Innervation of an engineered muscle graft for reconstruction of muscle defects.

Autologous muscle flaps are commonly used to reconstruct defects that involve muscle impairment. To maintain viability and functionality of these flaps, they must be properly vascularized and innervated. Tissue-engineered muscles could potentially replace autologous muscle tissue, but still require establishment of sufficient innervation to ensure functionality. In this study, we explored the possibility of innervating engineered muscle grafts transplanted to an abdominal wall defect in mice, by transferring the native femoral nerve to the graft. Six weeks posttransplantation, nerve conduction studies and electromyography demonstrated increased innervation in engineered grafts neurotized with the femoral nerve, as compared to non-neurotized grafts. Histologic assessments revealed axonal penetration and formation of neuromuscular junctions within the grafts. The innervation process described here may advance the fabrication of a fully functional engineered muscle graft that will be of utility in clinical settings.

Improved Patency of ePTFE Grafts as a Hemodialysis Access Site by Seeding Autologous Endothelial Cells Expressing Fibulin-5 and VEGF.

Small caliber synthetic vascular grafts used for dialysis access sites have high failure rates due to neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. We tested the use of ePTFE grafts seeded with autologous ECs expressing fibulin-5 and vascular endothelial growth factor (VEGF), as a dialysis access site in a porcine model. We connected the carotid arteries and jugular veins of 12 miniature pigs using 7-mm ePTFE grafts; five grafts were seeded with autologous venous ECs modified to express fibulin-5 and VEGF, and seven unseeded grafts were implanted at the same location and served as controls. At 6 months, after completion of angiography, the carotid arteries and jugular veins with the connecting grafts were excised and fixed. Autologous EC isolation and transduction and graft seeding were successful in all animals. At 3 months, 4 of 5 seeded grafts and 3 of 7 control grafts were patent. At 6 months, 4 of 5 (80%) seeded grafts and only 2 of 7 (29%) control grafts were patent. Seeding ePTFE vascular grafts with genetically modified ECs improved long term small caliber graft patency. The biosynthetic grafts offer a novel therapeutic modality for vascular access in hemodialysis.

Elderly Patient-Derived Endothelial Cells for Vascularization of Engineered Muscle.

In vitro prevascularization of engineered tissue constructs promises to enhance their clinical applicability. We hypothesize that adult endothelial cells (ECs), isolated from limb veins of elderly patients, bear the vasculogenic properties required to form vascular networks in vitro that can later integrate with the host vasculature upon implantation. Here, we show that adult ECs formed vessel networks that were more developed and complex than those formed by human umbilical vein endothelial cells (HUVECs) seeded with various supporting cells on three-dimensional (3D) biodegradable polymer scaffolds. In parallel, secreted levels of key proangiogenic cytokines were significantly higher in adult EC-bearing scaffolds as compared to HUVEC scaffolds. As a proof of concept for applicability of this model, adult ECs were co-seeded with human myoblasts as well as supporting cells and successfully formed a branched network, which was surrounded by aligned human myotubes. The vascularized engineered muscle tissue implanted into a full-thickness defect in immunodeficient mice remained viable and anastomosed with the host vasculature within 9 days of implantation. Functional "chimeric" blood vessels and various types of anastomosis were observed. These findings provide strong evidence of the applicability of adult ECs in construction of clinically relevant autologous vascularized tissue.

Enhanced Host Neovascularization of Prevascularized Engineered Muscle Following Transplantation into Immunocompetent versus Immunocompromised Mice.

: Engineering of functional tissue, by combining either autologous or allogeneic cells with biomaterials, holds promise for the treatment of various diseases and injuries. Prevascularization of the engineered tissue was shown to enhance and improve graft integration and neovascularization post-implantation in immunocompromised mice. However, the neovascularization and integration processes of transplanted engineered tissues have not been widely studied in immunocompetent models. Here, we fabricated a three-dimensional (3D) vascularized murine muscle construct that was transplanted into immunocompetent and immunocompromised mice. Intravital imaging demonstrated enhanced neovascularization in immunocompetent mice compared to immunocompromised mice, 18 days post-implantation, indicating the advantageous effect of an intact immune system on neovascularization. Moreover, construct prevascularization enhanced neovascularization, integration, and myogenesis in both animal models. These findings demonstrate the superiority of implantation into immunocompetent over immunocompromised mice and, therefore, suggest that using autologous cells might be beneficial compared to allogeneic cells and subsequent immunosuppression. Taken together, these observations have the potential to advance the field of regenerative medicine and tissue engineering, ultimately reducing the need for donor organs and tissues.

Genetically engineered human muscle transplant enhances murine host neovascularization and myogenesis.

Engineered tissues are a promising tool for addressing the growing need for tissues and organs in surgical reconstructions. Prevascularization of implanted tissues is expected to enhance survival prospects post transplantation and minimize deficiencies and/or hypoxia deeper in the tissue. Here, we fabricate a three-dimensional, prevascularized engineered muscle containing human myoblasts, genetically modified endothelial cells secreting angiopoietin 1 (ANGPT1) and genetically modified smooth muscle cells secreting vascular endothelial growth factor (VEGF). The genetically engineered human muscle shows enhanced host neovascularization and myogenesis following transplantation into a mouse host, compared to the non-secreting control. The vascular, genetically modified cells have been cleared for clinical trials and can be used to construct autologous vascularized tissues. Therefore, the described genetically engineered vascularized muscle has the potential to be fully translated to the clinical setting to overcome autologous tissue shortage and to accelerate host neovascularization and integration of engineered grafts following transplantation.