Application of cell laden hydrogels with temporally tunable stiffness in biomedical research.

Extracellular matrix (ECM) is a dynamic and complex environment regulating the cell fate and behavior. It is characterized by biophysical and biochemical properties specific for each tissue. Interestingly, hydrogels can serve as exceptional artificial cellular microenvironments as they can be designed to mimic the key features of the native ECM. They are valuable tools to understand how cells respond to complex microenvironments in normal and pathologic conditions. However, unlike the highly dynamic structure of ECM, nearly all of the conventional hydrogel platforms are primarily static and lack the dynamic properties of native extracellular matrices. Thus, it is necessary to develop dynamic hydrogels to better understand the mechanisms by which dynamic changes of ECM contribute to biological processes. Stiffness is one of the significant dynamic components of ECM which must be appropriately mimicked over time in vitro. In this review, we cover recent advances in engineering strategies to make cell laden hydrogels with temporally tunable stiffness. We also highlight the applications of these hydrogel systems in biomedicine focusing on specific examples in cancer, cardiovascular system, tissue fibrosis and stem cell research. Finally, the challenges regarding the development and application of cell laden hydrogels with temporally tunable stiffness are proposed.

Time dependency of morphological remodeling of endothelial cells in response to substrate stiffness.

Introduction: Substrate stiffness regulates cellular behavior as cells experience different stiffness values of tissues in the body. For example, endothelial cells (ECs) covering the inner layer of blood vessels are exposed to different stiffness values due to various pathologic and physiologic conditions. Despite numerous studies, cells by time span sense mechanical properties of the substrate, but the response is not well understood. We hypothesized that time is a major determinant influencing the behavior of cells seeded on substrates of varying stiffness. Methods: We monitored cell spreading, internal structure, 3D topography, and the viability of ECs over 24 hours of culture on polydimethylsiloxane (PDMS) substrates with two different degrees of elastic modulus. Results: Despite significant differences in cell spreading after cell seeding, cells showed a similar shape and internal structure after 24 hours of culture on both soft and stiff substrates. However, 3D topographical images confirmed existence of rich lamellipodia and filopodia around the cells cultured on stiffer PDMS substrates. Conclusion: It was concluded that the response of ECs to the substrate stiffness was time dependent with initial enhanced cellular spreading and viability on stiffer substrates. Results can provide a better comprehension of cell mechanotransduction for tissue engineering applications.

Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip.

Engineering cardiac tissues and organ models remains a great challenge due to the hierarchical structure of the native myocardium. The need of integrating blood vessels brings additional complexity, limiting the available approaches that are suitable to produce integrated cardiovascular organoids. In this work we propose a novel hybrid strategy based on 3D bioprinting, to fabricate endothelialized myocardium. Enabled by the use of our composite bioink, endothelial cells directly bioprinted within microfibrous hydrogel scaffolds gradually migrated towards the peripheries of the microfibers to form a layer of confluent endothelium. Together with controlled anisotropy, this 3D endothelial bed was then seeded with cardiomyocytes to generate aligned myocardium capable of spontaneous and synchronous contraction. We further embedded the organoids into a specially designed microfluidic perfusion bioreactor to complete the endothelialized-myocardium-on-a-chip platform for cardiovascular toxicity evaluation. Finally, we demonstrated that such a technique could be translated to human cardiomyocytes derived from induced pluripotent stem cells to construct endothelialized human myocardium. We believe that our method for generation of endothelialized organoids fabricated through an innovative 3D bioprinting technology may find widespread applications in regenerative medicine, drug screening, and potentially disease modeling.

Laterally Confined Microfluidic Patterning of Cells for Engineering Spatially Defined Vascularization.

A biofabrication strategy for creating planar multiscale protein, hydrogel, and cellular patterns, and simultaneously generating microscale topographical features is developed that laterally confines the patterned cells and direct their growth in cell permissive hydrogels.

Mechanical characterization of human mesenchymal stem cells subjected to cyclic uniaxial strain and TGF-ß1.

Human mesenchymal stem cells (hMSCs) have shown promising potential in the field of regenerative medicine particularly in vascular tissue engineering. Optimal growing of MSCs into specific lineage requires a thorough understanding of the role of mechanobiology in MSC metabolism. Although effects of external physical cues (mechanical stimuli through external loading and scaffold properties) on regulation of MSC differentiation into Smooth muscle (SM) lineage have attracted widespread attention, fewer studies are available on mechanical characterization of single engineered MSCs which is vital in tissue development through proper mechanotransductive cell-environment interactions. In this study, we investigated effects of uniaxial tensile strain and transforming growth factor-ß1 (TGF-ß1) stimulations on mechanical properties of engineered MSCs and their F-actin cytoskeleton organization. Micropipette aspiration technique was used to measure mechanical properties of MSCs including mean Young׳s modulus (E) and the parameters of standard linear viscoelastic model. Compared to control samples, MSCs treated by uniaxial strain either with or without TGF-ß1 indicated significant increases in E value and considerable drop in creep compliance curve, while samples treated by TGF-ß1 alone met significant decreases in E value and considerable rise in creep compliance curve. Among treated samples, uniaxial tensile strain accompanied by TGF-ß1 stimulation not only caused higher stimulation in MSC differentiation towards SM phenotype at transcriptional level, but also created more structural integrity in MSCs due to formation of thick bundled F-actin fibers. Results can be applied in engineering of MSCs towards functional target cells and consequently tissue development.

Effect of uniaxial stretch on morphology and cytoskeleton of human mesenchymal stem cells: static vs. dynamic loading.

Abstract Human mesenchymal stem cells (hMSCs) are capable of self-renewal and differentiation into various cell lineages. Mechanical stimuli have been shown to regulate function of stem cells through alteration in morphology and structure. The aim of this study was to evaluate and compare effects of uniaxial static stretch and combined static-dynamic stretch on the orientation, regulation and cytoskeletal structure of hMSCs. Mean values of topological were calculated before and after loadings. Moreover, fractal dimension (FD) was employed to quantify alterations in shape complexity of the cells. Internal cytoskeletal structure of cells was observed by actin filament staining. Results demonstrated a statistically significant change in cell topology and FD due to 10% static-dynamic stretch after 24 h. Static stretch was not as influential as dynamic loading. Whereas for combined static-dynamic stretch systemic alignment of cells was detected, in the static test group local alignment of actin fibers was observed, although the entire cell network was not totally aligned in a specific direction. It was concluded that dynamic stretch leads to cytoskeletal alignment and repolarization of hMSCs, whereas static stretch does not. Under static stretch hMSCs proliferated more than under dynamic stretch. Results can be applied in tissue engineering when functionalization of stem cells is required.