MODULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND PULSE WIDTH UTILIZATION

Abstract

Described is precise bioelectrical stimulation of a subject's cellular tissue with a selected bioelectric signal to modulate the expression of a peptide by the cellular tissue. More specifically, the application relates to a device that delivers a programmed bioelectric signal or signals, and associated methods for the controlled modulation of a peptide, such as vascular endothelial growth factor (VEGF) and/or hypoxia-inducible factor 1-alpha (HIF1α), or manipulation of stem cells, via precise bioelectrical signals and sequences useful in, for example, orthodontic and other procedures. The bioelectric signals have preferably been further optimized by selecting a desired pulse width for the peptide(s) to be expressed.

Claims

1. A bioelectric stimulator comprising an electric signal generator and electrodes, which electric signal generator is programmed to produce a bioelectric signal that modulates expression and/or release of vascular endothelial growth factor (“VEGF”) in a target cellular tissue, wherein the bioelectric signal has a pulse width of (a) from about 50 microseconds (“μsec”) to about 200 μsec or (b) from about 400 μsec to about 10,000 μsec.

2. The bioelectric stimulator of claim 1, wherein the bioelectric signal has a pulse width from about 50 μsec to about 200 μsec.

3. The bioelectric stimulator of claim 1, wherein the bioelectric signal has a pulse width from greater than 400 μsec to about 10,000 μsec.

4. The bioelectric stimulator of claim 1, wherein the bioelectric stimulator upregulates VEGF in the target cellular tissue and the bioelectric signal has a frequency selected from the group consisting of 25 Hz, 50 Hz, 75 Hz, 100 Hz, 500 Hz, 750 Hz, and 1,000 Hz.

5. The bioelectric stimulator of claim 1, wherein the bioelectric signal has a pulse width of about 100 μsec.

6. The bioelectric stimulator of claim 1, wherein the bioelectric signal has a pulse width of about 500 μsec.

7. The bioelectric stimulator of claim 1, wherein the bioelectric signal has a pulse width of about 10,000 μsec.

8. The bioelectric stimulator of claim 1, wherein the bioelectric stimulator downregulates VEGF in the target cellular tissue and the bioelectric signal has a frequency of about 250 Hz.

9. The bioelectric stimulator of claim 8, wherein the bioelectric signal has a pulse width of about 2,000 μsec.

10. The bioelectric stimulator of claim 1, wherein the bioelectric stimulator is programmed to produce a plurality of bioelectric signals.

11. A method of using the bioelectric stimulator of claim 1 to stimulate cellular tissue to modulate the expression and/or release of vascular endothelial growth factor (VEGF) in the cellular tissue, the method comprising: using the bioelectric stimulator to apply the programmed bioelectric signal(s) to the cellular tissue, so as to modulate the expression and/or release of VEGF by the cellular tissue.

12. The method according to claim 11, wherein the cellular tissue is selected from the group consisting of bone, dental arch, dental gum tissue, and any combination(s) thereof.

13. A method of treating a cell, the method comprising: stimulating the cell to express and/or release vascular endothelial growth factor (VEGF) by applying a bioelectric signal to the cell, wherein the bioelectric signal comprises, within 15%, a square, biphasic waveform at 50% duty cycle, at a frequency selected from the group consisting of 25 Hz, 50 Hz, 75 Hz, 100 Hz, 500 Hz, 750 Hz, and 1,000 Hz and 1.0 V as may be measured at the level of the cell.

14. The method according to claim 13, wherein the bioelectric signal has a pulse width selected from the group consisting of 100 μsec, 500 μsec, and 10,000 μsec.

15. The method according to claim 14, wherein the bioelectric signal has a pulse width of 100 μsec and a frequency of 1,000 Hz.

