Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.
Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1 . The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3 . Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4
Cardiac fibrosis is one of the most devastating effects of cardiac disease. Current in vitro models of cardiac fibrosis do not sufficiently mimic the complex in vivo environment of the cardiomyocyte. We determined the local composition and mechanical properties of the myocardium in established mouse models of genetic and acquired fibrosis and tested the effect of myocardial composition on cardiomyocyte contractility in vitro by systematically manipulating the number of fibroblasts and collagen concentration in a platform of engineered cardiac microtissues. The in vitro results showed that while increasing collagen content had little effect on microtissue contraction, increasing fibroblast density caused a significant reduction in contraction force. In addition, the beating frequency dropped significantly in tissues consisting of 50% cardiac fibroblasts or higher. Despite apparent dissimilarities between native and in vitro fibrosis, the latter allows for the independent analysis of local determinants of fibrosis, which is not possible in vivo. Electronic supplementary materialThe online version of this article (doi:10.1007/s12265-017-9737-1) contains supplementary material, which is available to authorized users.
Cardiac tissue is composed of muscle and non-muscle cells, surrounded by extracellular matrix (ECM) and spatially organized into a complex three-dimensional (3D) architecture to allow for coordinated contraction and electrical pulse propagation. Despite emerging evidence for cardiomyocyte turnover in mammalian hearts, the regenerative capacity of human cardiac tissue is insufficient to recover from damage, e.g. resulting from myocardial infarction (MI). Instead, the heart 'repairs' lost or injured tissue by ongoing synthesis and remodeling of scar tissue. Conventional therapies and timely (stem) cell delivery to the injured tissue markedly improve short-term function and remodeling, but do not attenuate later stage adverse remodeling, leading to functional deterioration and eventually failure of the heart. Material-based therapies have been successfully used to mechanically support and constrain the post-MI failing heart, preventing it from further remodeling and dilation. When designed to deliver the right microenvironment for endogenous or exogenous cells, as well as the mechanical and topological cues to guide neo-tissue formation, material-based therapies may even reverse remodeling and boost cardiac regeneration. This paper reviews the up-to-date status of material-based cardiac regeneration with special emphasis on 1) the use of bare biomaterials to deliver passive constraints that unload the heart, 2) the use of materials and cells to create engineered cardiac constructs for replacement, support, or regeneration of damaged myocardium, and 3) the development of bio-inspired and bioactive materials that aim to enhance the endogenous regenerative capacity of the heart. As the therapies should function in the infarcted heart, the damaged host environment and engineered in vitro test systems that mimic this environment, are reviewed as well.
Engineered cardiac tissue models become increasingly important for understanding normal and diseased cardiac physiology. The use of in-vitro engineered disease models can give more insight in the changing structurefunction properties during pathological condition; therefore, contributing to the development of new cardiac therapies. It is hypothesized that during cardiac disorders of impaired mechanotransduction, the ratios of cardiomyocytes, fibroblasts and their supporting endogenous extracellular matrix (ECM) change. Furthermore, these changes are comparable and predictable of the different stages of diseased cardiac tissue. The aim of this study is to investigate the effect of different cardiomyocyte/fibroblast ratios on tissue morphology and function. Co-cultures of the HL-1 cardiomyocyte cell line and mouse embryonic fibroblasts (MEFs) at different ratios were used in 2D feasibility studies. Cyclic mechanical straining was applied to mimic cardiac tissue deformation during contraction. Both HL-1 and MEFs survived in co-culture although clustering of HL-1 cells was observed. The cluster size of HL-1 was dependent on the amount of MEFs. Mechanical stimulation of cultures showed strain avoidance response of MEFs while co-culture with HL-1 prevented this response. The data obtained provides new insights in the usefulness of cardiac cell-line-derived HL-1 and MEFs in the development of cardiac tissue models.
Engineered cardiac tissue models become increasingly important for understanding normal and disease cardiac physiology [1]. Where clinical diagnostic tools usually measure overall function of the heart, cardiac tissue models make it possible to focus on single CMs and their microenvironment. The use of in-vitro cardiac disease models can give more insight in the functionality changes of CMs during disease and thereby speed up the development of new therapies. Therefore, we aim to develop a model for healthy and diseased myocardium to study the effect of diseased microenvironments on the mechanical performance of CMs. The platform consists of 3D engineered microtissues with matrix, CMs and fibroblasts (FBs) on an array of polydimethylsiloxane (PDMS) microposts and allows for real-time characterization of CMs and their surrounding matrix. The design was adapted from Legant et. al. [2] and enables us to measure inhomogeneous tissue forces which may occur if not all cells contract equally. Here we focus on optimization and validation of the platform to measure contraction forces and gain insight in CM mechanical functioning.
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