DEVICE AND METHOD FOR BIOELECTRIC STIMULATION ACCELERATING BONE INTEGRATION INTO IMPLANT HEALING

Abstract

Described are a device, system, and method for bioelectric stimulation to accelerate bone integration of an implant and/or bone graft in order to aid healing. Specifically described are devices and methods for enhancing bone integration of a body implant (e.g., a dental implant) or bone graft by modulating (e.g., stimulating) the controlled expression and/or release of selected proteins, which proteins promote osteogenesis and osseointegration via specific bioelectric signals delivered via, e.g., a specialized mouthpiece, electrodes, and/or wireless means. The description particularly relates to the acceleration of the healing of dental implants and/or bone grafts via promoting bone osteointegration more quickly with bioelectric stimulation.

Claims

1. A method of accelerating the healing of an implant in a subject, the method comprising: applying to the implant a bioelectric signal selected from the group consisting of a biphasic pulse of between 2,500 Hz and 750,000 Hz and a biphasic pulse of about 3,000,000 Hz, with a voltage of between 0.001 volts and 4 volts, so as to promote bone osteointegration of the implant.

2. The method according to claim 1, further comprising: stimulating the implant by applying a bioelectric signal to the implant to upregulate expression of bone morphogenic protein 9 (BMP9), wherein the bioelectric signal is a biphasic pulse of between 100 Hz and 300 Hz with a voltage of between 0.1 volts and 4 volts as measured at the cell.

3. A method of accelerating the healing of an implant in a subject, the method comprising: applying to the implant a bioelectric signal selected from the group consisting of a biphasic pulse of between 2,000 Hz and 750,000 Hz and a biphasic pulse of about 3,000,000 Hz, with a voltage of between 0.001 volts and 4 volts, and stimulating the implant by applying a bioelectric signal to the implant to upregulate expression of bone morphogenic protein 9 (BMP9), wherein the bioelectric signal is a biphasic pulse of between 100 Hz and 300 Hz with a voltage of between 0.1 volts and 4 volts as measured at the cellular level, so as to promote bone osteointegration of the implant.

4. A method of treating a cell, the method comprising: stimulating the cell to modulate expression of hypoxia-inducible factor 1-alpha (“HIF1α”) by the cell by applying a bioelectric signal to the cell, wherein the bioelectric signal is selected from the group consisting of 0.25 mA to 0.75 mA (3.0 V), 80 to 100 Hz, 80 to 110 μs pulse width, square wave and 30 Hz, 3.5 mV, with the voltage measured at the cell.

5. The method according to claim 4, wherein the bioelectric signal is 0.25 mA to 0.75 mA (3.0V), 80 to 100 Hz, 80 to 110 μs pulse width, square wave, and HIF1α expression is upregulated.

6. The method according to claim 4, wherein the bioelectric signal is 30 Hz, 3.5 mV, and HIF1α expression is downregulated.

7. A method of accelerating the healing of a bone graft in a subject, the method comprising: applying to the bone graft and cellular tissue surrounding the bone graft a bioelectric signal selected from the group consisting of a biphasic pulse of between 2,000 Hz and 750,000 Hz and a biphasic pulse of about 3,000,000 Hz, with a voltage of between 0.001 volts and 4 volts, and stimulating the implant by applying a bioelectric signal to the implant to upregulate expression of bone morphogenic protein 9 (BMP9), wherein the bioelectric signal is a biphasic pulse of between 100 Hz and 300 Hz with a voltage of between 0.1 volts and 4 volts as measured at the cellular level.

8. A method of treating a cell, the method comprising: stimulating the cell to upregulate expression of osteoprotegerin (OPG) by the cell by applying a bioelectric signal to the cell, wherein the bioelectric signal is selected from the group consisting of a biphasic pulse of between 2,500 Hz and 750,000 Hz and a biphasic pulse of about 3,000,000 Hz, with a voltage of between 0.001 volts and 4 volts as measured at the cell.

9. The method according to claim 8, wherein the bioelectric signal is a biphasic pulse of between 10,000 Hz and 500,000 Hz with a voltage of between 0.1 volts and 2 volts as measured at the cell.