16. A method of modulating expression of a desired peptide by a target cellular tissue of the type comprising applying at least one bioelectric signal to the target cellular tissue for a sufficient time to modulate mRNA expression of the desired peptide, the method comprising: selecting a bioelectric signal with a frequency particular for modulating the desired peptide; determining a desired pulse width of the selected bioelectric signal for expression of the mRNA encoding the desired peptide; and applying the bioelectric signal to the target cellular tissue at a voltage/amperage sufficient to overcome resistance to safely reach the target cellular tissue so as to modulate expression of the desired peptide in the target cellular tissue.

17. The method according to claim 16, wherein the desired peptide is selected from the group consisting of Activin B, bone morphogenic protein 9 (BMP9), COL17A1, C-X-C motif chemokine 5 (CXCL5), epidermal growth factor (EGF), follistatin, hepatocyte growth factor (HGF), hypoxia-inducible factor 1-alpha (HIF-1α), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6), klotho, osteoprotegerin (OPG), platelet-derived growth factor (PDGF), receptor activator of nuclear factor kappa-B ligand (RANKL), stromal cell-derived factor 1 (SDF1), Sonic Hedgehog, tissue necrosis factor (TNF), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-β1), and any combination thereof.

18. The method according to claim 16, wherein the frequency is selected from the group consisting of 1 Hz, 5 Hz, 10 Hz, 15 Hz, 20 Hz, 22 Hz, 25 Hz, 30 Hz, 50 Hz, 75 Hz, 80 to 100 Hz, 100 Hz, 150 Hz, 250 Hz, 300 Hz, 500 Hz, 1,000 Hz, 2,000 Hz, 3 MHz, 2/100 Hz (frequency modulated biphasic signal), 50 Hz to 100 Hz (frequency modulated biphasic signal), and any combination thereof.

19. The method according to claim 16, wherein determining a desired pulse width of the selected bioelectric signal for expression of the mRNA encoding the desired peptide comprises measuring relative mRNA expression associated with the desired peptide utilizing different bioelectric signals having different pulse widths.

20. A method of treating a cell, the method comprising: stimulating the cell to express and/or release hypoxia-inducible factor 1-alpha (HIF1α) by applying a bioelectric signal to the cell, wherein the bioelectric signal comprises, within 15%, a square, biphasic waveform at 50% duty cycle, at a frequency selected from the group consisting of 25 Hz, 50 Hz, 75 Hz, and 100 Hz and 1.0 V as may be measured at the level of the cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is an exemplary electric signal generator.

[0045] FIG. 2 is an intraoral device having electrodes for applying bioelectric signals to the gums of a subject.

[0046] FIG. 3 is Table 1, a one-sample T-Test of pulse width (pec), frequency, and volts of mouse osteoblast stimulated at various conditions

[0047] FIG. 4 is a graph depicting mRNA expression of VEGF-A of osteoblast stimulated at 1V, 50 Hz, and 50-20,000 μsec.

[0048] FIG. 5 depicts a model bioelectric stimulation system.

[0049] FIG. 6 is a graph depicting HIF-1alpha and VEGF-A mRNA expression in porcine heart due to bioelectric stimulation (bioelectric signal application0.

DETAILED DESCRIPTION

[0050] Referring now to FIG. 1, depicted is a biostimulator for use in the treatment of a, for example, human subject. A micro voltage signal generator for use herein may be produced utilizing the same techniques to produce a standard heart pacemaker well known to a person of ordinary skill in the art. Programmable microvoltage generator are commercially available.

[0051] A micro voltage signal generator for use herein may be produced utilizing the same techniques to produce a standard heart pacemaker well known to a person of ordinary skill in the art. An exemplary microvoltage generator is available from Mettler Electronics Corp. of Anaheim, Calif., US or HTM Electrônica of Amparo, BR. The leading pacemaker manufacturers are Medtronic, Boston Scientific Guidant, Abbott St. Jude, BioTronik and Sorin Biomedica.

[0052] Construction of the electric signal generators and pacemakers, are known in the art and can be obtained from OEM suppliers as well as their accompanying chargers and programmers. The electric signal generators are programmed to produce specific signals to lead to specific protein expressions at precisely the right time for, e.g., optimal treatment or regeneration.

