Deok Ho Kim

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs

Heart tissue possesses complex structural organization on multiple scales, from macro- to nano-, but nanoscale control of cardiac function has not been extensively analyzed. Inspired by ultrastructural analysis of the native tissue, we constructed a scalable, nanotopographically controlled model of myocardium mimicking the in vivo ventricular organization. Guided by nanoscale mechanical cues provided by the underlying hydrogel, the tissue constructs displayed anisotropic action potential propagation and contractility characteristic of the native tissue. Surprisingly, cell geometry, action potential conduction velocity, and the expression of a cell–cell coupling protein were exquisitely sensitive to differences in the substratum nanoscale features of the surrounding extracellular matrix. We propose that controlling cell–material interactions on the nanoscale can stipulate structure and function on the tissue level and yield novel insights into in vivo tissue physiology, while providing materials for tissue repair.

Matrix nanotopography as a regulator of cell function

Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.The architecture of the extracellular matrix (ECM) directs cell behavior by providing spatial and mechanical cues to which cells respond. In addition to soluble chemical factors, physical interactions between the cell and ECM regulate primary cell processes, including differentiation, migration, and proliferation. Advances in microtechnology and, more recently, nanotechnology provide a powerful means to study the influence of the ECM on cell behavior. By recapitulating local architectures that cells encounter in vivo, we can elucidate and dissect the fundamental signal transduction pathways that control cell behavior in critical developmental, physiological, and pathological processes.

Microengineered platforms for cell mechanobiology

Mechanical forces play important roles in the regulation of various biological processes at the molecular and cellular level, such as gene expression, adhesion, migration, and cell fate, which are essential to the maintenance of tissue homeostasis. In this review, we discuss emerging bioengineered tools enabled by microscale technologies for studying the roles of mechanical forces in cell biology. In addition to traditional mechanobiology experimental techniques, we review recent advances of microelectromechanical systems (MEMS)-based approaches for cell mechanobiology and discuss how microengineered platforms can be used to generate in vivo-like micromechanical environment in in vitro settings for investigating cellular processes in normal and pathophysiological contexts. These capabilities also have significant implications for mechanical control of cell and tissue development and cell-based regenerative therapies.

Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients

In this report, we describe using ultraviolet (UV)-assisted capillary force lithography (CFL) to create a model substratum of anisotropic micro- and nanotopographic pattern arrays with variable local density for the analysis of cell-substratum interactions. A single cell adhesion substratum with the constant ridge width (1 microm), and depth (400 nm) and variable groove widths (1-9.1 microm) allowed us to characterize the dependence of cellular responses, including cell shape, orientation, and migration, on the anisotropy and local density of the variable micro- and nanotopographic pattern. We found that fibroblasts adhering to the denser pattern areas aligned and elongated more strongly along the direction of ridges, vs. those on the sparser areas, exhibiting a biphasic dependence of the migration speed on the pattern density. In addition, cells responded to local variations in topography by altering morphology and migrating along the direction of grooves biased by the direction of pattern orientation (short term) and pattern density (long term), suggesting that single cells can sense the topography gradient. Molecular dynamic live cell imaging and immunocytochemical analysis of focal adhesions and actin cytoskeleton suggest that variable substratum topography can result in distinct types of cytoskeleton reorganization. We also demonstrate that fibroblasts cultured as monolayers on the same substratum retain most of the properties displayed by single cells. This result, in addition to demonstrating a more sophisticated method to study aspects of wound healing processes, strongly suggests that even in the presence of adhesive cell-cell interactions, the cues provided by the underlying substratum topography continue to exercise substantial influence on cell behavior. The described experimental platform might not only further our understanding of biomechanical regulation of cell-matrix interactions, but also contribute to bioengineering of devices with the optimally structured design of cell-material interface.

3D bioprinting for engineering complex tissues.

Bioprinting is a 3D fabrication technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. While still in its early stages, bioprinting strategies have demonstrated their potential use in regenerative medicine to generate a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of high-resolution cell deposition, controlled cell distributions, vascularization, and innervation within complex 3D tissues. While no one-size-fits-all approach to bioprinting has emerged, it remains an on-demand, versatile fabrication technique that may address the growing organ shortage as well as provide a high-throughput method for cell patterning at the micrometer scale for broad biomedical engineering applications. In this review, we introduce the basic principles, materials, integration strategies and applications of bioprinting. We also discuss the recent developments, current challenges and future prospects of 3D bioprinting for engineering complex tissues. Combined with recent advances in human pluripotent stem cell technologies, 3D-bioprinted tissue models could serve as an enabling platform for high-throughput predictive drug screening and more effective regenerative therapies.

