BMP9 MODULATION

Abstract

Described is a low voltage, pulsed electrical stimulation device for modulating expression of BMP9 protein(s) by cellular tissues.

Claims

1. A bioelectric stimulator programmed to produce a bioelectric signal that modulates expression and/or release of bone morphogenetic protein 9 (BMP9) in a cell.

2. The bioelectric stimulator of claim 1, wherein the produced bioelectric signal upregulates the expression and/or release of BMP9 in the cell.

3. The bioelectric stimulator of claim 2, wherein the bioelectric signal is 100 Hz or 300 Hz.

4. The bioelectric stimulator of claim 3, wherein the produced bioelectric signal is 100 Hz.

5. The bioelectric stimulator of claim 3, wherein the produced bioelectric signal is 300 Hz.

6. The bioelectric stimulator of claim 1, wherein the programmed bioelectric signal downregulates the expression and/or release of BMP9 in the cell.

7. The bioelectric stimulator of claim 6, wherein the produced bioelectric signal is 400 Hz.

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

9. A method of using the bioelectric stimulator of claim 1, to stimulate cellular tissue, the method comprising: connecting the bioelectric stimulator to the cellular tissue, and actuating the bioelectric stimulator to produce the programmed bioelectric signal(s) so as to modulate expression and/or release of bone morphogenetic protein 9 (BMP9) in the cellular tissue.

10. The method according to claim 9, wherein the tissue is selected from the group consisting of bone, dental arch, dental gum tissue, adipose tissue, derived stromal fraction, amniotic membranes, amniotic secretome, platelet rich fibrin (“PRF”), and any combination(s) thereof.

11. The method according to claim 9, wherein the bioelectric signal is selected from the group consisting of 100 Hz (within 15%), 300 Hz (within 15%), 400 Hz (within 15%), and a combination thereof.

12. A method of treating a cell, the method comprising: stimulating the cell to express and/or release of bone morphogenetic protein 9 (BMP9) by applying a bioelectric signal to the cell, wherein the bioelectric signal comprises, within 15%, a biphasic pulse of 100 Hz and/or 300 Hz.

13. The method according to claim 12, wherein the bioelectric signal is 100 Hz.

14. The method according to claim 12, wherein the bioelectric signal is 300 Hz.

15. A method of treating a cell, the method comprising: stimulating the cell to downregulate expression and/or release of bone morphogenetic protein 9 (BMP9) by applying a bioelectric signal to the cell, wherein the bioelectric signal is, within 15%, a biphasic pulse of 400 Hz.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 depicts a programmed bioelectric stimulator for delivery to a subject connected to multiple soft conductive electrode pads.

[0018] FIG. 2 is a graph depicting the modulation of BMP9 as described in the Example hereof.

DETAILED DESCRIPTION

[0019] Referring now to FIG. 1, depicted is a biostimulator for use in the treatment of a, for example, human subject. A bioelectric stimulator typically includes a low voltage electrical signal generator programmed to produce the selected bioelectric signal(s) associated with electrodes for delivering the bioelectric signal(s) to the patient's cellular tissue.

[0020] 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 Electronica of Amparo, BR. The leading pacemaker manufacturers are Medtronic, Boston Scientific Guidant, Abbott St. Jude, BioTronik and Sorin Biomedica.

[0021] 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.

[0022] The bioelectric stimulator of FIG. 1 is depicted as a programmed electric signal generator with leads connecting it to multiple soft conductive electrode pads. Electrodes may be used to deliver a bioelectric signal to the subject.

[0023] When the patient's treated cellular tissue is dental gum, bone, or dental arch, the electrodes may be placed for administration to the patient using the orthodontic devices described in U.S. Pat. No. 10,695,563 to Leonhardt et al. (Jun. 30, 2020) for “Orthodontic Treatment” or US 20200330753 Al to Leonhardt et al. for “Orthodontic treatment,” published on Oct. 22, 2020, the contents of each of which is incorporated herein by this reference.

[0024] 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, e.g., BMP9 protein for delivery to the subject.

[0025] Typical subjects to be treated are mammals such as humans.

[0026] In certain embodiments, the bioelectric stimulator is programmed to produce further 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. Other useful bioelectric signals for use herein are described in the incorporated U.S. Pat. No. 10,695,563 to Leonhardt et al. and US 20200330753 A1 to Leonhardt et al.

[0027] The invention is further described with the aid of the following illustrative Example.

EXAMPLES

Exampe —Controlling Expression and/or Release of BMP9

[0028] Purpose: The purpose of this Example was to analyze the effects of bioelectric signal stimulation on BMP9 in platelet rich fibrin (“PRF”) stimulated at 100 Hz, 200 Hz, 300 Hz, and 400 Hz at 1 V for 30 minutes and compare its expression against a control (unstimulated) condition using the basic enzyme-linked immunosorbent assay (ELISA).

