Time‐dependent effect of electrical stimulation on osteogenic differentiation of bone mesenchymal stromal cells cultured on conductive nanofibers

Abstract

Bone tissue engineering using bone mesenchymal stromal cells (BMSCs) is a multidisciplinary strategy that requires biodegradable scaffold, cell, various promoting cues to work simultaneously. Electrical stimulation (ES) is known able to promote osteogenic differentiation of BMSCs, but it is interesting to know how can it play the strongest promotion effect. To strengthen local ES on BMSCs, parallel-aligned conductive nanofibers were electrospun from the mixtures of poly(L-lactide) (PLLA) and multi-walled carbon nanotubes (MWCNTs), and used for cell culture. Osteogenic differentiation of BMSCs was conducted by applying ES (direct current, 1.5 V, 1.5 h/day) perpendicular to the fiber direction during the day 1-7, day 8-14, or day 15-21 period of the osteoinductive culture. In comparison with ES-free groups, bone-related markers and genes were found significantly up-regulated when ES was applied on BMSCs growing on nanofibers having higher conductivity. When the ES was applied at the earlier stage of osteoinductive culture, the promotion effect on osteogenic differentiation would be stronger. In the presence of a BMP blocker, the down-regulated expressions of bone-related genes were able to be slightly recovered by ES, especially when the ES was applied at the beginning of osteoinductive culture (i.e. day 1-7). The promotion effect generated by ES in the early stage was found sustainable to later stages of differentiation, while those ES applied at later stages of differentiation should have missed the optimal point. In other words, later ES was not so necessary in inducing the osteogenic differentiation of BMSCs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3369-3383, 2017.

Roles of electrical stimulation in promoting osteogenic differentiation of BMSCs on conductive fibers

“…And the residual BSA concentration in solution was tested with BCA protein assay kit (Thermo, USA). To perform protein adsorption under ES, a homebuilt electric field was set up as reported in our previous study …”

“…The cell lysis contained 1% Triton X‐100, 20 mmol/L Tris (pH 7.5), and 150 m M NaCl. Referring to our previous study, subsequently, ALP activity was determined using the p ‐nitrophenyl phosphate ( p ‐NPP) method with an ALP assay kit (Sigma), COL‐I synthesis was measured using a COL‐I ELISA kit (R&D, USA), and calcium deposition was determined by the o ‐cresolphthalein complexon (OCPC, Sigma) method. Manufacturers’ protocols were abode by in carrying out these assays.…”

“…Some researches point out that the conductive substrates can mimic the cells’ niche in an appropriate manner and translate signals between cells through electricity . In order to supply a favor electric field for cells, conductive scaffolds emerge in various forms like three‐dimensional hydrogel, two‐dimensional fibers or sometimes particles …”

“…The mechanism by which ES promotes cell activities has been widely studied. For bone regeneration, the enhanced osteogenic differentiation of multipotential mesenchymal stromal cells (MSCs) under ES draws concerns . One of the most accepted mechanisms is that ES can increase the influx of Ca 2+ into cells via voltage‐gated Ca 2+ channel, and subsequently the elevated cytosolic Ca 2+ concentration can enhance cellular osteogenic differentiation via activating Ca 2+ ion signaling pathway .…”

“…[16][17][18] In order to supply a favor electric field for cells, conductive scaffolds emerge in various forms like threedimensional hydrogel, two-dimensional fibers or sometimes particles. [19][20][21] The mechanism by which ES promotes cell activities has been widely studied. For bone regeneration, the enhanced osteogenic differentiation of multipotential mesenchymal stromal cells (MSCs) under ES draws concerns.…”