[0053] Electrodes may be used to deliver a bioelectric signal sequence to the subject's cellular tissue (e.g., skin, gums, or muscle).

[0054] In use, such electrodes are connected to the subject's body and placed for administration of the described bioelectric signal sequence.

[0055] The biostimulator of FIG. 1 may be used with, for example, multiple soft conductive electrode pads to apply the bioelectric signal to the target tissue. Electrodes may be used to deliver a bioelectric signal to the subject. In certain embodiments, other electrodes that may be used to deliver bioelectric signals are depicted in the incorporated US 20200330753 A1 to Leonhardt, Marchetto et al.

[0056] As depicted in FIGS. 1 and 2 and the incorporated US 20200330753 A1, a system useful in an orthodontic procedure comprises a bioelectric stimulator (FIG. 1) programmed to produce sequential electrical signals in electrical association with (e.g., via stem portion) a mouth piece comprised of a polymer and constructed to fit about and/or over the subject's teeth, braces or clear aligners and in proximity of the subject's gums.

[0057] The bioelectric signal generator (FIG. 1) is used to generate the specific bioelectric signal(s) typically transmitted via electrodes (e.g., contact pins/points) on the mouthpiece (FIG. 2) that cause the specific cells and proteins to be expressed by the cells associated with the gums and bone. The bioelectric stimulator is programmed with selected signals in a designed sequence to facilitate bone resorption/demineralization (softening). In the depicted embodiment, the bioelectric stimulator sends preprogrammed bioelectric signals via the intraoral electrode mouthpiece during an orthodontic procedure to the subject's gum and bone tissue.

[0058] The biostimulator is actuated and runs through programmed signals to modulate the production of a bioelectric signal or signals that can induce a subject to increase or decrease the expression of a peptide for delivery to the subject. For an orthodontic procedure, the peptides typically include RANKL, VEGF, and OPG.

[0059] A particularly preferred bioelectric signal for upregulating the expression and/or release of VEGF in mammalian cellular tissue has a frequency of 50 pulses per second at a pulse width of 500 μseconds in a continuous delivery mode.

[0060] In addition to the bioelectric signals identified herein for VEGF, the frequencies of the bioelectric signals typically associated with various peptides are: [0061] 1. Activin B (150 Hz), [0062] 2. bone morphogenic protein 9 (BMP9) (100 Hz or 300 Hz), [0063] 3. COL17A1 (25 Hz upregulate) (50 Hz downregulate), [0064] 4. C-X-C motif chemokine 5 (CXCL5) (1 Hz), [0065] 5. epidermal growth factor (EGF) (500 Hz), follistatin (50 Hz, then 100 Hz), [0066] 6. hepatocyte growth factor (HGF) (50 Hz), [0067] 7. hypoxia-inducible factor 1-alpha (HIF-1α) (80 to 100 Hz), [0068] 8. insulin-like growth factor 1 (IGF-1) (22 Hz), [0069] 9. interleukin-6 (IL-6) (50 Hz), [0070] 10. klotho (20 Hz), [0071] 11. osteoprotegerin (OPG) (3 MHz or 2,000 Hz), [0072] 12. platelet-derived growth factor (PDGF) (10 Hz or 100 Hz), [0073] 13. receptor activator of nuclear factor kappa-B ligand (RANKL) (2/100 Hz frequency modulated biphasic signal), [0074] 14. stromal cell-derived factor 1 (SDF1) (100 Hz), [0075] 15. Sonic Hedgehog (a biphasic pulse at from 50 Hz to 100 Hz; 5 Hz, 10 Hz, 20 Hz, 50 Hz, 100 Hz), [0076] 16. stem cell differentiation (20 Hz), [0077] 17. tissue necrosis factor (TNF) (2/100 Hz, alternating frequency, followed by 15 Hz, followed by 30 Hz), [0078] 18. tropoelastin (50 Hz), and [0079] 19. Transforming growth factor beta 1 (TGF-β1) (≥75 Hz).

