Gokhan Bahcecioglu

In this article we review the microfabrication approaches, with a focus on bioprinting and organ-on-chip technologies, used to engineer cardiac tissue. First, we give a brief introduction to heart anatomy and physiology, and the developmental stages of the heart from fetal stages to adulthood. We also give information on the cardiac tissue microenvironment, including the cells residing in the heart, the biochemical composition and structural organization of the heart extracellular matrix, the signaling factors playing roles in heart development and maturation, and their interactions with one another. We then give a brief summary of both cardiovascular diseases and the current treatment methods used in the clinic to treat these diseases. Second, we explain how tissue engineering recapitulates the development and maturation of the normal or diseased heart microenvironment by spatially and temporally incorporating cultured cells, biomaterials, and growth factors (GF). We briefly expand on the cells, biomaterials, and GFs used to engineer the heart, and the limitations of their use. Next, we review the state-of-the-art tissue engineering approaches, with a special focus on bioprinting and heart-on-chip technologies, intended to (i) treat or replace the injured cardiac tissue, and (ii) create cardiac disease models to study the basic biology of heart diseases, develop drugs against these diseases, and create diagnostic tools to detect heart diseases. Third, we discuss the recent trends in cardiac tissue engineering, including the use of machine learning, CRISPR/Cas editing, exosomes and microRNAs, and immune modeling in engineering the heart. Finally, we conclude our article with a brief discussion on the limitations of cardiac tissue engineering and our suggestions to engineer more reliable and clinically relevant cardiac tissues.

Tissue engineered cardiac patches have great potential as a regenerative therapy for myocardial infarction. Yet, the mutual interaction of cardiac patches with healthy tissue has not been completely understood. Here, we investigated the impact of acellular and cellular patches on a beating two-dimensional (2D) cardiac cell layer, and the effect of the beating of this layer on the cells encapsulated in the patch. We cultured human-induced pluripotent stem cell-derived cardiomyocytes (iCMs) on a coverslip and placed gelatin methacryloyl hydrogel alone or with encapsulated iCMs to create acellular and cellular patches, respectively. When the acellular patch was placed on the cardiac cell layer, the beating characteristics and Ca⁺² handling properties reduced, whereas placing the cellular patch restored these characteristics. To better understand the effects of the cyclic contraction and relaxation induced by the beating cardiac cell layer on the patch placed on top of it, a simulation model was developed, and the calculated strain values were in agreement with the values measured experimentally. Moreover, this dynamic culture induced by the beating 2D iCM layer on the iCMs encapsulated in the cellular patch improved their beating velocity and frequency. Additionally, the encapsulated iCMs were observed to be coupled with the underlying beating 2D iCM layer. Overall, this study provides a detailed investigation on the mutual relationship of acellular/cellular patches with the beating 2D iCM layer, understanding of which would be valuable for developing more advanced cardiac patches.

Aging is one of the inherent risk factors for breast cancer. Although the influence of age-related cellular alterations on breast cancer development has been extensively explored, little is known about the alterations in the aging breast tissue microenvironment, specifically the extracellular matrix (ECM). Here, for the first time in literature, we have identified tissue resident matrix bound vesicles (MBVs) within the healthy mouse breast ECM, investigated and compared their characteristics in young and aged healthy breast tissues, and studied the effects of these MBVs on normal (KTB21) and cancerous (MDA-MB-231) human mammary epithelial cells with respect to the tissue age that they are extracted from. Using vesicle labeling technology, we were able to visualize cellular uptake of the MBVs directly from the native decellularized tissue sections, showing that these MBVs have regulatory roles in the tissue microenvironment. We mimicked the ECM by embedding the MBVs in collagen gels, and showed that MBVs could be taken up by the cells. The miRNA and cytokine profiling showed that MBVs shifted towards a more tumorigenic and invasive phenotype with age, as evidenced by the more pronounced presence of cancer-associated cytokines, and higher expression levels of oncomiRs miR-10b, miR-30e, and miR-210 in MBVs isolated from aged mice. When treated with MBVs or these upregulated factors, KTB21 and MDA-MB-231 cells showed significantly higher motility and invasion compared to untreated controls. Treatment of cells with a cocktail of miRNAs (miR-10b, miR-30e, and miR-210) or with the agonist of adiponectin (AdipoRon), which both were enriched in the aged MBVs, recapitulated the effect of aged MBVs on cells. This study shows for the first time that the MBVs have a regulatory role in the tissue microenvironment and that the MBV contents change towards cancer-promoting upon aging. Studying the effects of MBVs and their cargos on cellular behavior could lead to a better understanding of the critical roles of MBVs played in breast cancer progression and metastasis.

Aging is a risk factor for cardiovascular disease, the leading cause of death worldwide. Cardiac fibrosis is a harmful result of repeated myocardial infarction that increases risk of morbidity and future injury. Interestingly, rates of cardiac fibrosis are different between young and aged individuals, as well as men and women. Here, for the first time, we identify and isolate matrix-bound extracellular vesicles from the left ventricles (LVs) of young or aged men and women. These LV vesicles (LVVs) show differences in morphology and content between these four cohorts. LVVs effects on fibrosis were also investigated in vitro , and it was shown that aged male LVVs were pro-fibrotic, while other LVVs were anti-fibrotic. miRNAs identified from these LVVs could partially recapitulate these effects together, but not individually, and confer other benefits. These data suggest that synergistic effects of matrix-resident exosomal miRNAs may influence the differential clinical response to MI.

Current biomarkers for myocardial infarction (MI) diagnosis are typically late markers released upon cell death, incapable of distinguishing between ischemic and reperfusion injury and can be symptoms of other pathologies. Circulating microRNAs (miRNAs) have recently been proposed as alternative biomarkers for MI diagnosis; however, detecting the changes in the human cardiac miRNA profile during MI is extremely difficult. Here, to study the changes in miRNA levels during acute MI, a heart‐on‐chip model with a cardiac channel, containing human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes in human heart decellularized matrix and collagen, and a vascular channel, containing hiPSC‐derived endothelial cells, is developed. This model is exposed to anoxia followed by normoxia to mimic ischemia and reperfusion, respectively. Using a highly sensitive miRNA biosensor that the authors developed, the exact same increase in miR‐1, miR‐208b, and miR‐499 levels in the MI‐on‐chip and the time‐matched human blood plasma samples collected before and after ischemia and reperfusion, is shown. That the surface marker profile of exosomes in the engineered model changes in response to ischemic and reperfusion injury, which can be used as biomarkers to detect MI, is also shown. Hence, the MI‐on‐chip model developed here can be used in biomarker discovery.

Age is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer‐like phenotype, while promoting motility and invasiveness in MDA‐MB‐231 cells. Decellularized breast matrix from aged mice leads to loss of E‐cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer‐related proteins. The aged matrix upregulates cancer‐related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial‐mesenchymal transition‐related genes. Lysyl oxidase knockdown reverts the aged matrix‐induced changes to the young levels; it relocalizes E‐cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer‐like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention.