1111111111111111111inuuu1111111111u~ (12) United States Patent (io) Patent No.: US 9,896,681 B2 Goodwin et al. (45) Date of Patent: *Feb. 20, 2018 (54) GENETIC REGULATION OF BONE AND 4,993,413 A 2/1991 McLeod CELLS BY ELECTROMAGNETIC 5,002,890 A 3/1991 Morrison STIMULATION FIELDS AND USES 5,026,650 A 6/1991 Schwarz 5,153,132 A 10/1992 Goodwin THEREOF 5,153,133 A 10/1992 Schwarz 5,155,034 A 10/1992 Wolf (71) Applicant: The United States of America as 5,155,035 A 10/1992 Schwarz Represented by the Administrator of 5,308,764 A 5/1994 Goodwin the National Aeronautics and Space 5,627,021 A 5/1997 Goodwin 5,846,807 A 12/1998 Goodwin Administration, Washington, DC (US) 6,485,963 B1 11/2002 Wolf 6,673,597 B2 1/2004 Wolf (72) Inventors: Thomas J. Goodwin, Kemah, TX 6,730,498 B1 5/2004 Goodwin (US); Linda C. Shackelford, Webster, 6,919,205 B2 7/2005 Brighton TX (US) 7,160,024 B2 1/2007 Dougherty, Sr. 7,179,217 B2 2/2007 Goodwin 7,456,019 B2 11/2008 Goodwin (73) Assignee: The United States of America as 2006/0229487 Al 10/2006 Goodwin represented by the National 2007/0105769 Al 5/2007 Simon Aeronautics and Space 2008/0138415 Al 6/2008 Hussain Administration, Washington, DC (US) 2009/0234417 Al 9/2009 Pastena 2011/0105959 Al 5/2011 OConnor (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 0 days. An, et al„ "Interactive effects of surface topography and pulsatile This patent is subject to a terminal dis- electrical field stimulation on orientation and elongation of fibro- claimer. blasts and cardiomyocytes" , Biomaterials, 28(29):4277-93 (2007). Hammond, et al., "Optimized suspension culture: the rotarinf-wall (21) Appl. No.: 14/445,995 cessel" , Am J Physiol Renal Physiol, 281:1`12-25 (2001). Reddy, et al., "Micronuclei in blood and bone marrow cells of mice (22) Filed: Jul. 29, 2014 exposed to specific complex time-varying pulsed magnetic fields" , Bioelectromagnetics, 31:445-53 (2010). (65) Prior Publication Data US 2014/0342428 Al Nov. 20, 2014 Primary Examiner Catherine S Hibbert Related U.S. Application Data (74) Attorney, Agent, or Firm Benjamin Aaron Adler (63) Continuation-in-part of application No. 12/899,815, (57) ABSTRACT filed on Oct. 7, 2010, now Pat. No. 8,795,147. The present invention provides methods to modify the genetic regulation of mammalian tissue, bone, cells or any (51) Int. Cl. combination thereof by preferential activation, up-regula- A61 2/00 (2006.01) tion and/or down-regulation. The method comprises steps of C12N 13/00 (2006.01) tuning the predetermined profiles of one or more time- A61 2/02 (2006.01) varying stimulation fields by manipulating the B-Field mag- (52) U.S. Cl. nitude, rising slew rate, rise time, falling slew rate, fall time, CPC ............... C12N 13/00 (2013.01); A61 2/02 frequency, wavelength, and duty cycle, and exposing mam- (2013.01) malian cells or tissues to one or more tuned time-varying (58) Field of Classification Search stimulation fields with predetermined profiles. Examples of None mammalian cells or tissues are chondrocytes, osteoblasts, See application file for complete search history osteocytes, osteoclasts, nucleus pulposus, associated tissue, or any combination. The resulted modification on gene (56) References Cited regulation of these cells, tissues or bones may promote the U.S. PATENT DOCUMENTS retention, repair of and reduction of compromised mamma- lian cartilage, bone, and associated tissue. 4,839,046 A 6/1989 Chandler 4,988,623 A 1/1991 Schwarz 19 Claims, 8 Drawing Sheets U.S. Patent Feb. 20, 2018 Sheet 1 of 8 US 9,896,681 B2 10 - B -Field f = 9-200Hz 21 2C A= 500ms FIG. 1 U.S. Patent Feb. 20, 2018 Sheet 2 of 8 US 9,896,681 B2 280ps B-Field A= f = 15H7 FIG. 2 U.S. Patent Feb. 20, 2018 Sheet 3 of 8 US 9,896,681 B2 30 ~51 FIG. 3 41 FIG. 4 U.S. Patent Feb. 20, 2018 Sheet 4 of 8 US 9,896,681 B2 32 "` .- 34 20 FIG. 6 U.S. Patent Feb. 20, 2018 Sheet 5 of 8 US 9,896,681 B2 50 €:€ at istre predetermined profile firt lyst ti rte„ varying stimulation field. Controllingat least one tr ryaed tiroe ,varyin stimulation, field to 12 produce at feats t one tuned predeterminedfined €rode. Exposing mammalian chondrocytres, Oster;:Wasts,f ost ocytes, osteoclasts, nucleus pulposuss associated tissue, or any 11 combinationation to at least one tuned tl€ e—v rying stimulation field comprised of at least one tuned predetermined profile for a predetermined tuned exposure Lime= ar plurality of tuned exposure Vrne sequences. FIG. 7 11 Exposing marnrr#aliarl Cf et i E irVE ~sr ~ FC i1 aE$, E Providing 3 Ci#mulatior, 0E' d g:: ,n .