Background Research on skeletal muscle injury repair represents a critical frontier in tissue regeneration. A deeper understanding of the cellular and molecular mechanisms involved in muscle repair is pivotal for developing effective strategies to address sports injuries, challenges associated with aging, and degenerative diseases.

Objective To evaluate the therapeutic potential of engineered macrophages that stably express IGF-1 while lacking transforming growth factor-β (TGF-β), a key driver of fibrosis, investigate their efficacy in promoting skeletal muscle repair and explore their potential for clinical application.

Methods The RAW264.7 mouse macrophage cell line was used. The TGF-β gene in macrophages was knocked out using CRISPR-Cas9 technology, and then lentiviral vectors were used to mediate the stable expression of insulin-like growth factor-1 (IGF-1). Thus, engineered macrophages (IGF1^secTGFβ^koM) that stably expressing IGF-1 but lacking TGF-β were constructed. The cells were identified using Western blotting and enzyme-linked immunosorbent assay (ELISA), and transcriptomic analysis was performed to depict the differential expression between them and control cells. A total of 120 C57BL/6 mice were randomly divided into normal control group (NC), PBS control group, wild-type macrophage group (M), TGF-β knockout macrophage group (M-TGF^ko), IGF1^secTGFβ^koM treatment group (IGF1M-TGF^ko), and recombinant IGF-1 protein group. At 7 d and 14 d after injury, parameters such as the standing time, swing time, footprint area, and intensity of the mice were measured using the CatWalk system. The mice were then sacrificed, and muscle tissues were collected for hematoxylin-eosin (HE) and Masson staining, followed by quantitative analysis. Real-time fluorescence quantitative PCR was used to verify the expression of IGF-1, Pax7, TNF, and IL-1β.

Results The IGF1^secTGFβ^koM cells were successfully constructed. Western blot showed that the expression of TGF-β protein was absent in TGFβ^koM cells, while the expression of IGF-1 in IGF1^secTGFβ^koM cells was significantly enhanced. ELISA results indicated that the secretion of IGF-1 in IGF1^secTGFβ^koM cells was significantly increased by 32-fold compared with that in the M group (P < 0.001). Transcriptomic analysis revealed that compared with the M group, the expression of IGF-1 was up-regulated and the expression of TGF-β was down-regulated in the IGF1^secTGFβ^koM group. The differentially expressed genes were enriched in pathways such as JAK-STAT, PPAR, and ECM-receptor interaction (P_adj < 0.05). qPCR results showed that compared with the PBS group, the expression of IGF-1 (P < 0.001) and Pax7 (P=0.038) in the IGF1M-TGF^ko group was increased at 7 d, and the expression of TNF (P=0.007) and IL-1β (P < 0.001) was transiently increased. At 14 d, the expression of IGF-1 (P < 0.001) and Pax7 (P < 0.028) continued to increase, and the levels of inflammatory factors returned to normal (P > 0.05). Gait analysis showed that compared with the PBS group, the support duration (0.189±0.025 s vs 0.154±0.040 s, P=0.043), swing duration (0.140±0.017 s vs 0.109±0.016 s, P=0.017), and footprint area (0.385±0.057 cm² vs 0.485±0.079 cm², P=0.009) of the IGF1M-TGF^ko group were significantly improved at 7 d. The functional recovery at 14 d was still better than that of the control group. Histological examination showed that the myofiber diameter (51.09±17.95 μm vs 39.39±17.65 μm, P=0.002) and cross-sectional area (1287.48±734.42 μm² vs 730.23±599.48 μm², P=0.040) of the IGF1M-TGF^ko group were significantly increased at 7 d. At 14 d, the myofiber diameter (57.47±13.86 μm vs 46.79±11.54 μm, P=0.002) and cross sectional area (1 698.86±458.11 μm² vs 999.24±150.81 μm², P=0.040) further increased, and the collagen fiber area (0.69%±0.56% vs 6.66%±2.78%, P=0.043) significantly decreased.

Conclusion The injection of engineered macrophages accelerates the functional recovery of skeletal muscles in mice and reduces the fibrosis of injured muscle tissues.