Abstract
Introduction
Migration of bone cells and precursor cells to the site of a bone defect can accelerate bone regeneration. Therefore, guidance of these cells by direct current (DC) is an interesting approach to improve implant ingrowth or fracture healing. To allow a better understanding of DC-induced directed migration, a specific stimulation chamber was established and the influence of DC on calcium channel expression in osteoblasts was investigated.
Methods
Human osteoblasts were isolated from femoral heads of patients undergoing total hip arthroplasty after patient”s consent. The study was approved by the local ethical committee (AZ: 2010–10). Differentiation into osteoblasts was ensured by cultivation in standard cell culture medium enriched with β-glycerophosphate, ascorbic acid and dexamethasone. 2×103 osteoblasts were seeded into custom-made chambers for DC field application. After 12 h DC was applied to chambers via Ag/AgCl electrodes set into separate reservoirs coupled to cell culture area by 2% agarose bridges in order to prevent cytotoxic impact of electrochemical reactions proceeding at the electrodes. Electric fields ranging from 150 to 450 V/m were applied to cells for 7 h. Several cell images were taken over time and used for evaluation of migration direction and speed with ImageJ software. Subsequently, cells were lysed in Trizol for RNA isolation and semiquantitative real-time polymerase chain reaction of voltage-gated calcium channels Cav1.4 and Cav3.2 as well as stretch-activated magnesium and calcium channel TRPM7 was performed.
Results
Migration velocity of DC stimulated bone cells was 6.4 ± 2.1 µm/h whereas unstimulated control cells migrated significantly slower with a velocity of 3.6 ± 1.1 µm/h (p<0.001). No correlation between magnitude of electric field and migration velocity was found. Migration of osteoblasts was directed towards the anode during DC application while unstimulated cells migrated undirectedly. Gene expression analysis showed significant correlation of electric field strength and TRPM7 expression (p<0.01) appearing in increased TRPM7 expression after exposure to higher electric fields. Voltage-gated calcium channels Cav1.4 and Cav3.2 were not regulated by DC fields.
Conclusion
A chamber for DC field application on human osteoblasts was established and migration velocity and direction was found to be influenced by DC fields. Regulation of selected calcium channels by DC was observed for stretch-activated channel TPRM7 that is known to be involved in osteoblast differentiation and migration induced by platelet-derived growth factor. Future studies will concentrate on investigation of involvement of specific calcium channels in osteoblast migration by using specific calcium channel inhibitors and calcium deprivation from cell culture medium.