10. The method according to claim 8, further comprising: stimulating the cell to upregulate expression of bone morphogenic protein 9 (BMP9) by applying a bioelectric signal to the cell, wherein the bioelectric signal is a biphasic pulse of between 100 Hz and 300 Hz with a voltage of between 0.1 volts and 4 volts as measured at the cell.

11. The method according to claim 8, wherein the cell is comprised within a subject.

12. The method according to claim 11, wherein the subject is undergoing a bone graft.

13. The method according to claim 8, wherein the stimulation causes OPG release to enhance osteoblast formation and bone formation/re-mineralization to increase stability of a dental implant.

14. The method according to claim 8, further comprising: Applying a bioelectric signal of a continuous current of 10 μA (as measured at the cellular level), for 5 minutes, where the continuous current has a biphasic waveform, with a frequency of 50 Hz.

15. A method of treating a cell, the method comprising: stimulating the cell to downregulate expression of osteoprotegerin by the cell by applying a bioelectric signal to the cell, wherein the bioelectric signal is selected from the group consisting of a biphasic pulse of between 100 Hz and 1,000 Hz and a biphasic pulse of about 1,000,000 Hz, with a voltage of between 0.001 volts and 4 volts as measured at the cell.

16. The method according to claim 15, wherein the bioelectric signal is a biphasic pulse of between 100 Hz and 500 Hz with a voltage of between 0.1 volts and 2 volts as measured at the cell.

17. The method according to claim 15, wherein the cell is comprised within a subject.

18. A method of treating a cell, the method comprising: stimulating the cell to upregulate expression of transforming growth factor beta 1 (TGF-β1) by the cell by applying a bioelectric signal to the cell, wherein the bioelectric signal is a square, biphasic waveform at 50% duty, wherein the frequency is at least 75 Hz and the signal amplitude is about 1.0 V as measured at the cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 depicts a bioelectric stimulator.

[0025] FIG. 2 depicts a mouthpiece, which connects to a bioelectric stimulator, having electrodes for applying bioelectric signals to the patient's gums and treated area.

[0026] FIG. 3 depicts a bioelectric stimulator and associated mouthpiece and electrodes for applying bioelectric signals to the patient's oral cavity.

[0027] FIG. 4 depicts a dental implant.

[0028] FIG. 5 is a graph depicting cumulative results of OPG expression for specific bioelectric signal application protocols as applied to osteoblasts.

DETAILED DESCRIPTION

[0029] 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. An exemplary microvoltage generator is available from Mettler Electronics Corp. of Anaheim, California, US or HTM Electrônica of Amparo, BR. The leading pacemaker manufacturers are Medtronic, Boston Scientific Guidant, Abbott St. Jude, BioTronik and Sorin Biomedica.

[0030] 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 bioelectric signals to lead to specific protein expressions at precisely the right time for, e.g., optimal treatment or for tissue regeneration.

[0031] Described is a system for selectively administering electrical stimuli to a patient undergoing an implant and/or bone graft procedure, the system comprising: a programmable source of variable frequency, variable amplitude electrical signals, wherein the frequency is capable of being varied within a range of approximately 0.1 to 970 Hertz and the amplitude is capable of being varied within a range of approximately 20 to 400 microamps; and a plurality of electrodes to connect the source to a patient, wherein the variable frequency, variable amplitude electrical signals are applied as electrical stimuli to a region proximate to, e.g., the mouth of the patient to perform a predetermined protocol.

[0032] In certain embodiments, the biostimulator is electrically connected via wires to, for example, electrodes, which electrodes contact or are positioned for contact to the implant or to the subject's cellular tissue for application of at least one bioelectric signal. In certain embodiments, the electrodes are appropriately placed in a mouthpiece (e.g., FIG. 2) and connected to the biostimulator as shown in FIG. 3. The mouthpiece is typically made of a biocompatible flexible material and contains the leads connecting the electrodes with the biostimulator. The mouthpiece is sized to fit as comfortably as possible in the patient's mouth.

[0033] The bioelectric stimulator is programmed to produce particular bioelectric signals, such as those disclosed in U.S. Pat. No. 10,960,206 to Leonhardt et al. for “Bioelectric Stimulator” (Mar. 20, 2021), the contents of the entirety of which are incorporated herein by this reference. Described therein are bioelectric signals to 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, and any combination thereof.

