02 Apr 2018
Engineering of cardiac tissues in vitro through the micropatterning approach
In the United States, each year 600,000 people die from some form of a cardiovascular disease [ref]. Due to heart’s limited ability to regenerate and complications from organ transplants, there is a strong rationale to find an alternative solution to treat heart injuries. Tissue engineering aims to repair and restore damaged tissues through a combination of cellular biology and engineering approach. A widely used approach to engineer cardiac tissues in vitro is through use of a scaffold and a cell source. Scaffolds are designed to provide cells with a suitable microenvironment (physiological surrounding of a cell) for attachment, growth and differentiation into desired tissue types. For the purposes of cardiac tissue engineering, a fundamental requirement is the ability to control the cellular microenvironment as cells are highly susceptible to their microenvironment [ref].
Cells in vivo, within organs or tissues, are surrounded by a highly organized microenvironment. Different biochemical and biophysical cues within the microenvironment tend to influence cell behavior. In context of in vitro culture systems, similar biophysical cues are presented by the substrate on which the cells are cultured. Researchers have exploited surface topography as a biomimetic cue through micropatterning techniques for purposes of cardiac tissue engineering.
Microfabrication technologies have enabled the generation of micropatterned substrates with controlled dimensions to closely mimic the pattern within cardiac tissues. Implementation of micropatterned substrates with cardiomyocytes is a strategy adopted for engineering cardiac tissues in vitro [ref].
In this review, I highlight the microenvironment of a cardiac tissue, importance of topography as a biophysical cue, micropatterning as a technique for engineering cardiac tissues, and the applications of engineered cardiac tissues.
Cell microenvironment is comprised by a cell or groups of cell whose behavior is influenced by different biophysical and biomechanical factors within the surrounding [ref]. Many of these factors are imparted by the extracellular matrix (ECM) that functions as a cellular scaffold for cell adhesion, growth, and differentiation. The native heart matrix is comprised of cardiomyocytes, fibroblasts, blood, and the surrounding tissues. Understanding the microenvironment of cardiomyocytes within a cardiac tissue is vital for cardiac tissue engineering.
Native myocardium is a differentiated muscle comprised of cardiomyocytes and fibroblasts with a cell density of approximately 2-10 x 108 cells cm-3 [ref]. Cardiomyocytes are cylindrical in shape with the following dimensions of 10-20μm in diameter, 100-110μm in length with a thickness of 8-10μm. The extracellular matrix within the cardiac tissues in vivo is comprised of collagen with a dense network of blood vessels. Fibroblasts present within the cardiac tissue secrete ECM factors and play a role in transduction of mechanical forces (Sussman et al 2002). Cardiomyocytes are involved in propagation of electrical signals to ensure synchronized contraction mechanism for pumping of blood forward. Continuous blood flow within the cardiac microenvironment exerts shear-stress and strain on the cardiomyocytes which is an important consideration while engineering cardiac tissues. Biomimetic approach to cardiac tissue engineering requires mimicking the cardiac environment in vitro to generate physiologically relevant models of cardiac tissues.
Engineering cardiac tissues in vitro involves the use of cardiomyocytes within a scaffold and the microenvironment of cardiomyocytes varies with factors governing the cardiac tissue development (Table 1). The cell type within the in vitro culture system is only cardiomyocytes, oxygen supply is through the flowing culture medium, and a modified extracellular matrix to promote cell adhesion, growth and differentiation into a functional cardiac tissue.
Different strategies have been implemented to copy nature into the lab to ensure successful translation of the cells into the desired tissue. In context of cardiac tissue engineering, surface topography has been identified as effective strategy based on the anisotropic structure of the cardiac tissue. A promising technological advancement in form of micropatterning technique, has enabled recapitulation of the cardiac microenvironment for effective engineering of cardiac tissues in vitro.
Cardiomyocytes, fibroblasts and endothelial cells
Striated muscle fibers within the tissue
Micropatterned channels on the substrate
Nutrient and gas exchange
Through blood flow
Through the culture medium
Electrical signal propogation
Table 1: Major elements involved in development of cardiac tissues both in vivo and vitro . Differences in the microenvironment conditions translate to varying factors that play a role in cardiac tissue development.
