The Journal of Bone and Joint Surgery (American). 2006;88:1053-1065.
doi:10.2106/JBJS.E.00443
© 2006 The Journal of Bone and Joint Surgery, Inc.
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Up-Regulation of Bone Morphogenetic Proteins in Cultured Murine Bone Cells with Use of Specific Electric Fields

Zhenyu Wang, MD, PhD1, Charles C. Clark, PhD1 and Carl T. Brighton, MD, PhD1

1 Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, 424 Stemmler Hall, Philadelphia, PA 19104-6081. E-mail address for C.T. Brighton: ctb{at}mail.med.upenn.edu

Investigation performed at the McKay Laboratory of Orthopaedic Surgery Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

NOTE: The authors thank Dr. Paul Billings for his help with the BMP-2 enzyme-linked immunosorbent assay.

The authors did not receive grants or outside funding in support of their research for or preparation of this manuscript. One or more of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (royalties from Biolectron, Inc., to perform the research). No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Background: Capacitively coupled electric stimulation has been successfully used in the treatment of bone nonunions and to effect spinal fusions. However, the pathway of biologic events whereby this is accomplished has not been fully elucidated. To determine whether bone morphogenetic proteins (BMPs) could be involved, the effect of electrical stimulation on BMP gene expression was investigated.

Methods: Postconfluent cultures of MC3T3-E1 bone cells were exposed to a series of capacitively coupled signals in which the duration, amplitude, frequency, and duty cycle were sequentially and systematically varied. The cellular response was measured by quantifying the mRNA levels of BMP-2 through BMP-8, as well as the BMP antagonists gremlin and noggin, with use of reverse transcription followed by real-time quantitative polymerase chain reaction. BMP-2 protein was measured by enzyme-linked immunosorbent assay, and alkaline phosphatase activity was measured by a specific colorimetric assay.

Results: The results showed that BMP-2 through BMP-8, gremlin, and noggin were all normally expressed by MC3T3-E1 cells, and could be significantly up-regulated by specific and selective capacitively coupled electric fields (p < 0.05). However, mRNA expression for BMP-2, 4, 5, 6, and 7 was consistently up-regulated several times higher than that for BMP-3 and BMP-8, gremlin, and noggin under identical conditions. Concomitantly, BMP-2 protein production and alkaline phosphatase activity were both significantly increased in the same electrically stimulated cultures (p = 0.001 and p < 0.01, respectively).

Conclusions: These data clearly show that our optimal capacitively coupled signal (60 kHz, 20 mV/cm at a 50% duty cycle for twenty-four hours) can specifically, selectively, and simultaneously up-regulate the expression of a number of osteoinductive BMPs; other BMPs and antagonists are only moderately affected.

Clinical Relevance: Electrical stimulation may be a useful treatment modality for in vivo situations where bone induction is required, since it is noninvasive, safe, effective, can be easily targeted to a variety of anatomic sites, can provide a controlled production of BMPs, and can be used repeatedly. The optimal duration (continuous stimulation at 100% duty cycle) and frequency (60 kHz) determined in this in vitro study are the same—and the amplitude (20 mV/cm) is in the same range (12 mV/cm)—as are used clinically.


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