[0034] The application of the bioelectric signals aids in the rapid accelerated healing in implant dentistry. The described combinations of bioelectric and biologics therapy keys on highly precise controlled protein expressions, such as OPG, SDF1, PDGF, VEGF, and IGF1, providing a treatment that activates, amplifies, extends, enhances and accelerates the natural healing process.

[0035] Particularly preferred proteins for use herein are OPG, Klotho, RANKL, IGF-1, a combination of bioelectrics and biologics for accelerated healing, and PRF.

[0036] Controlled release of OPG promotes osseointegration (bone in growth). SDF1 and PDGF promote stem cell homing and healing. The system promotes rapid regeneration of tissues.

[0037] For example, as described in the incorporated U.S. Pat. No. 10,960,206 to Leonhardt et al., the cellular expression of stromal cell-derived factor 1 (“SDF1,” also known as CXCL12) (a stem cell recruiting signal) is upregulated by the application of the following bioelectric signal to a subject's cellular tissue: 30 Hz with a voltage of 3.5 mV, and successively alternating currents of 700 to 1500 picoamps for one minute, and again with 700 to 1500 picoamps for one minute and stimulated with current of 0.25 mA, pulse duration of 40 Hz, pulse width of 100 μs, and frequency of 100 Hz.

[0038] As also described in the incorporated U.S. Pat. No. 10,960,206, the cellular expression of platelet-derived growth factor (“PDGF”) is upregulated by the application of the following bioelectric signal to a subject's cellular tissue: 3 V/cm, 10 Hz, 2 μA (0.000002 amps), and pulse duration of 0.2 ms. Another bioelectric signal that upregulates expression of PDGF is 20 V/cm, 100 Hz, 0.25 mA (2.5e-7 amps), and pulse duration of 40 Hz, width of 100 μs. A third such bioelectric signal described in U.S. Pat. No. 10,960,206 is 20 V for 1 minute, 20 mV for 10 minutes, current of 0.25 mA, pulse duration of 40 Hz, pulse width of 100 μs, and frequency of 100 Hz for 5 minutes followed by 528 Hz for 3 minutes and 432 Hz for 3 minutes and 50 Hz for 3 minutes.

[0039] As also described in the incorporated U.S. Pat. No. 10,960,206, the cellular expression of vascular endothelial growth factor (VEGF) is upregulated by the application of the following bioelectric signal to a subject's cellular tissue: 3 V/cm, 10 Hz, 2 μA (0.000002 amps), and pulse duration of 0.2 ms. As described in U.S. Patent Application Ser. No. 63/215,841 (filed Jun. 28, 2021) to Leonhardt et al. for “Modulation of Vascular Endothelial Growth Factor (VEGF) and Pulse Width Utilization,” other bioelectric signals upregulate the expression of VEGF.

[0040] As further described in the incorporated U.S. Pat. No. 10,960,206, the cellular expression of insulin-like growth factor 1 (“IGF-1”) is upregulated by the application of the following bioelectric signal to a subject's cellular tissue: within 15%, 3 mV with a frequency of about 22 Hz, and a current of about 1 mA, followed by 3 mA.

[0041] As further described in the incorporated US Publication No. 2020/033079 A1, the cellular expression of osteoprotegerin (“OPG”) may be upregulated by the application of the following bioelectric signal to a subject's cellular tissue: range 3 mV to 5 mV at frequency range 1 to 3 MHz duration range 30 to 40 mW/cm.sup.2 for a minimum of 20 to 45 minutes.

[0042] The bioelectric stimulator may be programmed to produce a bioelectric signal of, within 15%, a biphasic current of frequency 20 Hz and a 7.8 ms pulse duration and/or produce at least one bioelectric signal having a frequency selected from the group consisting of 5 Hz, 10 Hz, 20 Hz, 25 Hz, 50 Hz, 75 Hz, 100 Hz, 250 Hz, 500 Hz, 750 Hz, 2,500 Hz, 100,000 Hz, 500,000 Hz, and 1 MHz. As described in US Patent Application Publication US 2020-0289826-A1 to Leonhardt et al. (Sep. 17, 2020) for “Klotho Modulation,” and U.S. patent application Ser. No. 17/473,809 to Leonhardt, filed Sep. 13, 2021, application of such bioelectric signals to a subject's tissue upregulates expression of Klotho.