Micropatterning is a technique that allows us to engineer microtopographical features of controlled dimensions and potentially address some of the challenges facing the field of tissue engineering. Despite advancements in the field of tissue engineering that have enabled successful generation of tissues such as skin and cartilage, engineering of cardiac tissues in vitro still remains a challenge [ref]. About 40 years ago, microfabrication technique emerged as a powerful tool in the semiconductor and microelectronic device industry [ref]. A few of the earlier techniques that focused in producing microtopographical features through mechanical means include sandblasting, grinding and abrasion. The main drawback of these techniques was lack of uniformity in the features produced. It is not until the past few years (figure 1), microfabrication techniques became accessible to the field of tissue engineering to facilitate fabrication of substrates with controlled features such as grooves, wells, and pillars.
Biomedical research has involved the use of the micro-fabrication process to pattern biomaterials to study biomaterial-cell interaction [ref]. The fabrication process produces patterns that enable researchers to investigate cellular behavior at the micro or nanoscale environment in response to different surface morphology. Micropatterning approaches are particularly suited for designing and defining cell-microenvironment interactions in the order of micro to nanometer scale. Microfabrication technologies have allowed for studying the effects of biophysical cues such as topography on cell behavior at the cell-substrate interface.
Figure 1: Time line highlighting the emergence of microfabrication techniques following its introduction into the field of cell biology and tissue engineering.
In context of topography, recent advancements in microfabrication technologies have led to development of surface features with controlled dimensions and shapes to mimic tissue-like conditions for in vitro cell culture system. Topography is also involved in controlling the cellular processes such as cell adhesion [Dent et al 2003 & Karuri et al 2011], migration [Haga et al 2005& Provenzano et al 2008], and differentiation into tissues [Khademhosseini et al 2008]. The ability to understand the cellular properties through micropatterning techniques at a microscale level is vital for understanding tissue organization and development. In the current time, designing substrates with a well-controlled microenvironment has become important factor in engineering of tissues in vitro.
Cardiac tissue has an anisotropic structural organization that is vital for transduction of electrical signals which in turn is important in maintaining cardiac function. Cardiomyocytes are arranged in a parallel pattern to ensure the propagation of signals at a high velocity along the fibers [ref]. Micropatterning techniques have enabled replication of the aligned structures onto the substrates used for engineering cardiac tissues. Topographical cues can be presented to guide elongation and alignment of cardiomyocytes in vitro through creating nanopatterns on polyethylene-glycol (PEG) hydrogel substrates [kim et al 2010], nanofibers assembly through rotary jet-spinning [Reza et al 2010], and patterning ECM protein lanes on poly(dimethylsiloxane) substrate (PDMS) [Alford et al 2010]. These studies show the importance of surface topography in inducing cardiomyocytes to form cardiac tissues with resembling in vivo phenotypic and contractile properties.
3.2 Micropatterning techniques implemented in cardiac tissue engineering
Microengineering of cardiac is based on simulating the native myocardial architecture present in vivo. Contractile properties within the cardiac tissues is dependant on cellular elongation and orientation of cardiomyocytes within the muscle fibers. Micropatterning techniques have been utilized to mimic the organized anisotropic architecture of cardiac tissues to direct cellular organization and tissue development.
Photolithography, a method that involves transfer of features from a photomask onto a substrate using ultra violet light (UV). The first step involves coating the substrate with a photoresist (light-sensitive polymer) that is exposed to UV through a photomask. Following the photolithographic patterning, the unexposed areas can be etched isotropically or anisotropically using etching processes. Selection of etching processes depends on the final desired features, wet etching processes for isotropic features while dry etching process for anisotropic microfeatures. Photolithography is one of the widely used techniques for micropatterning features for biomedical applications.
3.2.2 Soft lithography
Soft lithography is a technique which uses elastomeric stamps created using hot embossing or molding techniques to generate desired pattern onto a substrate. It is a maskless technique which does not require the expensive clean room facilities and user
friendly for patterning purposes. Poly(dimethylsiloxane) (PDMS) is a commonly used phriolymeric elastomer for developing patterns due to its transparency, permeability to oxygen, and biocompatibility [weibel et al 2006].
3.2.3 Microcontact printing
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