Spatially patterned scaffolds enhances vascular organization and functional integration in volumetrc muscle loss

We report the application of bioengineered skeletal muscle composed of murine skeletal myoblasts interspersed with human vascular endothelial cells (ECs) using spatially patterned scaffolds for the treatment of volumetric muscle loss. To gain insights into the molecular mechanisms by which spatial patterning promoted myo-angiogenesis, we performed mRNA sequencing of engineered muscle and endothelialized engineered muscle on either randomly-oriented or aligned scaffolds. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from four groups: 1) randomly-oriented scaffold seeded with myoblasts; 2) randomly-oriented scaffolds seeded with myoblasts + ECs; 3) aligned scaffold seeded with myoblasts only; 4) aligned scaffold seeded with myoblasts + ECs. Samples were generated in triplicate and the NovaSeq 6000 (Illumina) sequencing platform was utilized. 150-bp paired-end reads were generated (20-30 million reads per sample). The raw data were checked for quality with FastQC (Version 0.11.7) and results were aggregated with MultiQC and were aligned to the mouse genome (GRCm38) using STAR (Version 2.5.3a) with ENCODE options for long RNA-seq pipeline. The alignment results were assessed using Samtools and aggregated with MultiQC (Version 1.5) and the differential gene expression analysis of the uniquely mapped reads/raw counts wwas performed using the DESeq2 package (Version 1.20.0). Each DE analysis was composed of a pairwise comparison between an experimental group and the control group. Differentially expressed genes were identified after false discovery rate (FDR = 0.05) correction. Examination of differences in transcriptome profiling between 4 different bioengineered skeletal muscle