[0043] For pain relief and inflammation control, the bioelectric stimulator may be further programmed to produce a bioelectric signal of a continuous current of 10 μA (as measured at the cellular level), for 5 minutes, where the continuous current has a biphasic waveform, with a frequency of 50 Hz, which downregulates the expression of interleukin-10 (IL-10).

[0044] Hypoxia-inducible factor 1-alpha (“HIF1α”) expression can be upregulated by the application of the following bioelectric signal to the subject's tissue: 0.25 mA to 0.75 mA (3.0V), 80 to 100 Hz, 80 to 110 μs pulse width, square wave (voltage/amperage being measured at the level of the cell being stimulated). Upregulation of expression of 285% has been observed.

[0045] HIF1α can be downregulated by the application of the following bioelectric signal to the subject's tissue: 30 Hz, 3.5 mV in Retinal Pigment Epithelium (RPE—purchased from ATCC) cells for 30 minutes.

[0046] The pharmacologic activation of the HIF-1 complex can be desirable in ischemic and inflammatory disorders. In contrast, HIF-1 blockade may be beneficial to prevent tumor angiogenesis and tumor growth. See, e.g., Wig-Bill-gel “Hypoxia-Inducible Factor-1 (HIF-1): A Novel Transcription Factor in Immune Reactions” Journal Of Interferon & Cytokine Research 25:297-310 (2005). Hypoxia-inducible factor-1 alpha has further uses, such as for liver treatment, repair, and regeneration. Tajima et al. “HIF-1alpha is necessary to support gluconeogenesis during liver regeneration.” Biochem Biophys Res Commun. 2009 Oct. 2; 387(4):789-94. doi: 10.1016/j.bbrc.2009.07.115. Epub 2009 Jul. 28. PMID: 19643083; Nath et al. “Hypoxia and hypoxia inducible factors: diverse roles in liver diseases.” Hepatology (Baltimore, Md.) vol. 55, 2 (2012): 622-33. doi:10.1002/hep.25497; Lin et al. “Hypoxia-Inducible Factor 2 Alpha Is Essential for Hepatic Outgrowth and Functions via the Regulation of leg1 Transcription in the Zebrafish Embryo” PLoS ONE 9(7): e101980; doi.org/10.1371/journal.pone.0101980. It also has uses in preventing kidney injury (Bhatt et al. “MicroRNA-687 Induced by Hypoxia-Inducible Factor-1 Targets Phosphatase and Tensin Homolog in Renal Ischemia-Reperfusion Injury” JASN July 2015, 26 (7) 1588-1596; DOI: doi.org/10.1681/ASN.2014050463) and treating ischemic hearts (Guimaraes-Camboa, N. and Evans, S. “Redox Paradox: Can Hypoxia Heal Ischemic Hearts?” Developmental Cell 39(4): 392-394 (Nov. 21, 2016); DOI: doi.org/10.1016/j.devcel.2016.11.007 and Cerrada et al. “Hypoxia-inducible factor 1 alpha contributes to cardiac healing in mesenchymal stem cells-mediated cardiac repair” Stem Cells Dev. 2013 Feb. 1; 22(3):501-11. doi: 10.1089/scd.2012.0340. Epub 2012 Sep. 14. PMID: 22873764).

[0047] In certain embodiments, the described methods include use of a bioelectric stimulator, which is programmed to produce bioelectric signals that cause the patient to upregulate the expression of OPG so as to enhance osteoblast formation and bone formation/re-mineralization for implant stability. In certain embodiments, controlled release of OPG (which promotes osseointegration (bone in growth)) is combined with upregulation of expression of SDF1 and PDGF to promote stem cell homing and healing, which promotes rapid regeneration of tissues.

