Volumetric muscle loss (VML) injury is the traumatic loss of skeletal muscle fibers, along with associated vascular and neural structures, resulting in long‐term functional disability. To date, several strategies have been developed to target skeletal muscle and vascular regeneration and recovery of muscle function, with only limited improvements reported. Our objective, using a standardized unilateral VML injury to the rat tibialis anterior (TA) muscle, was two‐fold: (1) examine the rate of cell population and distribution of cell phenotypes in a bioengineered aligned collagen scaffold; and (2) evaluate the in vivo transfection efficiency of aligned collagen scaffolds loaded with firefly luciferase (Fluc) using bioluminescence imaging. We hypothesize that a modified mRNA (mmRNA)‐incorporated aligned collagen scaffold could be an ideal platform to stimulate vascular and muscle fiber regeneration following VML injury. In total, 25 adult male Lewis rats were used across studies. In Study 1, following unilateral VML injury to the TA muscle (~82mg removed), rats received implantation of an aligned collagen scaffold (6mm in diameter, 0.5–3.0mm thickness), consisting of two interconnected bundles of BioBridge threads that were cross‐linked, lyophilized, and sterilized. The TA muscle was evaluated histologically at 1, 2, and 4 weeks post‐VML to examine cell population and distribution, fibrotic content, and extent of scaffold degradation within the remaining TA muscle and VML defect area. In Study 2, in vivo bioluminescence imaging quantified localized RNA‐transfected cell expression in rats up to 2 weeks post‐Fluc injection within the TA muscle, with or without VML injury, or implantation of an aligned collagen scaffold loaded with Fluc‐RNA following VML injury to evaluate transfection efficiency for potential mmRNA cargo. Scaffolds exhibited minimal degradation through 4 weeks following implantation. Quantification of cellular distributions within the VML defect area indicated significant macrophage infiltration and inflammation up to 4 weeks post‐VML. Intramuscular Fluc injections indicated peak bioluminescence signal (3.0x105 p/s/cm2/sr) occurs at 7 days post‐injection. These results serve as a reference for our ongoing development of a mmRNA‐loaded scaffold delivery system. Our long‐term goal is to evaluate candidate growth factors to co‐deliver in the mmRNA‐loaded aligned collagen scaffolds to facilitate organized, functional skeletal muscle and vascular regeneration following VML injury.Support or Funding InformationThis work is supported by: US Army Medical Research and Materiel Command under Contract Number W81XWH19C0021; T32AR050938; P217A170051.