Functional evaluation of prevascularization in one-stage versus two-stage tissue engineering approach of human bio-artificial muscle.

A common shortcoming of current tissue engineered constructs is the lack of a functional vasculature, limiting their size and functionality. Prevascularization is a possible strategy to introduce vascular networks in these constructs. It includes among others co-culturing target cells with endothelial (precursor) cells that are able to form endothelial networks through vasculogenesis. In this paper, we compared two different prevascularization approaches of bio-artificial skeletal muscle tissue (BAM) in vitro and in vivo. In a one-stage approach, human muscle cells were directly co-cultured with endothelial cells in 3D. In a two-stage approach, a one week old BAM containing differentiated myotubes was coated with a fibrin hydrogel containing endothelial cells. The obtained endothelial networks were longer and better interconnected with the two-stage approach. We evaluated whether prevascularization had a beneficial effect on in vivo perfusion of the BAM and improved myotube survival by implantation on the fascia of the latissimus dorsi muscle of NOD/SCID mice for 5 or 14 d. Also in vivo, the two-stage approach displayed the highest vascular density. At day 14, anastomosis of implanted endothelial networks with the host vasculature was apparent. BAMs without endothelial networks contained longer and thicker myotubes in vitro, but their morphology degraded in vivo. In contrast, maintenance of myotube morphology was well supported in the two-stage prevascularized BAMs. To conclude, a two-stage prevascularization approach for muscle engineering improved the vascular density in the construct and supported myotube maintenance in vivo.

Vascularization of tissue-engineered skeletal muscle constructs.

Skeletal muscle tissue can be created in vitro by tissue engineering approaches, based on differentiation of muscle stem cells. Several approaches exist and generally result in three dimensional constructs composed of multinucleated myofibers to which we refer as myooids. Engineering methods date back to 3 decades ago and meanwhile a wide range of cell types and scaffold types have been evaluated. Nevertheless, in most approaches, myooids remain very small to allow for diffusion-mediated nutrient supply and waste product removal, typically less than 1 mm thick. One of the shortcomings of current in vitro skeletal muscle organoid development is the lack of a functional vascular structure, thus limiting the size of myooids. This is a challenge which is nowadays applicable to almost all organoid systems. Several approaches to obtain a vascular structure within myooids have been proposed. The purpose of this review is to give a concise overview of these approaches.

Human tissue-engineered skeletal muscle: a novel 3D in vitro model for drug disposition and toxicity after intramuscular injection.

The development of laboratory-grown tissues, referred to as organoids, bio-artificial tissue or tissue-engineered constructs, is clearly expanding. We describe for the first time how engineered human muscles can be applied as a pre- or non-clinical model for intramuscular drug injection to further decrease and complement the use of in vivo animal studies. The human bio-artificial muscle (BAM) is formed in a seven day tissue engineering procedure during which human myoblasts fuse and differentiate to aligned myofibers in an extracellular matrix. The dimensions of the BAM constructs allow for injection and follow-up during several days after injection. A stereotactic setup allows controllable injection at multiple sites in the BAM. We injected several compounds; a dye, a hydrolysable compound, a reducible substrate and a wasp venom toxin. Afterwards, direct reflux, release and metabolism were assessed in the BAM constructs in comparison to 2D cell culture and isolated human muscle strips. Spectrophotometry and luminescence allowed to measure the release of the injected compounds and their metabolites over time. A release profile over 40 hours was observed in the BAM model in contrast to 2D cell culture, showing the capacity of the BAM model to function as a drug depot. We also determined compound toxicity on the BAMs by measuring creatine kinase release in the medium, which increased with increasing toxic insult. Taken together, we show that the BAM is an injectable human 3D cell culture model that can be used to measure release and metabolism of injected compounds in vitro.