[0048] In certain embodiments, the described methods include use of a bioelectric stimulator, which is programmed to produce a bioelectric signal that causes the patient to upregulate the expression of transforming growth factor beta 1 (TGF-β1). TGF-β1 is a polypeptide member of the transforming growth factor beta superfamily of cytokines, which is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation, and apoptosis. A bioelectric signal that upregulates expression of TGF-β1 in a cell is a square, biphasic waveform at 50% duty, wherein the frequency is at least 75 Hz and the signal amplitude is typically about 1.0 V as measured at the cellular level.

[0049] In certain embodiments, rapid accelerated healing in, for example, implant dentistry is attained with combination bioelectric and biologics therapy keyed on highly precise controlled protein expressions, such as OPG, SDF1, PDGF, VEGF, and IGF1, so as to activate, amplify, extend, enhance, and accelerate the natural healing process.

[0050] Described is a bioelectric stimulator that is programmed to upregulate expression of OPG, klotho, RANKL, IGF1, and bioelectric PRF combination.

[0051] In certain exemplary embodiments, the following sequence of bioelectric signals for promoting rapid dental implant healing and pain reduction is applied to a patient, and includes a bioelectric signal for SDF1 for about 5 minutes, a bioelectric signal for PDGF for about 5 minutes, a bioelectric signal for IGF1 for about 5 minutes, a bioelectric signal for BMP 9 for about 2.5 minutes, a bioelectric signal for OPG for about 5 minutes, a bioelectric signal for klotho for about 5 minutes, and a bioelectric signal for managing inflammation and pain for about 5 minutes. See, e.g., the incorporated U.S. Pat. No. 11,110,274 to Leonhardt (Sep. 7, 2021) for “System and method for treating inflammation” for bioelectric signals useful to manage pain and inflammation.

[0052] In certain embodiments, the described bioelectric signals are applied wirelessly to the patient. In certain such embodiments, a micro coil receiver crown is placed on the top of an implanted dental implant, which receiver communicates with an external electromagnetic energy generator that serves to allow the implant to deliver specific bioelectric signals to the surrounding tissues to control release of the specific proteins that accelerate dental implant healing, osseointegration (bone in growth) and reduce associated pain and discomfort.

[0053] In certain embodiments (e.g., FIG. 3), a bioelectric mouthpiece is electrically connected to a FDA cleared stimulator, which delivers specific bioelectric signals for modulating (upregulating or downregulating) the expression of proteins selected to accelerate, for example, dental implant healing, osseointegration (bone in growth), and to reduce associated pain and discomfort. For example, when programmed as described herein, the system depicted in FIG. 3 decreases the required time for a dental implant to be fully utilized by a patient in normal activities such as chewing food without pain.

[0054] As shown in FIG. 3, in some embodiments, a system includes a housing, an emitter, and an electronic circuit. The housing (or mouthpiece) is configured to fit within a patient's mouth. The emitter is configured to emit an effective amount of bioelectric signals either wirelessly or via wires and conductive electrode contact to the alveolar soft tissue when the apparatus is disposed within the mouth. The electronic circuit is operatively coupled to the bioelectric signaling emitter. The electronic circuit is configured to control the emitter when the housing is disposed within the mouth and the apparatus is in use during treatment. The system and is used for accelerated healing, reduced risk of infection, alleviating inflammation, and pain relief. Specific bioelectric signal sequences cause the release of the selected protein expressions for specific purposes.

[0055] As shown in FIG. 4, after successful implantation, a dental implant typically contains three portions, the post implant 10, the abutment 12, and the crown 14. The post implant 10 is typically a titanium or titanium alloy implant that is surgically fused with the jawbone (e.g., screwed into the jawbone to resemble a tooth root). Other materials such as other metals and ceramics may also be used as an The abutment 12 is typically made of titanium, gold, or porcelain, and fitted over the portion of the post that protrudes from the gum line after the patient's gum tissues are healed. The depicted abutment has an implant abutment interface/implant collar 16.

[0056] Initially, the implant is placed or inserted into the bone of the patient's jawline, and a removable cover screw is fitted into the implant abutment interface 16. The healing process of the bone then begins. Without the bioelectric signal therapy described herein, this healing process can take from 3 to 18 months. This healing period is reduced with the application of the described bioelectric signals in the schedule described herein.

