Danh Truong

PNIPAAm-based biohybrid injectable hydrogel for cardiac tissue engineering

UNLABELLED Injectable biomaterials offer a non-invasive approach to deliver cells into the myocardial infarct region to maintain a high level of cell retention and viability and initiate the regeneration process. However, previously developed injectable matrices often suffer from low bioactivity or poor mechanical properties. To address this need, we introduced a biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with excellent bioactivity as well as mechanical robustness for cardiac tissue engineering. A unique feature of our work was that we performed extensive in vitro biological analyses to assess the functionalities of cardiomyocytes (CMs) alone and in co-culture with cardiac fibroblasts (CFs) (2:1 ratio) within the hydrogel matrix. The synthesized hydrogel exhibited viscoelastic behavior (storage modulus: 1260 Pa) and necessary water content (75%) to properly accommodate the cardiac cells. The encapsulated cells demonstrated a high level of cell survival (90% for co-culture condition, day 7) and spreading throughout the hydrogel matrix in both culture conditions. A dense network of stained F-actin fibers (∼ 6 × 10(4) μm(2) area coverage, co-culture condition) illustrated the formation of an intact and three dimensional (3D) cell-embedded matrix. Furthermore, immunostaining and gene expression analyses revealed mature phenotypic characteristics of cardiac cells. Notably, the co-culture group exhibited superior structural organization and cell-cell coupling, as well as beating behavior (average ∼ 45 beats per min, co-culture condition, day 7). The outcome of this study is envisioned to open a new avenue for extensive in vitro characterization of injectable matrices embedded with 3D mono- and co-culture of cardiac cells prior to in vivo experiments. STATEMENT OF SIGNIFICANCE In this work, we synthesized a new class of biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with suitable bioactivity and mechanical properties for cardiac tissue engineering. A significant aspect of our work was that we performed extensive in vitro biological analyses to assess the functionality of cardiomyocytes alone and in co-culture with cardiac fibroblasts encapsulated within the 3D hydrogel matrix.

Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment

In this study, to model 3D chemotactic tumor-stroma invasion in vitro, we developed an innovative microfluidic chip allowing side-by-side positioning of 3D hydrogel-based matrices. We were able to (1) create a dual matrix architecture that extended in a continuous manner, thus allowing invasion from one 3D matrix to another, and (2) establish distinct regions of tumor and stroma cell/ECM compositions, with a clearly demarcated tumor invasion front, thus allowing us to quantitatively analyze progression of cancer cells into the stroma at a tissue or single-cell level. We showed significantly enhanced cancer cell invasion in response to a transient gradient of epidermal growth factor (EGF). 3D tracking at the single-cell level displayed increased migration speed and persistence. Subsequently, we analyzed changes in expression of EGF receptors, cell aspect ratio, and protrusive activity. These findings show the unique ability of our model to quantitatively analyze 3D chemotactic invasion, both globally by tracking the progression of the invasion front, and at the single-cell level by examining changes in cellular behavior and morphology using high-resolution imaging. Taken together, we have shown a novel model recapitulating 3D tumor-stroma interactions for studies of real-time cell invasion and morphological changes within a single platform.

Electrically conductive hydrogel-based micro-topographies for the development of organized cardiac tissues

The dense uniaxially aligned cardiac cytoarchitecture of the myocardium along with electrical and mechanical coupling between the cardiac cells are key factors in the synchronous contractility of the heart. Following myocardial infarction, the organized architecture of the myocardium is disrupted and non-functional scar tissues are produced. Engineered cardiac tissues, developed in hydrogel-based biomaterials, featuring biomimetic topographical cues have shown significant promise for regeneration and repair of injured myocardium. However, currently engineered tissues still do not exhibit an electrically conductive matrix integrated with highly oriented cellular constructs to promote tissue-level functionalities. To address this limitation, we utilized integrated micro- and nano-technologies to develop gelatin methacrylate (GelMA) hydrogel constructs comprised of surface micro-topographies (microgrooves with 50 μm width and depth) incorporated with electrically conductive gold nanorods (GNRs) to provide simultaneous electrical and topographical cues that mimic physiological relevant myocardium function. F-Actin stained images and fluorescent area coverage data revealed the formation of uniform, dense, and highly aligned cardiac tissues on GelMA–GNR hydrogels compared to discrete and disconnected cellular organization on pure GelMA constructs (control group). Immunostaining images of cardiac markers, specifically sarcomeric α-actinin and connexin 43 also showed higher cytoskeletal alignment and enhanced cellular connectivity on GelMA–GNR hydrogels. Tissues formed on both GelMA and GelMA–GNR constructs demonstrated spontaneous contractility from day 4 to 7 of culture. However, only electrically conductive GelMA–GNR cardiac tissues showed a consistent response in changing beat rate as a result of external stimulation. Overall, we demonstrate the enhanced formation of cardiac tissues with superior cellular organization, connectivity, and electrical properties with use of GNRs and microgrooved GelMA hydrogel that may have potential as a functional cardiac patch in the setting of infarcted myocardium.

Microfluidic Tumor–Vascular Model to Study Breast Cancer Cell Invasion and Intravasation

Cancer is a major leading cause of disease-related death in the world. The severe impact of cancer can be attributed to poor understanding of the mechanisms involved in earliest steps of the metastatic cascade, specifically invasion into the surrounding stroma and intravasation into the blood capillaries. However, conducting integrated biological studies of invasion and intravasation have been challenging, within in vivo models and traditional in vitro assay, due to difficulties in establishing a precise tumor microenvironment. To that end, in this work, a novel 3D microfluidic platform comprised of concentric three-layer cell-laden hydrogels for simultaneous investigation of breast cancer cell invasion and intravasation as well as vasculature maturation influenced by tumor-vascular crosstalk is developed. It was demonstrated that the presence of spontaneously formed vasculature enhance MDA-MB-231 invasion into the 3D stroma. Following invasion, cancer cells are visualized intravasating into the outer vasculature. Additionally, invading cancer cells significantly reduce vessel diameter while increasing permeability, consistent with previous in vivo studies. Major signaling cytokines involved in tumor-vascular crosstalk that govern cancer cell invasion and intravasation are further identified. Taken together, this platform will enable unique insights of critical biological events within the metastatic cascade, with significant potential for developing efficient cancer therapeutics.