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Regional Differentiation of Adipose-Derived Stem Cells Proves the Role of Constant Electric Potential in Enhancing Bone Healing

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Abstract

Mesenchymal stem cells (MSCs) have a great potential in the field of tissue engineering and regenerative medicine on account of their ability to self-renew and differentiate into various lineages. MSCs could be differentiated by a number of ways. Electric field is known to bring about differentiation, migration, proliferation, and reorientation of MSCs. Hence, we aim to create a bioreactor to attain osteodifferentiation of human-derived MSCs in the presence of osteoinduction medium (OIM) in combination with or without alternating current (AC) fields. A stimulation bioreactor was specially designed for the exposure of adipose-derived stem cells (ASCs) to an electric field of 20 mV/cm, 60 kHz. The electric field potential (E) within the chamber was simulated using COMSOL. The morphology, proliferation, and osteogenic differentiation of ASCs under the influence of electrical stimulation were studied. By week three, electrically stimulated ASCs exhibited their typical spindle-shaped morphology. Stimulated ASCs were more intensely stained with alkaline phosphatase and alizarin red, the markers of osteogenic differentiation, as compared to the unstimulated control groups. Darker stained regions correlated with the COMSOL simulation which showed constant electric potential at the same place. The results depicted a clear difference between the effect of constant and varying electric potential on osteodifferentiation of ASCs. Picogreen assay revealed lower DNA contents of electrically stimulated ASCs compared to the control group. In this study, we have additively enhanced the osteodifferentiation potential of ASCs by electrical stimulation and have proved that it is constant electric field potential which specifically augments osteogenic differentiation. We have successfully developed a bioreactor to improve the osteodifferentiation of ASCs by an electrical field, which could be applied in regenerative therapy strategies of bone fracture treatment.

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Acknowledgements

The authors would like to thank Dr. Renu John and Dr. Vimal Prabhu for help with the equipment required for stimulation studies.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, NH 65, Sangareddy, Telangana, 502285, India

    Sindhuja D. Eswaramoorthy, Sundeep Bethapudi, Shahdab I. Almelkar & Subha N. Rath

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  1. Sindhuja D. Eswaramoorthy
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  2. Sundeep Bethapudi
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  3. Shahdab I. Almelkar
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  4. Subha N. Rath
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Correspondence to Subha N. Rath.

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No potential conflict of interest was reported by the authors.

Ethics Approval

The lipoaspirate and umbilical cord were obtained from the patient after informed consent. It was carried out under the approval of Institutional Ethics Committee.

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40846_2018_373_MOESM1_ESM.tif

Supplementary Fig. S1: Characterization of ASCs a) After 3 weeks of osteoinduction, the cells are stained by alkaline phosphatase; b) after adipo-induction of 2 weeks, the cells are stained by oil red O; c) after chondrogenic induction for 3 weeks, cells at high density are stained with alcian blue in a 24 well plate d) FACS analysis of surface markers of ASCs. Supplementary material 1 (TIFF 3934 kb)

40846_2018_373_MOESM2_ESM.tif

Supplementary Fig. S2: Characterization of HUVECs. After ten days of culture in EGM-2, a) HUVECs forming tube-like structures on Matrigel and b) stained with Lectin. Supplementary material 2 (TIFF 1040 kb)

40846_2018_373_MOESM3_ESM.tif

Supplementary Fig. S3: HUVECs and hASCs under electrical stimulation on week 3: a) Group UC, b) Group UG, c) Group UE, d) Group AC, e) Group AG, f) Group AE. Scale bar in all images is 100 μm. Supplementary material 3 (TIFF 3138 kb)

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Eswaramoorthy, S.D., Bethapudi, S., Almelkar, S.I. et al. Regional Differentiation of Adipose-Derived Stem Cells Proves the Role of Constant Electric Potential in Enhancing Bone Healing. J. Med. Biol. Eng. 38, 804–815 (2018). https://doi.org/10.1007/s40846-018-0373-2

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