Udi Sarig

Engineering cell platforms for myocardial regeneration

Introduction: Various engineered ‘cell-platforms’ have been reported in recent years for the possible treatment of myocardial infarction (MI) and end-stage heart failure. These engineered platforms rely on two key factors: cells and/or biomaterial scaffolds for the regeneration of the infarcted heart tissue. Areas covered: Two major cell-platform approaches are described and broadly categorized as ‘injectable cell platforms’ and ‘patch-based cell platforms’. The recent advancements in these cell-platforms in terms of their relative successes in-vivo as well as their clinical feasibility are summarized. Natural as well as synthetic scaffolds, with or without the cellular component, are compared with cell based therapy alone. This review focuses on achievements, as well as the gaps that are presently checking any progress towards producing clinically relevant panacea for myocardial regeneration. Expert opinion: Cardiac and induced pluripotent stem cells will probably be the focus of future research. The combined cell-biomaterial scaffold therapy is superior to cell therapy alone. Nevertheless, encouraging pre-clinical successes have limited translation into clinical practice due to limited cell survival post transplantation, inadequate construct thicknesses for human-sized hearts and the traditional use of ‘flat (2D) tissue culture’ techniques. The development of complementary dynamic 3D cultivation platforms will probably lead to improved outcomes and enable fast screening of various therapeutic approaches.

Pushing the envelope in tissue engineering: ex vivo production of thick vascularized cardiac extracellular matrix constructs.

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 isolated thick acellular porcine cardiac ventricular ECM (pcECM). We now unveil this scaffolds potential 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.

Biohybrid cardiac ECM-based hydrogels improve long term cardiac function post myocardial infarction

Injectable scaffolds for cardiac tissue regeneration are a promising therapeutic approach for progressive heart failure following myocardial infarction (MI). Their major advantage lies in their delivery modality that is considered minimally invasive due to their direct injection into the myocardium. Biomaterials comprising such scaffolds should mimic the cardiac tissue in terms of composition, structure, mechanical support, and most importantly, bioactivity. Nonetheless, natural biomaterial-based gels may suffer from limited mechanical strength, which often fail to provide the long-term support required by the heart for contraction and relaxation. Here we present newly-developed injectable scaffolds, which are based on solubilized decellularized porcine cardiac extracellular matrix (pcECM) cross-linked with genipin alone or engineered with different amounts of chitosan to better control the gels mechanical properties while still leveraging the ECM biological activity. We demonstrate that these new biohybrid materials are naturally remodeled by mesenchymal stem cells, while supporting high viabilities and affecting cell morphology and organization. They exhibit neither in vitro nor in vivo immunogenicity. Most importantly, their application in treating acute and long term chronic MI in rat models clearly demonstrates the significant therapeutic potential of these gels in the long-term (12weeks post MI). The pcECM-based gels enable not only preservation, but also improvement in cardiac function eight weeks post treatment, as measured using echocardiography as well as hemodynamics. Infiltration of progenitor cells into the gels highlights the possible biological remodeling properties of the ECM-based platform. STATEMENT OF SIGNIFICANCE This work describes the development of new injectable scaffolds for cardiac tissue regeneration that are based on solubilized porcine cardiac extracellular matrix (ECM), combined with natural biomaterials: genipin, and chitosan. The design of such scaffolds aims at leveraging the natural bioactivity and unique structure of cardiac ECM, while overcoming its limited mechanical strength, which may fail to provide the long-term support required for heart contraction and relaxation. Here, we present a biocompatible gel-platform with custom-tailored mechanical properties that significantly improve cardiac function when injected into rat hearts following acute and chronic myocardial infarction. We clearly demonstrate the substantial therapeutic potential of these scaffolds, which not only preserved heart functions but also alleviated MI damage, even after the formation of a mature scar tissue.

Collagen-cellulose composite thin films that mimic soft-tissue and allow stem-cell orientation.

Mechanical properties of collagen films are less than ideal for biomaterial development towards musculoskeletal repair or cardiovascular applications. Herein, we present a collagen–cellulose composite film (CCCF) compared against swine small intestine submucosa in regards to mechanical properties, cell growth, and histological analysis. CCCF was additionally characterized by FE-SEM, NMR, mass spectrometry, and Raman Microscopy to elucidate its physical structure, collagen–cellulose composition, and structure activity relationships. Mechanical properties of the CCCF were tested in both wet and dry environments, with anisotropic stress–strain curves that mimicked soft-tissue. Mesenchymal stem cells, human umbilical vein endothelial cells, and human coronary artery smooth muscle cells were able to proliferate on the collagen films with specific cell orientation. Mesenchymal stem cells had a higher proliferation index and were able to infiltrate CCCF to a higher degree than small intestine submucosa. With the underlying biological properties, we present a collagen–cellulose composite film towards forthcoming biomaterial-related applications.Graphical Abstract

Contact guidance for cardiac tissue engineering using 3D bioprinted gelatin patterned hydrogel

