Cardiovascular Regenerative Medicine

We identified and addressed substantial and crucial gaps in the cardiovascular tissue engineering field, bringing this field ever more close to clinical applications. We developed a unique decellularization procedure resulting in ECM that highly resembles human tissue, and can promote tissue regeneration without evoking an immune response. The ECM was shown to support the static and dynamic cultivation of MSCs, as model cells for cardiomyocytes, and maintained their multi-potency. This work was highlighted by the Journal of Tissue Engineering for its impact on the field. A similar decellularization procedure was also used to produce vascular grafts that exhibited better performance than clinically approved synthetic grafts. We also reported on the use of mathematical modelling to analyze ECM mechanical properties and the population dynamics of MSCs co-cultured with different cells.


Re-ceullarization of thick vascularized porcine cardiac extra-cellular matrix construct:
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SELECTED PUBLICATIONS:

Acellular cardiac extracellular matrix as a scaffold for tissue engineering: in vitro cell support, remodeling, and biocompatibility. Tissue engineering. Part C, Methods 16, 671-683, 2010.

We have developed an efficient decellularization process for the isolation of extracellular matrix (ECM) from native cardiac tissue. The isolated ECM exhibited desirable mechanical properties in terms of elasticity, strength and durability-properties required from scaffolds used for cardiac tissuerepair. This study further investigates the potential use of this scaffold for cardiac tissue engineering in terms of interactions with seeded cells and biocompatibility. We used the commonly studied fibroblasts, cardiomyocytes, and mesenchymal stem cells, which were isolated and seeded onto thescaffold. Cell density and distribution were followed by 3,3′-dioctadecyloxacarbocyanine perchlorate staining, and their proliferation and viability were assessed by AlamarBlue assay and fluorecein-diacetate/propidium iodide staining. Fibroblast-seeded scaffolds shrank to 1-2 mm(3) spheroids, and their glycosaminoglycans significantly increased by 23%. The expression of ECM remodeling-related mRNAs of collagens I and III, matrixmetalloproteinase 2, and type 1 tissue inhibitor of metalloproteinases was quantified by real-time polymerase chain reaction, and was found significantly elevated in fibroblast-seeded scaffold, compared with the control cells on plates. Fibroblast-seeded scaffolds lost some flexibility, yet gained strength compared with the acellular scaffolds, as shown by mechanical testing. Scaffold seeded with cardiomyocyte began to beat in concert few days after seeding, and the myocytes expressed typical functional cardiac markers such as alpha-actinin, troponin I, and connexin43. The cellsrevealed aligned elongated morphology, as presented by immunofluorescent staining and scanning electron microscopy. Mesenchymal stem cell-seeded scaffolds maintained viability over 24 days in culture. These findings further strengthen the potential use of acellular cardiac ECM as a biomaterial for heart regeneration.

Thick acellular heart extracellular matrix with inherent vasculature: A potential platform for myocardial tissue regeneration. Tissue Engineering Part A, 18: 2125-37, 2012.

This work focused on optimizing the decellularization procedure for thicker tissue slabs (10-15 mm), while retaining their inherent vasculature, and on characterizing the resulting pcECM. The trypsin/Triton-based perfusion procedure that resulted in a non-immunogenic and cell-supportive pcECM was found to be more effective in cell removal and in the preservation of fiber morphology and structural characteristics than stirring, sonication, or sodium dodecyl sulfate/Triton-based procedures. Mass spectroscopy revealed that the pcECM is mainly composed of ECM proteins with no apparent cellular protein remains. Mechanical testing indicated that the obtained pcECM is viscoelastic in nature and possesses the typical stress-strain profile of biological materials. It is stiffer than native tissue yet exhibits matched mechanical properties in terms of energy dissipation, toughness, and ultimate stress behavior. Vascular network functionality was maintained to the first three-four branches from the main coronary vessels. Taken together, these results reaffirm the efficiency of the decellularization procedure reported herein for yielding thick non-immunogenic cell-supportive pcECM scaffolds, preserving both native tissue ultra-structural properties and an inherent vascular network. When reseeded with the appropriate progenitor cells, these scaffolds can potentially serve as ex vivo screening platforms for new therapeutics, as models for human cardiac ECM, or as biomedical constructs for patch or transmural transplantation strategies.

Porcine small diameter arterial ECM matrix supports endothelium formation and media remodeling forming a promising vascular bio-graft. Tissue Engineering Part A, 18:411-22, 2012.

Small caliber arteries were decellularized using an enzymatic and detergent procedure, which enabled preservation of major extracellular matrix proteins as well as the mechanical properties of the native artery. The decellularized artery was reseeded with human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) and cultured under static or dynamic conditions in a costume-made perfusion bioreactor. Dynamic co-culturing of SMC and HUVEC in this bioreactor led to a higher infiltration, migration and proliferation of SMC toward the media and to a more confluent endothelium formation on the luminal surface when compared with static culturing. In addition, SMC remodeled the vascular media and HUVEC remodeled several basement membrane proteins.

A mathematical model predicting the coculture dynamics of endothelial and mesenchymal stem cells for tissue regeneration. Tissue engineering. Part A 19, 1155-1164, 2013.

In most tissue engineering applications, understanding the factors affecting the growth dynamics of coculture systems is crucial for directing the population toward a desirable regenerative process. This work suggests a modification of the Lotka-Volterra model to analyze the population dynamics of cocultured cells and predict their growth profiles for tissue engineering applications. This model, was found to fit our empirical data on cocultures of endothelial cells (ECs) and mesenchymal stem cells (MSCs) that have been widely investigated for their regenerative potential. Applying this model to cocultures of this sort allows us to quantify the effect that culturing conditions have on the way cell growth is affected by the same cells or by the other cells in the coculture. The principles resulting from this analysis can be used in various applications to guide the population toward a desired direction while shedding new light on the fundamental interactions between ECs and MSCs.

Pushing the envelope in tissue engineering: ex vivo production of thick vascularized cardiac extracellular matrix constructs. Tissue engineering. Part A 21, 1507-1519 (2015). 

Functional vascularization is a prerequisite for cardiac tissue engineering of constructs with physiological thicknesses. We previously reported the successful preservation of main vascular conduits in. This work unveil the  potential of isolated thick acellular porcine cardiac ventricular ECM (pcECM) in supporting human cardiomyocytes and promoting new blood vessel development ex vivo, providing long-term cell support in the construct bulk. A custom-designed perfusion bioreactor was developed to remodel such vascularization ex vivo, demonstrating, for the first time, functional angiogenesis in vitro with various stages of vessel maturation supporting up to 1.7 mm thick constructs. A robust methodology was developed to assess the pcECM maximal cell capacity, which resembled the human heart cell  density. Taken together, these results demonstrate feasibility of producing physiological-like constructs such as the thick pcECM suggested here as a prospective treatment for end-stage heart failure. Methodologies reported herein may also benefit other tissues, offering a valuable in vitro setting for “thick-tissue” engineering strategies toward large animal in vivo studies.