[0057] As described herein, the described bioelectric signals are applied via electrodes or wirelessly to the post implant 10, the cover screw, and/or the abutment 12 to aid and hasten the healing process. In embodiments where the crown is electrically conductive, the bioelectric signals may alternatively or also be applied to the crown.

[0058] After healing, the cover screw is typically removed, and is replaced with the abutment 12. The crown (which is typically a high quality porcelain, metal material, metal fused porcelain, or ceramic resin) is fitted onto the abutment for use and for a natural appearance.

[0059] In certain embodiments such as a wireless embodiment, a micro coil receiver crown or micro coil receiver screw is placed on the top of the post implant 10, which micro coil receiver communicates with an external electromagnetic energy generator that serves to induce the implant to deliver specific bioelectric signals to surrounding cellular tissues to modulate expression of the selected proteins so as to accelerate implant healing, osseointegration (bone in growth), and reduction of associated pain and discomfort.

[0060] In certain embodiments, the described bioelectric signal therapy is combined with repeat delivery of biologics via a slow infusion re-fillable micro infusion pump placed on top of the dental implants (e.g., incorporated into the crown, abutment, or screw). This micro pump is re-filled daily or weekly with an implant healing acceleration composition comprising a combination selected from the group consisting of tooth pulp stem cells, bioelectric PRF, amniotic fluid, micro RNA gel, bone matrix, selected growth factors such as BMPs, nutrient hydrogel, oxygenated nanoparticles, and bone matrix.

[0061] For example, use of the bioelectrically stimulated implant device may be combined with the administration of any or all of these biologic agents: stem cells, selected exosomes, platelet rich fibrin PRF, platelet rich plasma PRP, amniotic fluid, placental fluid, selected growth factors such as OPG and BMP9, nutrient hydrogel, micron RNA gel, Wharton's Jelly, oxygenated nanoparticles, fibrin gel, extracellular matrix and bone matrix.

[0062] The invention is further described with the aid of the following illustrative Examples.

EXAMPLES

Example I

[0063] A trial was conducted as follows. A biostimulator (Mettler) was programmed to produce bioelectric signals that upregulate expression of SDF1, PDGF1, VEGF, IGF1, and OPG by human cells. Controlled expression of OPG promotes osseointegration (bone in growth). SDF1 and PDGF promote stem cell homing and healing, which promotes rapid regeneration of tissues. A first therapy of bioelectric signals was applied to a dental implant patient on day one as follows: [0064] A bioelectric signal for SDF1 for 5 minutes at 5 mA [0065] A bioelectric signal for PDGF1 for 5 minutes [0066] A bioelectric signal for VEGF for 5 minutes [0067] A bioelectric signal for IGF1 for 5 minutes [0068] A bioelectric signal for OPG for 10 minutes

[0069] On day three, a series of bioelectric signals was applied to the implant as follows: [0070] A bioelectric signal for SDF1 for 5 minutes at 4 mA to 4.5 mA [0071] A bioelectric signal for PDGF1 for 5 minutes at 4 mA [0072] A bioelectric signal for VEGF for 5 minutes at 2 mA [0073] A bioelectric signal for IGF1 for 5 minutes at 200 μA to 685 μA [0074] A bioelectric signal for OPG for 10 minutes at 3 mA

[0075] On day five, a series of bioelectric signals was applied to the implant as follows: [0076] A bioelectric signal for SDF1 for 5 minutes at 3.2 mA to 4 mA [0077] A bioelectric signal for PDGF1 for 5 minutes at 3.6 to 3.2 mA [0078] A bioelectric signal for VEGF for 5 minutes at 2 mA [0079] A bioelectric signal for IGF1 for 5 minutes at 625 μA [0080] A bioelectric signal for OPG for 10 minutes at 3.4 mA

[0081] On day seven, a series of bioelectric signals was applied to the implant as follows: [0082] A bioelectric signal for SDF1 for 5 minutes at 3.2 mA to 4.0 mA [0083] A bioelectric signal for PDGF1 for 5 minutes at 2.4 mA [0084] A bioelectric signal for VEGF for 5 minutes at 2 mA [0085] A bioelectric signal for IGF1 for 5 minutes at 645 μA [0086] A bioelectric signal for OPG for 10 minutes at 3.4 mA