Nanotopography-guided tissue engineering and regenerative medicine ☆

Human tissues are intricate ensembles of multiple cell types embedded in complex and well-defined structures of the extracellular matrix (ECM). The organization of ECM is frequently hierarchical from nano to macro, with many proteins forming large scale structures with feature sizes up to several hundred microns. Inspired from these natural designs of ECM, nanotopography-guided approaches have been increasingly investigated for the last several decades. Results demonstrate that the nanotopography itself can activate tissue-specific function in vitro as well as promote tissue regeneration in vivo upon transplantation. In this review, we provide an extensive analysis of recent efforts to mimic functional nanostructures in vitro for improved tissue engineering and regeneration of injured and damaged tissues. We first characterize the role of various nanostructures in human tissues with respect to each tissue-specific function. Then, we describe various fabrication methods in terms of patterning principles and material characteristics. Finally, we summarize the applications of nanotopography to various tissues, which are classified into four types depending on their functions: protective, mechano-sensitive, electro-active, and shear stress-sensitive tissues. Some limitations and future challenges are briefly discussed at the end.

A biomimetic undulatory tadpole robot using ionic polymer–metal composite actuators

The development of a wireless undulatory tadpole robot using ionic polymer–metal composite (IPMC) actuators is presented. In order to improve the thrust of the tadpole robot, a biomimetic undulatory motion of the fin tail is implemented. The overall size of the underwater microrobot prototype, shaped as a tadpole, is 96 mm in length, 24 mm in width, and 25 mm in thickness. It has one polymer fin tail driven by the cast IPMC actuator, an internal (wireless) power source, and an embedded controller. The motion of the tadpole microrobot is controlled by changing the frequency and duty ratio of the input voltage. Experimental results show that this technique can accurately control the steering and swimming speed of the proposed underwater tadpole robot.

Fabrication of nanostructures of polyethylene glycol for applications to protein adsorption and cell adhesion

A simple method was developed to fabricate polyethylene glycol (PEG) nanostructures using capillary lithography mediated by ultraviolet (UV) exposure. Acrylate-containing PEG monomers, such as PEG dimethacrylate (PEG-DMA, MW = 330), were photo-cross-linked under UV exposure to generate patterned structures. In comparison to unpatterned PEG films, hydrophobicity of PEG nanostructure modified surfaces was significantly enhanced. This could be attributed to trapped air in the nanostructures as supported by water contact angle measurements. Proteins (fibronectin, immunoglobulin, and albumin) and cells (fibroblasts and P19 EC cells) were examined on the modified surfaces to test for the level of protein adsorption and cell adhesion. It was found that proteins and cells preferred to adhere on nanostructured PEG surfaces in comparison to unpatterned PEG films; however, this level of adhesion was significantly lower than that of glass controls. These results suggest that capillary lithography can be used to fabricate PEG nanostructures capable of modifying protein and cell adhesive properties of surfaces.

Biomimetic nanopatterns as enabling tools for analysis and control of live cells.

It is becoming increasingly evident that cell biology research can be considerably advanced through the use of bioengineered tools enabled by nanoscale technologies. Recent advances in nanopatterning techniques pave the way for engineering biomaterial surfaces that control cellular interactions from the nano- to the microscale, allowing more precise quantitative experimentation capturing multi-scale aspects of complex tissue physiology in vitro. The spatially and temporally controlled display of extracellular signaling cues on nanopatterned surfaces (e. g., cues in the form of chemical ligands, controlled stiffness, texture, etc.) that can now be achieved on biologically relevant length scales is particularly attractive enabling experimental platform for investigating fundamental mechanisms of adhesion-mediated cell signaling. Here, we present an overview of bio-nanopatterning methods, with the particular focus on the recent advances on the use of nanofabrication techniques as enabling tools for studying the effects of cell adhesion and signaling on cell function. We also highlight the impact of nanoscale engineering in controlling cell-material interfaces, which can have profound implications for future development of tissue engineering and regenerative medicine.