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 scaffold's 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.Israeli Science Foundation/1563/10Singapore National Research Foundatio

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 asin vivo generating custom designed cell impregnated constructs for tissue engineering and regenerative medicine applications.NRF (Natl Research Foundation, S’pore)Published versio

Distinct HAND2/HAND2-AS1 Expression Levels May Fine-Tune Mesenchymal and Epithelial Cell Plasticity of Human Mesenchymal Stem Cells

We previously developed several successful decellularization strategies that yielded porcine cardiac extracellular matrices (pcECMs) exhibiting tissue-specific bioactivity and bioinductive capacity when cultured with various pluripotent and multipotent stem cells. Here, we study the tissue-specific effects of the pcECM on seeded human mesenchymal stem cell (hMSC) phenotypes using reverse transcribed quantitative polymerase chain reaction (RT-qPCR) arrays for cardiovascular related gene expression. We further corroborated interesting findings at the protein level (flow cytometry and immunological stains) as well as bioinformatically using several mRNA sequencing and protein databases of normal and pathologic adult and embryonic (organogenesis stage) tissue expression. We discovered that upon the seeding of hMSCs on the pcECM, they displayed a partial mesenchymal-to-epithelial transition (MET) toward endothelial phenotypes (CD31+) and morphologies, which were preceded by an early spike (~Day 3 onward after seeding) in HAND2 expression at both the mRNA and protein levels compared to that in plate controls. The CRISPR-Cas9 knockout (KO) of HAND2 and its associated antisense long non-coding RNA (HAND2-AS1) regulatory region resulted in proliferation arrest, hypertrophy, and senescent-like morphology. Bioinformatic analyses revealed that HAND2 and HAND2-AS1 are highly correlated in expression and are expressed in many different tissue types albeit at distinct yet tightly regulated expression levels. Deviation (downregulation or upregulation) from these basal tissue expression levels is associated with a long list of pathologies. We thus suggest that HAND2 expression levels may possibly fine-tune hMSCs’ plasticity through affecting senescence and mesenchymal-to-epithelial transition states, through yet unknown mechanisms. Targeting this pathway may open up a promising new therapeutic approach for a wide range of diseases, including cancer, degenerative disorders, and aging. Nevertheless, further investigation is required to validate these findings and better understand the molecular players involved, potential inducers and inhibitors of this pathway, and eventually potential therapeutic applications

A mathematical model predicting the coculture dynamics of endothelial and mesenchymal stem cells for tissue regeneration

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. Yet, no comprehensive analysis method exists to quantify coculture population dynamics, let alone, a unifying model addressing the “environmental” factors influencing cell growth, all together. Here we suggest 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, commonly used to describe the population dynamics of a prey and predator sharing a closed ecological niche, 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. We found that in most cases, EC growth was inhibited by the same cells but promoted by MSCs. 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. Similar results were also demonstrated on complex substrates made from decellularized porcine cardiac extracellular matrix, where growth occurred only after coculturing ECs and MSCs together. Finally, this unique implementation of the Lotka-Volterra model may also be regarded as a roadmap for using such models with other potentially regenerative cocultures in various applications.Published Versio

Thick acellular heart extracellular matrix with inherent vasculature : a potential platform for myocardial tissue regeneration

The decellularization of porcine heart tissue offers many opportunities for the production of physiologically relevant myocardial mimetic scaffolds. Earlier, we reported the successful isolation of a thin porcine cardiac extracellular matrix (pcECM) exhibiting relevant bio-mechanical properties for myocardial tissue engineering. Nevertheless, since native cardiac tissue is much thicker, such thin scaffolds may offer limited regeneration capacity. However, generation of thicker myocardial mimetic tissue constructs is hindered by diffusion limitations (100 μm), and the lack of a proper vascular-like network within these constructs. In our present work, we 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 nonimmunogenic 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 nonimmunogenic 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.Published Versio

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.NRF (Natl Research Foundation, S’pore)Published versio

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 gel’s 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 (12 weeks 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.NRF (Natl Research Foundation, S’pore)Accepted versio

Robust Fabrication of Composite 3D Scaffolds with Tissue-Specific Bioactivity: A Proof-of-Concept Study

The basic requirement of any engineered scaffold is to mimic the native tissue extracellular matrix (ECM). Despite substantial strides in understanding the ECM, scaffold fabrication processes of sufficient product robustness and bioactivity require further investigation, owing to the complexity of the natural ECM. A promising bioacive platform for cardiac tissue engineering is that of decellularized porcine cardiac ECM (pcECM, used here as a soft tissue representative model). However, this platform's complexity and batch-to-batch variability serve as processing limitations in attaining a robust and tunable cardiac tissue-specific bioactive scaffold. To address these issues, we fabricated 3D composite scaffolds (3DCSs) that demonstrate comparable physical and biochemical properties to the natural pcECM using wet electrospinning and functionalization with a pcECM hydrogel. The fabricated 3DCSs are non-immunogenic in vitro and support human mesenchymal stem cells' proliferation. Most importantly, the 3DCSs demonstrate tissue-specific bioactivity in inducing spontaneous cardiac lineage differentiation in human induced pluripotent stem cells (hiPSC) and further support the viability, functionality, and maturation of hiPSC-derived cardiomyocytes. Overall, this work illustrates the technology to fabricate robust yet tunable 3D scaffolds of tissue-specific bioactivity (with a proof of concept provided for cardiac tissues) as a platform for basic materials science studies and possible future R and D application in regenerative medicine.Nanyang Technological UniversityNational Research Foundation (NRF)This research is supported by the Singapore National Research Foundation under the CREATE program: The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research, the Li Ka Shing Foundation research grant (grant number 2020LKSFG02A), the Israeli Science Foundation and School of Materials Science & Engineering, Nanyang Technological University, Singapore

Optimized‐Surface Wettability: A New Experimental 3D Modeling Approach Predicting Favorable Biomaterial–Cell Interactions

International audienceDespite several decades of research on biomedical implant materials, the identification of predictive and robust in vitro characteristics of cell support ability and viabilities—as indicators of biocompatibility and future implant-tissue integration—remain elusive. This study addresses the phenomenology of cell–implant interfaces based on experimental, theoretical, and numerical analysis of cell response to functionalized bioceramic coatings of commercial titanium implants, cp-Ti. A variable spectrum of coatings having differing surface wettabilities, with optimized solid tension values, is obtained. Measured values are modeled and correlated to cell support ability and viabilities. The contributions of different surface aspects to cell viability are decoupled, resulting in the identification of the polar component of the surface free energy as a significant and major cell–substrate effector. Furthermore, results of this study and the suggested model establish the thermodynamic interfacial free energy as an omnipotent measure that can be fully correlated to the morphology of an individual cell under numerical simulation matching empirical observations. Collectively, the 3D model reported herein can offer a new generic theoretical framework, using implementable mathematical simulation, toward the objective of rational biomaterial design that can improve next-generation metal and ceramic implants