An In Vitro Platform to Study Reversible Endothelial-to-Mesenchymal Transition.

Endothelial cells can acquire a mesenchymal phenotype in response to external stimuli through both mechanical and biological factors, using a process known as endothelial-to-mesenchymal (EndoMT) transition. EndoMT is characterized by the decrease in endothelial characteristics, increase in mesenchymal markers, and morphological changes. It has been recognized not only during development but also in different pathological conditions including organ/tissue fibrosis in adults. The ability to modulate the EndoMT process could have a therapeutic potential in many fibrotic diseases. An in vitro method is presented here to induce EndoMT with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) and angiotensin II (Ang II) followed by a protocol to study the reversibility of EndoMT. Using this method, we furnish evidence that the combination of L-NAME and Ang II can stimulate EndoMT in Human umbilical vascular endothelial cells (HUVECs) and this process can be reversed as observed using endothelial functionality assays. This method may serve as a model to screen and identify potential pharmacological molecules to target and regulate the EndoMT process, with applications in drug discovery for human diseases.

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&D application in regenerative medicine.

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.