Formation of endothelial networks in human tissue-engineered skeletal muscle

Skeletal muscle is a complex tissue with a capacity to regenerate upon injury. However, when skeletal muscle loss is beyond the regenerative capacity of skeletal muscle, the muscle repair process fails and scar tissue replaces the damaged area with resultant functional impairment. Current treatments have several disadvantages and new strategies are being explored, such as skeletal muscle tissue engineering. The aim is to reproduce the native structure and function of muscle in vitro and transplant this tissue in the damaged area. Besides applications in regenerative medicine, there are many other applications in which tissue-engineered skeletal muscle can be useful. Some examples are the use as an in vitro model for studying myogenesis, myopathology or molecular pathways or implementation as in vitro preclinical model for drug-screening and toxicity testing of compounds. The main goal of this project is development, characterization and optimization of 3D in vitro tissue-engineered human skeletal muscle tissue. Existing techniques involve culturing and differentiation of myogenic (stem) cells on natural or synthetic scaffolds. These techniques often result in non-aligned myofibers. Alignment of the myofibers is important for directional force generation, the principal function of skeletal muscle. In our lab, human muscle progenitor cells are engineered in a 3D extracellular fibrin matrix and grown in a custom-made mold with 2 attachment points, serving as artificial tendons. The resulting bio-artificial muscle (BAM) is about one millimeter thick and consists of aligned multinucleated myotubes. However, to create tissue-engineered constructs with a thickness exceeding the millimeter size, perfusion of the construct is essential to avoid cell death by lack of oxygen and nutrients. In this project, we have developed two approaches to introduce vascular networks in vitro in the 3D muscle tissue, the so-called prevascularization. In the one-stage approach, human muscle cells were directly cocultured with endothelial cells in 3D. In the two-stage approach, a one week old BAM containing differentiated myotubes was coated with a fibrin hydrogel containing endothelial cells. To prove functionality of in vitro engineered endothelial networks, prevascularized BAMs were implanted subcutaneously in mice for 14 days. In vivo anastomosis and perfusion of engineered endothelial networks with host vessels was shown. In addition, we have discussed how prevascularization in turn influences the survival and integration of a tissue-engineered construct in vivo. Furthermore, the BAM approach has been evaluated as a preclinical model for intramuscular drug injection. We have shown that the BAM is an adequate model to assess compound toxicity, retention, release and compound metabolism. To conclude, the BAM model is a promising tool, but several challenges need to be addressed in order to translate the BAM model into the clinic for regenerative medicine or towards drug development.status: publishe

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.status: publishe

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.status: publishe

Stem Cell-Based and Tissue Engineering Approaches for Skeletal Muscle Repair

Skeletal muscle tissue exhibits significant regeneration capacity upon injury or disease. This intrinsic regeneration potential is orchestrated by stem cells termed satellite cells, which undergo activation and differentiation in response to muscle insult, giving rise to fusion-competent myogenic progenitors responsible for tissue rejuvenation. Skeletal muscle diseases such as Duchenne muscular dystro-phy are characterized by progressive loss of muscle mass which precipitates reduced motility, paralysis, and in some occurrences untimely death. A manifold of muscle pathologies involve a failure to efficiently regenerate the muscle tissue, rendering stem cell-based approaches an attractive therapeutic strategy. Here we will present past and contemporary methods to treat skeletal muscle degeneration by stem cell therapy, covering prominent challenges facing this technology and potential means to overcome current hurdles. A primary focus of this chapter is directed toward illustrating innovative ways to utilize stem cells alone or in conjunction with biomaterials and tissue engineering techniques to remedy Duchenne muscular dystrophy or volumetric muscle loss