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. 2022 Apr 14;23(8):4331.
doi: 10.3390/ijms23084331.

Mechano Growth Factor Accelerates ACL Repair and Improves Cell Mobility of Mechanically Injured Human ACL Fibroblasts by Targeting Rac1-PAK1/2 and RhoA-ROCK1 Pathways

Yongqiang Sha  1   2 , Beibei Zhang  1 , Liping Chen  1 , Huhai Hong  1 , Qingjia Chi  2   3
Affiliations

Mechano Growth Factor Accelerates ACL Repair and Improves Cell Mobility of Mechanically Injured Human ACL Fibroblasts by Targeting Rac1-PAK1/2 and RhoA-ROCK1 Pathways

Yongqiang Sha et al. Int J Mol Sci. .

Abstract

Exceeded mechanical stress leads to a sublethal injury to anterior cruciate ligament (ACL) fibroblasts, and it will hinder cell mobility and ACL regeneration, and even induce osteoarthritis. The mechano growth factor (MGF) could be responsible for mechanical stress and weakening its negative effects on cell physiological behaviors. In this study, effects of MGF on cell mobility and relevant molecules expression in injured ACL fibroblasts were detected. After an injurious mechanical stretch, the analysis carried out, at 0 and 24 h, respectively, showed that the cell area, roundness, migration, and adhesion of ACL fibroblasts were reduced. MGF (10, 100 ng/mL) treatment could improve cell area, roundness and promote cell migration and adhesion capacity compared with the injured group without MGF. Further study indicated that cell mobility-relevant molecules (PAK1/2, Cdc42, Rac1, RhoA, and ROCK1) expression in ACL fibroblasts was down-regulated at 0 or 24 h after injurious stretch (except Rac1 and RhoA at 0 h). Similarly, MGF improved cell mobility-relevant molecule expression, especially the ROCK1 expression level in ACL fibroblasts at 0 or 24 h after injurious stretch. Protein expression of ROCK1 in injured ACL fibroblasts was also reduced and could be recovered by MGF treatment. In a rabbit partial ACL transection (ACLT) model, ACL exhibited poor regenerative capacity in collagen and extracellular matrix (ECM) synthesis after partial ACLT for 2 or 4 weeks, and MGF remarkably accelerated ACL regeneration and restored its mechanical loading capacity after partial ACLT for four weeks. Our findings suggest that MGF weakens the effects of pathological stress on cell mobility of ACL fibroblasts and accelerates ACL repair, and might be applied as a future treatment approach to ACL rupture in the clinic.

Keywords: anterior cruciate ligament; cell adhesion; cell migration; fracture strength; mechano growth factor.

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Conflict of interest statement

All authors have no conflict of interest to disclose.

Figures

Figure 1
Figure 1
Schematic presentation of the experimental procedure. (A) Schematic presentation of the mechanical stretch performed by six-well flexible silicone rubber BioFlexTM plates coated with collagen I. (B) Schematic diagram of equi-biaxial tension.
Figure 2
Figure 2
Mechano growth factor (MGF) improved cell morphology of human anterior cruciate ligament (ACL) fibroblasts withstanding injured stretch. Cell morphological changes in human ACL fibroblasts in non-stretch, 12% stretch, (10, 100 ng/mL) MGF + 12% stretch groups at (A) 0 or (B) 24 h after injurious stretch. The quantitative results of cell area detected at (C) 0 or (D) 24 h. The quantitative results of cell roundness detected at (E) 0 or (F) 24 h. Scar bar = 100 μm. Data are presented as mean ± SD. **, p < 0.01; ***, p < 0.001 compared to the control group (the 12% stretch without MGF group).
Figure 3
Figure 3
Mechano growth factor (MGF) promoted cell migration of human anterior cruciate ligament (ACL) fibroblasts withstanding injurious stretch. Effects of MGF on cell migration of human ACL fibroblasts were detected through (A) crystal violet and (B) DAPI staining at 0 or 24 h after injurious stretch. Quantitative results of migratory cells of human ACL fibroblasts at (C) 0 or (D) 24 h after injurious stretch. Scale bar = 200 μm. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the 12% stretch group without MGF.
Figure 4
Figure 4
Mechano growth factor (MGF) accelerated cell adhesion of human anterior cruciate ligament (ACL) fibroblasts withstanding injurious stretch. Cell adhesion of human ACL fibroblasts was detected using DAPI staining at (A) 0 or (B) 24 h after injurious stretch. Quantitative results of adherent cells of human ACL fibroblasts at (C) 0 or (D) 24 h after injurious stretch. Scale bar = 100 μm. Data are presented as mean ± SD. **, p < 0.01; ***, p < 0.001 compared to the 12% stretch without MGF group.
Figure 5
Figure 5
Mechano growth factor (MGF) regulated mRNA expression levels of cell motility-relevant genes in injured human anterior cruciate ligament (ACL) fibroblasts. Effects of MGF on mRNA expression of PAK1, PAK2, Cdc42, Rac1, RhoA, and ROCK1 in human ACL fibroblasts at (A) 0 or (B) 24 h after injurious stretch. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the 12% stretch group without MGF.
Figure 6
Figure 6
Mechano growth factor (MGF) increased ROCK1 protein expression level in human anterior cruciate ligament (ACL) fibroblasts withstanding injured stretch. (A,B) Western blotting analysis and (C,D) quantified ROCK1 protein expression levels in human ACL fibroblasts in non-stretch, 12% stretch, (10, 100 ng/mL) MGF + 12% stretch groups at 0 or 24 h after injurious stretch. (E) ROCK1 protein expression levels in human ACL fibroblasts in abovementioned four groups at 24 h after injurious stretch. Scale bar = 200 μm. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the 12% stretch group without MGF.
Figure 7
Figure 7
Mechano growth factor (MGF) accelerated ACL regeneration through up-regulating extracellular matrix synthesis after anterior cruciate ligament (ACL) transection surgery in vivo. (A) Macroscopically evaluation of ACL in the ACLT (the control group) and (0.1, 1 and 10 μg/mL) MGF + ACLT groups after ACL partial transection for 2 or 4 weeks. Red arrows: ACL transection sites. (B) Histological scores. (C) Hematoxylin-Eosin (HE) and (D) Masson staining of ACL tissue after ACL injured surgery for 2 or 4 weeks. H: host tissue; N: new-born tissue. Scale bar = 100 μm. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the ACLT group without MGF. #, p < 0.05; ##, p < 0.01 compared to the ACLT group with 0.1 μg/mL MGF.
Figure 8
Figure 8
Mechano growth factor (MGF) restored the mechanical capacity of anterior cruciate ligament (ACL) after partial transection surgery. (A) Stress-strain curve of ACL in the normal, ACLT and (0.1, 1 and 10 μg/mL) MGF + ACLT groups after ACL transection for 4 weeks. (B) Quantitative results of the largest fracture strength in abovementioned groups. Data are presented as mean ± SD. **, p < 0.01; ***, p < 0.001 compared to the ACLT group without MGF.
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