[0029] Electrical Signals

[0030] 100 Hz, 1 V

[0031] 200 Hz, 1 V

[0032] 300 Hz, 1 V

[0033] 400 Hz, 1 V

[0034] Target Protein: BMP9

[0035] Methods: Human blood without anticoagulants was collected and immediately centrifuged at 900 rpm for 5 minutes. PRF was collected and equally plated in a 6-well dish with 1 mL/per well DMEM (10% FBS). Samples were stimulated with a RIGOL biostimulator (Suzhou, China) at the described frequencies for 30 minutes and the other three wells were control (unstimulated) samples.

[0036] Post-stimulation, media was collected and Human BMP9 was quantified using QUANTIKINE® ELISA kits according to the manufacturer's instructions (R&D Systems, Minneapolis, Minn., US) on a Enspire 2300 multilabel microplate reader (Perkin Elmer, Wallac Oy, Turku, Finland).

[0037] Conclusions: After adjustments (4 tests), post hoc tests showed 100 Hz and 300 Hz increased expression of BMP9 and 400 Hz decreased expression of BMP9.

[0038] In summary, these data (shown graphically in FIG. 2) show that bioelectric signal treatment can be used to increase or decrease BMP9 protein concentration in platelet-rich fibrin and the sensitivity of the assay used.

[0039] Results

TABLE-US-00001 Summary: Fold Change ## Frequency N FoldChange sd se ci ## 1 0 4 1.00 0.06 0.03 0.09 ## 2 100 4 1.25 0.22 0.11 0.35 ## 3 200 4 1.15 0.09 0.05 0.15 ## 4 300 4 1.23 0.07 0.03 0.11 ## 5 400 4 0.65 0.01 0.00 0.01 ANOVA Fold Change ## [1] “F-statistic: 3.81 on 1 and 18 DF, p-value: 0.06667” Post Hoc Test ## Analysis of Variance Table ## ## Response: FoldChange ## Df Sum Sq Mean Sq F value Pr(>F) ## FrequencyFactor 4 0.98186 0.24546 18.809 1.037e−05 *** ## Residuals 15 0.19575 0.01305 ## ~~~ ## Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1 ## [1] “Comparisons” ## contrast estimate SE df t. ratio p. value p. value. adj ## 1  0-100 −0.248 0.081 15 −3.072 0.008 0.015 ## 2  0-200 −0.146 0.081 15 −1.804 0.091 0.130 ## 3  0-300 −0.233 0.081 15 −2.891 0.011 0.019 ## 4  0-400 0.351 0.081 15 4.348 0.001 0.001 ## 5 100-200 0.102 0.081 15 1.268 0.224 0.280 ## 6 100-300 0.015 0.081 15 0.182 0.858 0.858 ## 7 100-400 0.599 0.081 15 7.420 0.000 0.000 ## 8 200-300 −0.088 0.081 15 −1.086 0.294 0.327 ## 9 200-400 0.497 0.081 15 6.152 0.000 0.000 ## 10 300-400 0.585 0.081 15 7.239 0.000 0.000

REFERENCES

[0040] (The contents of the entirety of each of which is incorporated herein by this reference.)

[0041] Fujioka-Kobayashi et al. “Absorbable collagen sponges loaded with recombinant bone morphogenetic protein 9 induces greater osteoblast differentiation when compared to bone morphogenetic protein 2″ Clin Exp Dent Res 2017; 3:32-40.

[0042] Hustedt, Joshua W, and Daniel J Blizzard. “The controversy surrounding bone morphogenetic proteins in the spine: a review of current research.” The Yale Journal of Biology and Medicine vol. 87,4 549-61.12 Dec. 2014

[0043] Khorsand, Behnoush et al. “A Comparative Study of the Bone Regenerative Effect of Chemically Modified RNA Encoding BMP-2 or BMP-9.” The AAPS Journal vol. 19,2 (2017): 438-446. doi:10.1208/s12248-016-0034-8.

[0044] Liu et al. “BMP9 is a potential therapeutic agent for use in oral and maxillofacial bone tissue engineering” Biochem Soc Trans. 2020 Jun 30;48(3):1269-1285. doi: 10.1042/B5T20200376.

[0045] U.S. Pat. No. 10,695,563 to Leonhardt et al. (Jun. 30, 2020) for “Orthodontic Treatment”.

[0046] U.S. Pat. No. 10,960,206 to Leonhardt et al. for “Bioelectric Stimulator” (Mar. 20, 2021).

[0047] US 20200330753 A1 to Leonhardt et al. for “Orthodontic treatment,” published on Oct. 22, 2020.