[0080] The expression of transforming growth factor beta 1 (TGF-β1) is upregulated by a square, biphasic waveform at 50% duty, wherein the frequency is at least 75 Hz and the signal amplitude is, e.g., 1.0 V.

[0081] As used herein with respect to the frequency of a particular bioelectric signal, the term “about” means within 15% plus or minus.

[0082] As used herein with respect to the pulse width of a particular bioelectric signal, the term “about” means within 33% plus or minus.

[0083] Typical subjects to be treated are mammals such as humans.

[0084] In certain embodiments, the bioelectric stimulator is programmed to produce further bioelectric signals, such as those that induce expression by cellular tissue of osteoprotegerin or “OPG”, RANKL, SDF-1, PDGF, a signal for stem cell homing, PDGF, different signals for stem cell proliferation, activin-B, EGF, IGF-1, tropoelastin, VEGF, follistatin, HGF, TGF-β1, and any combination thereof.

[0085] For example in orthodontic procedures, upregulation of VEGF allows for an increase in the rate of tooth movement. Preferably, the process increases blood flow during active tooth movement of the teeth (e.g., RANKL is a key initiator), and the inorganic components of bone are able to be moved away from the compression side of the tooth, thus allowing the tooth to move. Conversely, when bone needs to be deposited, increased blood flow carries the inorganic components back into the area in need of stabilization.

[0086] The length of time that a bioelectric signal for VEGF is applied to the subject for a single treatment in a treatment regimen can vary dependent on, for example, the disease or condition being treated. For example, in assisting orthodontic movement of teeth, the length of time of application will typically be between 2 and 15 minutes (e.g., 5 minutes continuously) per application to the cellular tissue. For example, in treating stroke the length of time of application will typically be between 5 and 15 minutes (e.g., 10 minutes) per application. In treating erectile dysfunction, the length of time of application will typically be between 2 and 20 minutes (e.g., 5, 12, or 15 minutes) per application. In aiding hearing recovery, the length of time of application will typically be between 2 and 10 minutes (e.g., 5 minutes) per application. For pain reduction (e.g., in dental applications), the length of time of application will typically be between 2 and 10 minutes (e.g., 5 minutes) per application. In assisting hair growth, the length of time of application will typically be between 2 and 15 minutes (e.g., 5 minutes continuously) per application. In enhancing skin appearance, the length of time of application of the bioelectric signal to cellular tissue will typically be between 2 and 15 minutes (e.g., 5 minutes continuously) per application.

[0087] The invention is further described with the aid of the following illustrative Examples.

EXAMPLES

Example I—Effects of Pulse Width on MC3T3-E1 Cells on VEGF-A mRNA Expression

[0088] Purpose: The purpose of this study was to analyze the effects of pulse width of mouse osteoblast (MC3T3-E1) cells stimulated at 1V, 50 Hz, 50-20,000 μsec with RT-qPCR.

[0089] Methods: Mouse osteoblast cells were cultured and stimulated using pulse width ranging from 50 to 20,000 μsec at 1 V, 50 Hz for 30 minutes using a constant voltage waveform generator RIGOL. Gene expression was analyzed by extracting mRNA from osteoblasts and applying RT-qPCR assessment to quantify VEGF-A mRNA expression.

[0090] Electrical Signals: MC3T3-E1 cells were stimulated for 30 minutes at 1V, 50 Hz, 50-20,000 μsec with the RIGOL stimulator.

[0091] Results: Table 1 is depicted in FIG. 3, which is a one-sample T-Test on μsec, frequency, and volts of mouse osteoblast stimulated at various conditions. See, also, FIG. 4.

[0092] Conclusions: As described herein, pulse width plays a significant role on VEGF-A mRNA expression. mRNA expression was significantly, and substantially increased with pulse-width levels ranging from 50-500 μsec. At 10,000 μsec, expression increases were significant, albeit relatively small, while 2,000 μsec produced significant, but again small decreases in expression. Therefore, pulse width plays a major role in gene expression during bioelectric stimulation.

