Extracorporeal Shockwaves (ESW) Promote Proliferation and Differentiation of Keratinocytes In vitro-Histology and Immunohistochemistry

Abstract

The objective of presented study was to evaluate in vitro effects of extracorporeal shockwave therapy (ESWT) on the keratinocyte morphology, cytoskeleton and mitotic activity. To determine in vitro effects of ESWT on keratinocytes, we applied 100 pulses with an energy flux density 0.1 mJ/mm 2 and a frequency of 1 Hz at a distance of 5 cm between therapy head and culture flask. The treatment parameters were determined in a pilot study. Haematoxylin and Eosin staining, as well as immunohistochemistry for Ki-67, CK5, CK14 and CK10 was performed on the cultured keratinocytes and results were compared between ESW treated group and Controls. No pathomorphological abnormalities or alterations in the cells monolayer could be observed. When assessing histological images of all HE-stained sections of ESW-treated and untreated keratinocytes (control group), no morphological differences could be seen between or within the groups. The proliferation marker Ki-67 was found at a frequency of 14% for ESW-treated and 10% for untreated keratinocytes. There were also figures of 95% (CK5 staining) and 90% (CK14 staining) for ESW-treated, as well as 85% (CK5 staining) and 85% (CK14 staining) for untreated keratinocytes. CK10-positive cells showed a frequency of 24% in the ESWtreated and 33% in the untreated cell cultures. Immunohistochemical observations indicated an increased proliferation behaviour following treatment with extracorporeal shockwaves in vitro and maturation of the exposed keratinocytes. Our results suggest an increase in proliferation activity of keratinocytes after in vitro treatment with ESWT. Further investigations of the effects of ESWT on the keratinocyte expression of the chemokines and cytokines are underway.

In Vitro Effects of Extracorporeal Shockwave Therapy (ESWT) on Proliferation and Metabolic Activity of Adult Human Keratinocytes

“…In a previously published study, we have used 100 pulses with an energy flux density 0.1 mJ/mm 2 and a frequency of 1 Hz at a distance of 5 cm and showed using Ki-67 staining significant induction of proliferation and using IHC, promotion of mature phenotype [28]. Other authors used range 100-400 impulses applied to prostate carcinoma cells [27], 100 impulses for nerve cells [32], 50-500 impulses for tenocytes [33] and 300 to 2000 impulses for fibroblasts [22].…”

“…Using a trypsin / EDTA solution (Biochrom AG), the keratinocytes were then separated from the other epidermal cells in the cell complex. Cultivation and passaging of keratinocytes was done according to published protocol [28]. When confluent monolayer was achieved ESWT were applied in predetermined treatment parameters or left untreated in control flasks.…”

“…For the starting EFD=0.1 mJ/mm 2 on the experimental position of the flasks at 50, 60 and 70 mm from the applicator, respective actual EFD of 0.04, 0.025 and 0.015 mJ/mm 2 were determined. All the ESWT exposure was conducted in the modified heated water bath at constant temperature of 37 ° C filled with 2.500ml preheated water [28,29].…”

“…For the starting EFD=0.1 mJ/mm 2 on the experimental position of the flasks at 50, 60 and 70 mm from the applicator, respective actual EFD of 0.04, 0.025 and 0.015 mJ/mm 2 were determined. All the ESWT exposure was conducted in the modified heated water bath at constant temperature of 37 ° C filled with 2.500ml preheated water [28,29].Experimental groups were generated by combining predetermined experimental parameters: 1)number of shockwave impulses (n= 25, 50, 100, 300 and 600), 2) frequency (1, 3, 5 Hz) and 3) distance from the cell culture flasks to the applicator (5, 6, 7 cm). After shockwave treatment, the excess medium in the cell culture flasks was discarded and the keratinocytes were then cultured further under standard conditions.…”

“…We used secondpassage subcultured keratinocytes, which are comparable to primary culture [30], and did not change due to high number of passages [31] and we also avoided immortalized keratinocyte cell lines as they were unsuitable for the question we were addressing. We were able to show that ESWT has significant dose-and frequency-dependent effects on the keratinocytes, ranging from cytotoxic to proliferation-stimulating effects.In a previously published study, we have used 100 pulses with an energy flux density 0.1 mJ/mm 2 and a frequency of 1 Hz at a distance of 5 cm and showed using Ki-67 staining significant induction of proliferation and using IHC, promotion of mature phenotype [28]. Other authors used range 100-400 impulses applied to prostate carcinoma cells [27], 100 impulses for nerve cells [32], 50-500 impulses …”