Summary

Experimental in vivo study in twenty C57BL6 male mice. In this animal model, the local administration of ASCD after a muscle injury modulated the normal healing process by decreasing the expression of inflammatory, atrophic, and reparative extracellular matrix and myogenic markers.

Abstract

Background

Acellular Stem Cell Derivatives (ASCD), also known as secretomes, contain a wide range of growth factors demonstrated to be a potential treatment for muscle injuries. However, there is scarce evidence regarding their molecular mechanism. This study aims to evaluate the molecular effect of an ASCD local administration in the genetic expression of muscle healing markers. The hypothesis is that ASCD modulates the healing process by decreasing the gene expression of inflammatory, muscle atrophy, and reparative markers.

Methods

Experimental in vivo study in twenty C57BL6 male mice (age 3-4 months). A complete mid-section in the right quadriceps was done on all animals and then randomly assigned to two intervention groups: ASCD Group (n=10): ASCD (1mg) administered 14 days from the muscle injury, and Control Group (n=10): No intervention. All left quadriceps were left without injury nor intervention and were considered as a third study group: Healthy muscle group (n=20). Molecular evaluations were blinded and included mRNA expression (qRT-PCR) of the following markers: Inflammatory (TNF-a, FASL, ANX), muscle atrophy (ACT3, TGF-a, and MSTN), and reparative [extracellular matrix (MMP3 and Col6a5) and myogenic (IGFBP-2, CDKN2-a, CDK-1, APOE, KRT-8, KRT-18, FGF-1, FGF-6, Ki67, VEGF-a, VEGF-b, and Myog)]. Evaluations were performed at 21 and 28 days after the muscle section to assess the time frame progression of the molecular changes. Statistical analysis included the Kruskal-Wallis, ANOVA, and Bonferroni tests (p<0.05). The minimum necessary animals were considered for 80% of statistical power.

Results

Inflammatory: Control Group showed significantly more TNF-a and FASL than the ASCD group (p<0.05). The ASCD Group did not differ from the Healthy Group for any factor (p>0.05).
Muscle atrophy: ASCD Group showed significantly less TGF-a, ACT3, and MSTN expression than the Healthy Group (p<0.05).
Reparative (extracellular matrix): Control Group showed significantly more MMP3 and COL6a5 expression than the Healthy Group (p<0.05).
Reparative (myogenic): Control Group showed significantly more IGFBP, CDKN2a, APOE, KRT-8, KRT-18, Ki67, and Myog than the Healthy Group (p<0.05). The ASCD group showed significantly more IGFBP and KRT-8 expression than the Healthy Group only on day 28 (p=0.014).

Conclusion

In this animal model, the local administration of ASCD after a muscle injury modulated the normal healing process by decreasing the expression of inflammatory, atrophic, and reparative extracellular matrix and myogenic markers.