` ai.-or z o5.tt~ocytes', ostOoC4wtB, app;irrtt* in pro,xwnata ~-pat€al rFUflQr:ship i i > S;i ?rf?f~3u~#aft chondErocytes, ostir:llests, nucleus rulposus, as-wc#ateti I 1 tissue, or any Combination to tI o-Aeopylms, cstc~ctast , nucleus puiposus, at feast on FIuned BEET - ~ + Y4,Sociate ds~we, Dr Amy Cof7i1)#?7- Rio " corr,lorisr d of at mast one ~ t# l)Od predetermined pro if i t ; EIfT3 ilatEot3 €i id Gi t£?' .Safisaid _q';?Tit:""tilt r:#Eald 19 Or >:i }3md:':te mined tuned generator Ipparati,~, expa,wrf-'~ time or plurality of Wned exposure time, FIG. 8 U.S. Patent Feb. 20, 2018 Sheet 6 of 8 US 9,896,681 B2 pravidin, at km!st one: set a re11u€ar 51 5;3f? pies irE ;it 11 art t,mt N rotating wa€: vess;--A, Expo5ing Said aY- kvct oml , spl of ce€ £.lar samples to, at €na'st one exrperimimt;d Iime- 52 ii;arying stimuli€atfon 'idlld Y_arfli,?i'E'~SfSlr :::' least On£ ex ~trsr;ei3t4al are tlaretrf inns prafilz:, Conducting at iewa one gcmcE i:`>:pmssion araly'Ses is sa41 at least one set Of ce1€velar 53 sat pkls. 51 FA 3c1`y' 13? the gf?3"et?c anabr1i: and F:iit~ 'Ei1?C Ofe'.as f5'otf` the mt,ss +,s of 4c3; at least 54 p£IC' Lxprr;.§siara Fi Ytlf +~T -it ieaS: ono tuoni-ed predetermined profile for 1~ jemt One Li £ff ra Ft'E <3t least one crm €tsbn ft'£;m 55 tuned tithe-varyirtg 5a€ f stew a anxlyxlr:g. atlmulaion f€eidf 65 Doe" 1 ~ Ij i5tlf~, sa:s at least on a" least r~3rir„ axperimenta! pry>de 'vMioe-- 1 C-vtIC:ILF53at3 M; et .y. prede em nlr:k>cl ar~Jf>€e <3se°ri a ;caid 3 Inns: ci'Ite rla i one''07clia5ia£;. N Y \1..._' 56 SrtiC?i:ti"le said at least orie kwrr'~i tit e— varymgstirnlftation ff,A l;wmp,,isei Eli S3d at li;,asi one tl3: ed pYisdf?tetmined profile foe 3i 11?r£f £~tc:rrE33r f~if Nned expos n 57 t>m or said p€ur-<Oity csf tt_< ate $ifs t'. 5c"• t:l;,£c'?i3 C£~~i. FIG. 9 U.S. Patent Feb. 20, 2018 Sheet 7 of 8 US 9,896,681 B2 Selecting a predetermined set of genes or ~1 gene i'amilie;a. 54 Calculating a fold &Iane for t-z4 .h gene o said :iet ge( nes fr n, the r'taultw of said at 59 Analy?irig the yfed)eta- lca"c ^t one gene expres~ion anabalic, and effects from the results of 4f i$ said at least One sere Determining if en-h said foW expression artalyses, C.or"sisterat loth up-m"gulivk'w or uiwn- .e regWation for said l:adl 'gene, DeteYr1?Fni,ly if £'clt::h of ~aid 1o3Lz i. Eaf?y~ is consWent kv th t. p-regulc3?;ion co down.- 61 regWri tten for!i'I Zd each gene, FIG. 10 U.S. Patent Feb. 20, 2018 Sheet 8 of 8 US 9,896,681 B2 Establishing is relative val3 ;. e for r.'acli of si#d 62 55 regu#at€on or =ova n-mgwation for said each, ,;0- #Ir . -----------------------------valie Jm uo A~dusk,;,n from:tx~:~ ste, ~ .M. 63 of a tli yzi # 7 s 3 substaaitive net anabolic :effect, r .e~`b, it<ardvc ,., £~i? Ca#t t 3o iC c ff c", a net M FIG. 11 US 9,896,681 B2 2 GENETIC REGULATION OF BONE AND known as osteoid, which mineralizes to become bone. CELLS BY ELECTROMAGNETIC Osteoid is primarily composed of Type I collagen. STIMULATION FIELDS AND USES In the disease commonly known as osteoporosis, bone THEREOF demineralizes and becomes abnormally rarefied. Dimin- 5 ished bone density may lead to vertebrae collapse, fractures ORIGIN OF THE INVENTION of hips, lower arms, wrists, ankles as well as incapacitating pains. The invention described herein was made by employees Current therapies comprise invasive procedures (e.g. sur- of the United States Government and may be manufactured gery or drug administration) as opposed to the described and used by or for the Government of the United States of io innovation which is a non-invasive procedure. Alternative America for governmental purposes without the payment of nonsurgical therapies for such diseases include electrical any royalties thereon or therefor. bone growth stimulation comprised of electric and magnetic field therapies. However these therapies are moderately BACKGROUND OF THE INVENTION invasive as they rely on either skin contact or insertion of 15 probes/electrodes into the tissues.