Here, we have developed a 3D bioprinted microchanneled gelatin hydrogel that promotes human mesenchymal stem cell (hMSC) myocardial commitment and supports native cardiomyocytes (CMs) contractile functionality. Firstly, we studied the effect of bioprinted microchanneled hydrogel on the alignment, elongation, and differentiation of hMSC. Notably, the cells displayed well defined F-actin anisotropy and elongated morphology on the microchanneled hydrogel, hence showing the effects of topographical control over cell behavior. Furthermore, the aligned stem cells showed myocardial lineage commitment, as detected using mature cardiac markers. The fluorescence-activated cell sorting analysis also confirmed a significant increase in the commitment towards myocardial tissue lineage. Moreover, seeded CMs were found to be more aligned and demonstrated synchronized beating on microchanneled hydrogel as compared to the unpatterned hydrogel. Overall, our study proved that microchanneled hydrogel scaffold produced by 3D bioprinting induces myocardial differentiation of stem cells as well as supports CMs growth and contractility. Applications of this approach may be beneficial for generating in vitro cardiac model systems to physiological and cardiotoxicity studies as well as in vivo generating custom designed cell impregnated constructs for tissue engineering and regenerative medicine applications.

Endothelialization of Acellular Porcine ECM with Chemical Modification

Vascularization remains a critical requirement for the long term survival of engineered tissue constructs, especially thick ones. Such thick constructs for cardiac tissue engineering has been reported by our group and others based on decellularized porcine cardiac extracellular matrix (pcECM) that has been shown to resemble the native tissue both structurally and chemically. The network of inherent vasculature, which was largely retained within our pcECM, can be used as primers for re-endothelialization and neo-vascularization with regenerative cells. Endothelial cells alone, seeded onto the ECM, not only attached and survived but also rearranged into typical confluent monolayer with self-alignment. Sequential co-cultures of human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC) were shown to support the growth of both lineages on the surface and in the vasculature of reseeded pcECM. After ECM treatment with gelatin or fibronectin, cell proliferation increased significantly for both MSCs and HUVECs. Preliminary results showed that future efforts combining co-culture, treated scaffolds and dynamic culture environment may result in re-endothelialization leading to functional blood vessels in thick engineered tissue for partial cardiac replacement therapy.

Biological and mechanical interplay at the Macro- and Microscales Modulates the Cell-Niche Fate

Tissue development, regeneration, or de-novo tissue engineering in-vitro, are based on reciprocal cell-niche interactions. Early tissue formation mechanisms, however, remain largely unknown given complex in-vivo multifactoriality, and limited tools to effectively characterize and correlate specific micro-scaled bio-mechanical interplay. We developed a unique model system, based on decellularized porcine cardiac extracellular matrices (pcECMs)—as representative natural soft-tissue biomaterial—to study a spectrum of common cell–niche interactions. Model monocultures and 1:1 co-cultures on the pcECM of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) were mechano-biologically characterized using macro- (Instron), and micro- (AFM) mechanical testing, histology, SEM and molecular biology aspects using RT-PCR arrays. The obtained data was analyzed using developed statistics, principal component and gene-set analyses tools. Our results indicated biomechanical cell-type dependency, bi-modal elasticity distributions at the micron cell-ECM interaction level, and corresponding differing gene expression profiles. We further show that hMSCs remodel the ECM, HUVECs enable ECM tissue-specific recognition, and their co-cultures synergistically contribute to tissue integration—mimicking conserved developmental pathways. We also suggest novel quantifiable measures as indicators of tissue assembly and integration. This work may benefit basic and translational research in materials science, developmental biology, tissue engineering, regenerative medicine and cancer biomechanics.

Modelling early tissue formation biomechanics using adult meso- and endo-dermal cultures on decellularized ECM

the top half being extraembryonic ectoderm-trophoblast (ExE), the bottom epiblast. The ExE secretes proteases (Furin), which diffuse into the epiblast rim to activate Nodal, resulting in gastrulation. In most other mammals, the pregastrulation embryo resembles a hockey puck, with a disc-shaped epiblast overlain by a single layer of trophoblast cells termed Rauber’s layer (RL). RL is likely homologous to the ExE, as both derive from polar trophectoderm. Yet, before gastrulation, RL disappears. We propose that RL is involved in the induction of gastrulation and for this very reason has to disappear in mammals having a disc-shaped epiblast: The unusual shape of the mouse embryo results in the tip of the epiblast being sufficiently far from the ExE to avoid receiving gastrulation signals. A similar signalfree central area can only be obtained in disc-shaped epiblasts if most of RL disappears, thereby confining Furin-positive trophoblast to the edges of the disc. This idea would imply that if RL did not disappear, the epiblast would be exposed to excessive signalling. We tested this using cattle embryos. FURIN was indeed expressed in RL. We determined that RL disappears by apoptosis and not a reduction in cell proliferation and thus made transgenic cattle embryos overexpressing the antiapoptotic protein BCL2. BCL2-transgenes maintained RL for significantly longer than control transgenic embryos. When examining gastrulation-stage BCL2-overexpressing embryos for expression of the gastrulation marker BRACHYURY, we found evidence for an extended domain of gastrulation induction as well as double axis formation. This is reminiscent of mice, where ectopic Nodal activation leads to axis duplication. So, just as players competing in Rugby and Hockey have to adjust to the different trajectile shapes, so did evolution adapt to speciesspecific pregastrulation embryo shapes, while continuing to use the same molecular players.