[0087] On day nine, a series of bioelectric signals was applied to the implant as follows: [0088] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA to 3.2 mA [0089] A bioelectric signal for PDGF1 for 5 minutes at 2.4 mA to 2.2 mA [0090] A bioelectric signal for VEGF for 5 minutes at 2 mA to 1.6 mA [0091] A bioelectric signal for IGF1 for 5 minutes at 645 μA to 600 μA [0092] A bioelectric signal for OPG for 10 minutes at 3.0 mA

[0093] On day 11, a series of bioelectric signals was applied to the implant as follows: [0094] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA [0095] A bioelectric signal for PDGF1 for 5 minutes at 2.3 mA [0096] A bioelectric signal for VEGF for 5 minutes at 2 mA to 2.2 mA [0097] A bioelectric signal for IGF1 for 5 minutes at 650 μA [0098] A bioelectric signal for OPG for 10 minutes at 3.0 mA

[0099] On day 14, a series of bioelectric signals was applied to the implant as follows: [0100] A bioelectric signal for SDF1 for 5 minutes at 3.7 mA [0101] A bioelectric signal for PDGF1 for 5 minutes at 2.4 mA [0102] A bioelectric signal for VEGF for 5 minutes at 2.2 mA [0103] A bioelectric signal for IGF1 for 5 minutes at 650 μA [0104] A bioelectric signal for OPG for 10 minutes at 3.0 mA

[0105] On day 16, a series of bioelectric signals was applied to the implant as follows: [0106] A bioelectric signal for SDF1 for 5 minutes at 3.7 mA [0107] A bioelectric signal for PDGF1 for 5 minutes at 2.4 mA [0108] A bioelectric signal for VEGF for 5 minutes at 2.6 mA [0109] A bioelectric signal for IGF1 for 5 minutes at 650 μA [0110] A bioelectric signal for OPG for 10 minutes at 3.0 mA

[0111] On day 19, a series of bioelectric signals was applied to the implant as follows: [0112] A bioelectric signal for SDF1 for 5 minutes at 3.4 mA [0113] A bioelectric signal for PDGF1 for 5 minutes at 2.8 mA [0114] A bioelectric signal for VEGF for 5 minutes at 2.6 mA [0115] A bioelectric signal for IGF1 for 5 minutes at 670 μA [0116] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0117] On day 21, a series of bioelectric signals was applied to the implant as follows: [0118] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0119] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0120] A bioelectric signal for VEGF for 5 minutes at 3.0 mA [0121] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0122] A bioelectric signal for OPG for 10 minutes at 3.5 mA

