Mimicking Cardiac Fibrosis in a Dish: Fibroblast Density Rather than Collagen Density Weakens Cardiomyocyte Function.

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.

Colorful protein-based fluorescent probes for collagen imaging.

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.

Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

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), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Mechanoregulation of vascularization in aligned tissue-engineered muscle: a role for vascular endothelial growth factor.

Skeletal muscle tissue engineering has major promise for regenerative treatment of patients suffering from muscle loss due to, for example, traumatic injury, but faces considerable challenges to progress toward clinical application. In the present study the creation of an aligned prevascularized muscle tissue was addressed. We hypothesized that an aligned vascularized three-dimensional (3D) muscle tissue can be induced in vitro by merely using uniaxial stress. The present study showed that not only do endothelial cells and muscle cells independently align in the direction of uniaxial stress in a hydrogel-based 3D culture system, but also, more importantly, the endothelial cells in the co-cultured 3D constructs organized into vascular structures. Strikingly, in these cultures no additional growth factors were needed to induce vascular formation of the endothelial cells. Vascular endothelial growth factor (VEGF) production by the muscle cells was stimulated by the uniaxial stress that develops in the tissue when constrained in one direction. This stress accompanied by VEGF production appeared to play a key role in the organization of the endothelial cells into vessel-like structures.