Abstract
BackgroundElectric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood.MethodsThe purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs) undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF) and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR) were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP) and collagen type 1 (col1), and stress response markers, such as heat shock protein 27 (hsp27) and heat shock protein 70 (hsp70). Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG) imaging.ResultsQuantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p < 0.05) in samples exposed to the electric field. A delayed but two fold increase in ALP and col1 transcript was detected by week 2 (p < 0.05) in differentiating hMSCs exposed to an electric field in comparison to the nonstimulated controls. Upregulation in stress marker, hsp27, and type 1 collagen deposition were correlated with this response. Increases in NADH, FAD, and lipofuscin were traced in the stimulation group during the first week of field exposure with differences statistically significant on day 10 (p < 0.05). Changes in hsp27 expression correlate well with changes in lipofuscin detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28.ConclusionsElectrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.
Highlights
Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing
Electric field effects on cell morphology On day 0 following initial stem cell seeding and attachment, two-photon excited fluorescence (TPEF) images were taken for baseline morphological comparisons (Figure 2a)
At the first two time points, stem cells exposed to an electric field appeared rounder than those in differentiation medium only and had an eccentricity value (Figure 2b) that was statistically lower than the control group (p < 0.05)
Summary
Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. After one year post-injury, 5-10% of bone fractures show impaired healing and require additional orthopedic intervention [1]. Treatments include load-bearing techniques that range from mechanical stress to ultrasound [4,5]. Musculoskeletal tissues, including bone and cartilage, respond to biophysical inputs such as electric and electromagnetic fields [6]. To apply biophysical stimulation for clinical use, non-invasive and implantable electromagnetic devices are used, such as bone growth stimulators [7,8]. Clinical devices employ pulsed electromagnetic fields (PEMF), or direct currents (DC) for healing nonunions and spinal fusions [11,12]. In one clinical study, 76% of recalcitrant non-unions were healed during treatment employing capacitively coupled (CC) alternating current electric fields [13]
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