Tensile force impairs lip muscle regeneration under the regulation of interleukin-10

Abstract

Background: Orbicularis oris muscle, the crucial muscle in speaking, facial expression and aesthetics, is considered the driving force for optimal lip repair. Impaired muscle regeneration remains the main culprit for unsatisfactory surgical outcomes. However, there is a lack of study on how different surgical manipulations affect lip muscle regeneration, limiting efforts to seek effective interventions.

Methods: In this study, we established a rat lip surgery model where the orbicularis oris muscle was injured by manipulations including dissection, transection and stretch. The effect of each technique on muscle regeneration was examined by histological analysis of myogenesis and fibrogenesis. The impact of tensile force was further investigated by the in vitro application of mechanical strain on cultured myoblasts. Transcriptome profiling of muscle satellite cells from different surgical groups was performed to figure out the key factors mediating muscle fibrosis, followed by therapeutic intervention to improve muscle regeneration after lip surgeries.

Results: Evaluation of lip muscle regeneration till 56 days after injury revealed that the stretch group resulted in the most severe muscle fibrosis (n = 6, fibrotic area 48.9% in the stretch group, P < 0.001, and 25.1% in the dissection group, P < 0.001). There was the lowest number of Pax7-positive nuclei at Days 3 and 7 in the stretch group (n = 6, P < 0.001, P < 0.001), indicating impaired satellite cell expansion. Myogenesis was impaired in both the transection and stretch groups, as evidenced by the delayed peak of centrally nucleated myofibers and embryonic MyHC. Meanwhile, the stretch group had the highest percentage of Pdgfra⁺ fibro-adipogenic progenitors infiltrated area at Days 3, 7 and 14 (n = 6, P = 0.003, P = 0.006, P = 0.037). Cultured rat lip muscle myoblasts exhibited impaired myotube formation and fusion capacity when exposed to a high magnitude (ε = 2688 μ strain) of mechanical strain (n = 3, P = 0.014, P = 0.023). RNA-seq analysis of satellite cells isolated from different surgical groups demonstrated that interleukin-10 was the key regulator in muscle fibrosis. Administration of recombinant human Wnt7a, which can inhibit the expression of interleukin-10 in cultured satellite cells (n = 3, P = 0.041), exerted an ameliorating effect on orbicularis oris muscle fibrosis after stretching injury in surgical lip repair.

Conclusions: Tensile force proved to be the most detrimental manoeuvre for post-operative lip muscle regeneration, despite its critical role in correcting lip and nose deformities. Adjunctive biotherapies to regulate the interleukin-10-mediated inflammatory process could facilitate lip muscle regeneration under conditions of high surgical tensile force.

Keywords: inflammation mediators; mesenchymal stromal cells; orofacial cleft; satellite cells, skeletal muscle; surgical procedures, operative.

Figure 1

The orbicularis oris (OO) muscle exhibited fibrotic alterations after surgical manipulation. (A) Schematic overview of the surgical injury and muscle harvest timeline. (B) Illustration of the dissection, transection and stretch procedures in the OO muscle. The black boxes indicated the surgical field. The black dotted line indicated the incision of the muscle bundle. The space between the two white arrows in the blue dotted line indicated the OO muscle defect after muscle bundle removal. The white arrows pointed to the residual ends of the OO muscle, where sutures were to be made. (C) Masson trichrome staining of the OO muscle at the centre of the injury harvested at 7, 14, 21 and 56 dpi. (D) Quantification of the fibrosis area. Dpi, days post‐injury. Scale bar, 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Comparison of muscle satellite cell (MuSC)‐mediated muscle regeneration in different surgical groups. (A) Immunofluorescent staining of 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue) and Pax7 (red) in different groups. (B) Immunofluorescent staining of DAPI (blue), Pax7 (red) and Ki67 (green) in different groups. (C) Immunofluorescent staining of DAPI (blue) and laminin (blue) in different groups. (D–F) Quantification of Pax7⁺ satellite cells, Pax7⁺Ki67⁺ proliferative satellite cells and centrally nucleated myofibers. Scale bars in (A) and (B), 20 μm; scale bar in (C), 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Temporal dynamics of myogenesis and fibrogenesis in different surgical groups. (A) Immunofluorescent staining of laminin (green), 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue) and embryonic MyHC (emb‐MyHC) (red) in blank, dissection, transection and stretch groups. Scale bar, 200 μm. (B) Immunofluorescent staining of Pdgfra (green), DAPI (blue) and laminin (red) in blank, dissection, transection and stretch groups. Scale bar, 20 μm. (C, D) Quantification of emb‐MyHC and Pdgfra in immunofluorescence pictures. (E, F) Quantification of relative expression levels of Pdgfra and Col1a1 in blank, dissection, transection and stretch groups. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

High mechanical strain impaired myogenesis in vitro. (A) Immunofluorescent staining of Ki67 (green) and DAPI (blue) in different groups with low, middle and high mechanical strain. (B) Immunofluorescent staining of MyHC (red) and DAPI (blue). (C) Quantification of Ki67 positive nuclei. (D) Quantification of MyHC positive myotubes and MyHC positive area. (E) Quantification of fusion index. (F) Quantification of relative expression level of MyoG. Scale bar, 50 µm.

Figure 5

Muscle satellite cell (MuSC) transcriptome profiling reveals IL10 as a key regulator. (A) Venn diagram of genes differentially regulated between the injured groups (dissection, transection and stretch groups) and the blank group. (B) Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment in the 1877 genes commonly expressed in the injured groups. (C) Protein–protein interaction of the top five pathways in KEGG enrichment and the expression level of IL10 in different groups. (D) Quantitative real‐time PCR of Il10 and analysis of protein levels of IL‐10 in orbicularis oris (OO) muscle tissue and MuSCs from different groups.

Figure 6

Injection of rh‐Wnt7a ameliorates stretch‐induced OO muscle fibrosis. (A) Comparison of Il10 expression in blank or stretch‐induced OO muscle myoblast with or without rh‐Wnt7a administration. (B) Stretch injury was performed in rat OO muscle, followed by intramuscular rh‐Wnt7a injection twice, immediately and 2 days after injury. OO muscles were harvested at day 3, day 7 and day 14 post‐injury. (C) Immunofluorescent staining of DAPI (blue), Pax7 (red) and laminin (green)/Ki67(green) in different groups. (D) Quantification of Pax7⁺ satellite cells and Pax7⁺Ki67⁺ proliferative satellite cells. (E) Quantitative analysis of IL‐10 protein in different groups. (F) Immunofluorescent staining of DAPI (blue), laminin (red) and Pdgfra (green) and histological staining of Picro Sirius Red in different groups in the left panel. Quantification of Pdgfra⁺ FAP cells and of fibrotic area in the right panel. Scale bars in (C), 20 µm; scale bars in (F), 50 µm.*P < 0.05; **P < 0.01; ***P < 0.001.