[0123] On day 24, a series of bioelectric signals was applied to the implant as follows: [0124] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0125] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0126] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0127] A bioelectric signal for IGF1 for 5 minutes at 670 μA [0128] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0129] On day 26, a series of bioelectric signals was applied to the implant as follows: [0130] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0131] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0132] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0133] A bioelectric signal for IGF1 for 5 minutes at 670 μA [0134] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0135] On day 28, a series of bioelectric signals was applied to the implant as follows: [0136] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA [0137] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0138] A bioelectric signal for VEGF for 5 minutes at 2.4 mA to 2.2 mA [0139] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0140] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0141] On day 30, a series of bioelectric signals was applied to the implant as follows: [0142] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA decreased to 3.5 mA [0143] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA decreased to 2.8 mA [0144] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0145] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0146] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0147] On day 32, a series of bioelectric signals was applied to the implant as follows: [0148] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0149] A bioelectric signal for PDGF1 for 5 minutes at 2.8 mA [0150] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0151] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0152] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0153] On day 34, a series of bioelectric signals was applied to the implant as follows: [0154] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA [0155] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0156] A bioelectric signal for VEGF for 5 minutes at 2.4 mA to 2.2 mA [0157] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0158] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0159] On day 36, a series of bioelectric signals was applied to the implant as follows: [0160] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0161] A bioelectric signal for PDGF1 for 5 minutes at 2.8 mA [0162] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0163] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0164] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0165] On day 38, a series of bioelectric signals was applied to the implant as follows: [0166] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0167] A bioelectric signal for PDGF1 for 5 minutes at 2.8 mA [0168] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0169] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0170] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0171] On day 40, a series of bioelectric signals was applied to the implant as follows: [0172] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0173] A bioelectric signal for PDGF1 for 5 minutes at 2.8 mA [0174] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0175] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0176] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0177] On day 42, a series of bioelectric signals was applied to the implant as follows: [0178] A bioelectric signal for SDF1 for 5 minutes at 3.5 mA [0179] A bioelectric signal for PDGF1 for 5 minutes at 2.8 mA [0180] A bioelectric signal for VEGF for 5 minutes at 2.4 mA [0181] A bioelectric signal for IGF1 for 5 minutes at 675 μA [0182] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0183] On day 45, a series of bioelectric signals was applied to the implant as follows: [0184] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA [0185] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0186] A bioelectric signal for VEGF for 5 minutes at 2.5 mA [0187] A bioelectric signal for IGF1 for 5 minutes at 680 μA [0188] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0189] On day 47, a series of bioelectric signals was applied to the implant as follows: [0190] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA [0191] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0192] A bioelectric signal for VEGF for 5 minutes at 2.5 mA [0193] A bioelectric signal for IGF1 for 5 minutes at 680 μA [0194] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0195] On day 49, a series of bioelectric signals was applied to the implant as follows: [0196] A bioelectric signal for SDF1 for 5 minutes at 3.6 mA [0197] A bioelectric signal for PDGF1 for 5 minutes at 3.0 mA [0198] A bioelectric signal for VEGF for 5 minutes at 2.5 mA [0199] A bioelectric signal for IGF1 for 5 minutes at 680 μA [0200] A bioelectric signal for OPG for 10 minutes at 3.6 mA

[0201] The patient from the trial experienced enhanced osteointegration of the implant.

Example II

[0202] Four patients needing implants underwent therapy utilizing the described therapy (stimulation twice a week for 40 minutes for 8 weeks). All four patients demonstrated a 300% acceleration in bone osteointegration and implant stabilization time compared to historical control data as measured by an OssTell device. See, e.g., Soicu et al. “An evaluation of TimPlant using Osstell: A device for non-invasive assessment of dental implants stability” OHDMBSC, Vol. VIII. No. 4, pp. 28-33 (December 2009).

Example III

[0203] Osteoblasts were cultured in 6-well plates. Using carbon electrodes inside the culture, biphasic square pulses at frequencies between 20 Hz and 3 MHz were applied. OPG was quantified by OPG mRNA extracted immediately after the stimulation protocol through RTqPCR. Data from stimulated cells was normalized to mRNA from control sister dishes and GAPDH expression. Changes in expression were plotted against frequency.

[0204] FIG. 5 is a graph depicting cumulative results of OPG expression for the specific bioelectric signal application protocols as applied to osteoblasts. In the graph, * indicates a significant log fold change due to treatment. The right tick marks indicate fold change. The circles indicate predicted values from Generalized Additive Model.

[0205] The data (FIG. 5) demonstrates that below 2000 Hz, the expression of OPG mRNA was significantly reduced (70%), with a lowest reached at ˜200 Hz. More importantly, the expression of OPG mRNA was significantly increased at frequencies above 2000 Hz with a maximal expression (200%) at ˜100,000 Hz. This data was consistent whereas the voltage applied was of 0.1, 1, or 2 volts. Overall, the results support that the expression of OPG can be regulated by the frequency of squared pulses at low voltages.

[0206] The results depicted in FIG. 5 indicate that there are primarily two regions in the frequency spectrum. A first region that reduces expression OPG by a cell from the basal level of between 75 and 1,000 Hz. The second region corresponds to overexpression of OPG by application of a bioelectric signal of between 2,500 Hz and 3,000,000 Hz.

Example IV

[0207] A cell was treated by stimulating the cell with a bioelectric signal so as to upregulate expression of transforming growth factor beta 1 (TGF-β1) by the cell. The bioelectric signal stimulation was by applying a bioelectric signal to the cell, wherein the bioelectric signal was a square, biphasic waveform at 50% duty, wherein the frequency was 75 Hz and the signal amplitude was about 1.0 V as measured at the cell. The cell experienced upregulated expression of TGF-β1.

REFERENCES

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