Extracorporeal shock waves enhance normal fibroblast proliferation in vitro and activate mRNA expression for TGF-beta1 and for collagen types I and III

Laura Berta, Annamaria Fazzari, Anna Maria Ficco, Patrizia Maurici Enrica, Maria Graziella Catalano, Roberto Frairia, Laura Berta, Annamaria Fazzari, Anna Maria Ficco, Patrizia Maurici Enrica, Maria Graziella Catalano, Roberto Frairia

Abstract

Background and purpose: Extracorporeal shock waves (ESWs) are used to good effect in the treatment of soft tissue injuries, but the underlying mechanisms are still unknown. We therefore determined the effects of ESWs on normal fibroblasts in vitro, in order to assess treatment-induced cell response.

Methods: A normal human fibroblast cell line (NHDF-12519) was treated with ESWs generated by a piezoelectric device (Piezoson 100; Richard Wolfe) using different protocols of impulses (300, 1,000, or 2,000 shots) and energy (0.11 or 0.22 mJ/mm(2)). Untreated controls and treated cells were cultivated for 12 days following a single shock-wave treatment. Viability, growth rate, and expression of mRNA for TGFbeta-1 and collagen types I and III were evaluated at days 3, 6, 9, and 12.

Results: 1 hour after shock-wave treatment, cell viability showed a decrease related mainly to impulse numbers applied. Fibroblasts treated with energy of 0.22 mJ/mm(2) subsequently showed an increase in proliferation from day 6 to day 9 that was higher than in untreated controls, without interference with the normal cell kinetic profile. mRNA expression was also higher in treated fibroblasts than in untreated controls for TGFbeta-1 on day 6 and day 9, for collagen type I on day 6, and for collagen type III on day 9.

Interpretation: These in vitro data confirm that the main factors involved in the repair process of connective tissues are activated by ESWs. The study gives the rationale for, and may provide schedules for, ESW treatment of tendonopathies.

Figures

Figure 1.
Figure 1.
Effect of ESW treatment on cell viability. Viability is expressed as ratio between cells treated with shock waves and untreated controls (n = 16). a p < 0.05; b p < 0.001 relative to untreated controls.
Figure 2.
Figure 2.
Effect of ESW treatment on cell growth. Cell growth was determined by the MTT method. Day 0 was seeding day (n = 16).
Figure 3.
Figure 3.
Effect of ESW treatment on TGF−β1 expression. Relative expression of TGF−β1 after treatment with ESW (EFD = 0.22 mJ/mm2, 1,000 impulses) (n = 4). Significance compared to no treatment: a p < 0.05.
Figure 4.
Figure 4.
Effect of ESW treatment on collagen type I expression. Relative expression of collagen type I after treatment with ESW (EFD = 0.22mJ/mm2, 1,000 impulses) (n = 4). Significance compared to no treatment: b p < 0.001.
Figure 5.
Figure 5.
Effect of ESW treatment on collagen type III expression. Relative expression of collagen type III after treatment with ESW (EFD = 0.22mJ/mm2, 1,000 impulses) (n = 4). Significance compared to no treatment: b p < 0.001.
Figure 6.
Figure 6.
Effect on collagen type I/collagen type III ratio after treatment with ESW (EFD = 0.22mJ/mm2, 1,000 impulses) (n = 4).

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