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

Vlado Antonic PhD,Christian Ottomann MD

doi:10.4172/2155-9538.1000171

Abstract

Background: The objective of the presented study is to investigate in vitro effects of low energy extracorporeal shockwave therapy (ESWT) on the keratinocytes and their metabolic and proliferative properties for their therapeutic applications. Materials: Primary culture of keratinocytes was isolated from clinical samples and after passaging into 12 flasks exposed to the various number of ESWT impulses (n=25, 50 and100impulses) in combination with several frequencies (1, 3 or 5Hz) and distances from the applicator head (5, 6 or 7cm with corresponding energy flux densities (EFD)=0.04, 0.025 and 0.015 mJ/mm2, respectively) using modified water bath at constant temperature 37°C. Flasks were assigned to receive one combination of treatment parameters. The individually tested distances/EFD with the various impulse numbers and frequencies were summarised to experimental series. Cell viability was measured using trypan blue, cell cytotoxicity was measured using lactate dehydrogenase assay, and cell metabolic activity was measured by level of glucose metabolism. Results: Our results indicate that low energy ESWT has both cytotoxic and stimulating effects on the keratinocytes. These effects depend on number of impulses, distance from the applicator head, and frequency. A proliferationstimulating effect and a higher viable cell count could be observed for a distance of 5 cm with 100 impulses at 1 Hz and EFD=0.04 mJ/mm2. Conclusion: Our results indicate that ESWT (EFD=0.04 mJ/mm2, 100 impulses, 1Hz at 5 cm) augmented proliferative capacity of keratinocytes in vitro. These promising results grant further investigation and have practical potential in keratinocyte research and production.

Highlights

Modern wound dressings engineered to promote wound healing often contain living cells [1,2]
Our results indicate that extracorporeal shockwave therapy (ESWT) (EFD=0.04 mJ/mm2, 100 impulses, 1Hz at 5 cm) augmented proliferative capacity of keratinocytes in vitro
Perforations in keratinocyte monolayer with hanging remnants of cell complexes as well as altered configurations of keratinocytes within the cell complexes could be observed after application of high number of impulses

Summary

Introduction

Modern wound dressings engineered to promote wound healing often contain living cells [1,2]. In the animal study directly comparing cryopreserved allogenic keratinocytes and cadaver full thickness skin grafts, authors reported faster epithelialisation in the keratinocytetreated group, with no difference in comparison with full thickness grafts by the end of the study [6,7] These reports provide a rationale for the further investigation of keratinocytes in wound healing, as well as need for development of cultivating protocols that will decrease culturing time. Materials: Primary culture of keratinocytes was isolated from clinical samples and after passaging into 12 flasks exposed to the various number of ESWT impulses (n=25, 50 and100impulses) in combination with several frequencies (1, 3 or 5Hz) and distances from the applicator head (5, 6 or 7cm with corresponding energy flux densities (EFD)=0.04, 0.025 and 0.015 mJ/mm, respectively) using modified water bath at constant temperature 37°C. Cell viability was measured using trypan blue, cell cytotoxicity was measured using lactate dehydrogenase assay, and cell metabolic activity was measured by level of glucose metabolism

Methods

Results

Discussion

Conclusion