Example II—Modulation of VEGF-A and Hypoxia-Inducible Factor 1-Alpha (HIF1α)

[0093] Purpose: Investigated was a spectrum of noninvasive bioelectric stimulation (BES) parameters that would modulate HIF-1α and VEGF-A expression in porcine heart tissue.

[0094] Electrical Signals: The tissue was stimulated for 30 minutes with a square, biphasic waveform at 50% duty cycle. Frequency and voltages were fixed and set from 5 Hz to 2500 Hz (e.g., 5 Hz, 10 Hz, 25 Hz, 50 Hz, 75 Hz, 100 Hz, 250 Hz, 500 Hz, 750 Hz, 1,000 Hz, 2,500 Hz, and control) and 1.0 V.

[0095] Methods: Tissue received from the Midwest Swine Institute was kept in cold, ringer solution and viability of tissue was analyzed with a fluorometric detection method before performing this study. Bioelectric signals were applied to heart tissue in vitro using a commercially available Rigol DG1022Z function generator (Beijing, China), which was further programmed via a 6-well stimulating plate interface (IONOPTIX, Westwood, Mass., US). To induce uniform electric fields in all stimulation chambers, 1.3 mL of DMEM solution was added to each well before BES signal application.

[0096] FIG. 5 depicts a bioelectric stimulation system. Cells were placed in each dish and cultured to 80% to 100% confluency. Once confluent, cells were stimulated using an electrode array (shown at the top of panel A of the figure), which was inverted and introduced into the 6-well dish where cells were grown. Each well received uniform stimulation via a pair of carbon electrodes positioned at opposite sides (panel B of FIG. 5.)

[0097] Gene expression was determined by homogenizing the tissue, extracting mRNA according to the manufacturer's instructions. RNA quality was determined using a spectrophotometer and was reverse transcribed using a cDNA conversion kit. The cDNA and RT2 SYBR Green qPCR Master mix was used on a Custom RT2 Profiler Array.

[0098] The Qiagen data analysis software was used to calculate fold change/regulation using the delta-delta CT method, in which delta CT is calculated between the gene of interest (GOI) and an average of housekeeping genes (HKG), followed by delta-delta CT calculation (delta CT(experiment)-delta CT(control). Fold change is then calculated using the 2{circumflex over ( )}(-delta delta CT) formula.

[0099] Results: FIG. 6 shows mRNA fold change of HIF-1α and VEGF-A in response to specific stimulation signals. The mRNA expression for HIF-1α had a peak above the threshold at 50 Hz and VEGF-A at 1000 Hz. The expression of VEGF-A decreased to 0.888-fold at 250 Hz. The mRNA expression for HIF-1α had a peak above the threshold at 50 Hz and VEGF-A at 1,000 Hz.

[0100] The expression at several frequency conditions was less than 2-fold, which is below the threshold for gene expression. Further experiments would be useful to better optimize BES signals for HIF-1α and VEGF-A.

Example III—Further Downregulation of VEGF

[0101] A bioelectric signal (30 Hz, 3.5 mV) was tested in Retinal Pigment Epithelium (RPE) cells (ATCC), which cells were stimulated for 30 minutes. The expression of genes was assessed on a Qiagen qPCR array (QIAGEN Redwood City, Calif., US). It was found that the stimulation of RPE cells at 30 Hz and signal strength of 3.5 mV, resulted in a decrease in the gene for VEGF as well as for the following genes: stromal cell-derived factor (SDF-1), elastin (ELN), follistatin (FN1), hypoxia-inducible factor-1 alpha (HIF1A), insulin-like growth factor 1 (IGF1), matrix metallopeptidase 9 (MMP9), nitric oxide synthase 1 (NOS1), and transforming growth factor-beta 1 (TGF-B).

REFERENCES

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