysiological stimulation has been incorporated into the whole heart tissue-engineered concept from the very beginning, this study presents for the first time a concept for the transfer of electrical field stimulation to the whole heart tissue-engineered approach. Furthermore to the best knowledge of the authors, this is the first control-based study showing a comparative investigation of electrophysiological stimulation of whole heart constructs.