Transcriptome profiling of a synergistic volumetric muscle loss repair strategy

Abstract

Volumetric muscle loss overwhelms skeletal muscle's ordinarily capable regenerative machinery, resulting in severe functional deficits that have defied clinical repair strategies. In this manuscript we pair the early in vivo functional response induced by differing volumetric muscle loss tissue engineering repair strategies that are broadly representative of those explored by the field (scaffold alone, cells alone, or scaffold + cells) to the transcriptomic response induced by each intervention. We demonstrate that an implant strategy comprising allogeneic decellularized skeletal muscle scaffolds seeded with autologous minced muscle cellular paste (scaffold + cells) mediates a pattern of increased expression for several genes known to play roles in axon guidance and peripheral neuroregeneration, as well as several other key genes related to inflammation, phagocytosis, and extracellular matrix regulation. The upregulation of several key genes in the presence of both implant components suggests a unique synergy between scaffolding and cells in the early period following intervention that is not seen when either scaffolds or cells are used in isolation; a finding that invites further exploration of the interactions that could have a positive impact on the treatment of volumetric muscle loss.

Keywords: Biomaterial implant; Muscle injury and repair; Pre-clinical animal model research; Transcriptomic profiling; Volumeric muscle loss.

Fig. 1

Creation of VML injuries and subsequent repair strategies (A) Rat tibialis anterior muscles were ablated with an 8 mm diameter biopsy punch to create a VML defect removing approximately 20% of muscle mass [57]. VML defects were immediately subjected to implant repair strategies (A-B) including minced muscle (MM), decellularized skeletal muscle (DSM), and a combination of the two (DSM + MM).

Fig. 2

Muscle histology and electrophysiology data (A) Gross appearance of tibialis anterior muscles for groups at fourteen DPI. TA muscle cross-sections were stained with Masson’s Trichrome. (B) Representative 2-week uninjured, VML, DSM, MM and DSM + MM repair groups are presented. Magnified (100X) images are shown. Inset indicates approximate location of magnified image within the TA cross-section. Scale bar = 100 μm. (C) Tibialis anterior mass (g/kg rat body weight) and electrophysiological measurement of mean peak contractile force for all groups (D) at fourteen DPI, indicating deficits in functional outcome among VML, MM, DSM, and DSM + MM-treated muscles relative to the uninjured contralateral limb at 14 DPI (p = 0.0001, < 0.0001, < 0.0001 and 0.0127, respectively). The *, **, ***, and **** indicate statistically significant difference of p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 when comparing each group to uninjured controls. Error bars are presented as ± standard deviation, with N = 4–5 animals per treatment group. Scale-bar = 1 cm

Fig. 3

Global transcription within the VML cell community diverges profoundly based on repair strategy (A) Heatmap of log₂FC for all 5174 differentially expressed genes (DEGs) across all treatment groups relative to uninjured muscle, with hierarchical clustering of gene expression data visualized using one minus Pearson correlation with average linkage. Visualization of the top 30 DEG intersections for upregulated (B) and downregulated (C) DEGs was performed using UpsetR.

Fig. 4

Top IPA Canonical Pathways at 3 and 14 days post injury Heatmaps of Z-score for the top 10 canonical pathways from the IPA knowledge base for treatment groups relative to uninjured muscle at (A) 3 days post injury and (B) 14 days post injury. Pathway analysis was not performed for the MM group at 14 days post injury as no genes within that group met DEG criteria

Fig. 5

Expression of key genes for relevant biological processes Heatmaps of gene expression (log₂FC) for all treatment groups relative to uninjured muscle for key (A) myogenesis, (B) angiogenesis, (C) fibrosis, (D) inflammation, (E) phagocytosis, and (F) IL-10 signaling related genes. Asterisks (*) indicate measurements meeting differential gene expression criteria; abs(log₂FC > 1.5) and p < 0.05 when comparing to uninjured controls

Fig. 6

Neuritogenesis transcription is significantly increased in DSM + MM repair Heatmaps of gene expression (log₂FC) for all treatment groups relative to uninjured muscle for key (A) peripheral neuritogenesis related genes. Asterisks (*) indicate measurements meeting differential gene expression criteria (abs(log₂FC) > 1.5 and p < 0.05) when comparing to uninjured controls. (B) Reln (p = 0.008) and (C) Robo1 (p = 0.02) expression correlated moderately with animal limb torque outcomes, indicated through linear regression with R² > 0.50 for each