Assessment of distraction callus in rabbits by monitoring of the electrical impedance of bone

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1 628 Act Orthopedic 21; 81 (5): Assessment of distrction cllus in rbbits by monitoring of the electricl impednce of bone Tkshi Yoshid, Wook-Cheol Kim, Yoshinobu Ok, Notke Ymd, nd Toshikzu Kubo Deprtment of Orthopedics, Grdute School of Medicl Science, Kyoto Prefecturl University of Medicine, Kyoto, Jpn Correspondence TY: Submitted Accepted Bckground nd purpose Evlution of distrction cllus is importnt for determintion of the optiml time for removl of externl fixtion. We ttempted to determine whether there might be reltionship between the electricl impednce of bone nd cllus mturtion, with view to using impednce s wy of knowing when to remove fixtor. Methods We pplied n externl lengthener to the right tibi of 24 rbbits nd performed distrction t 1 mm/dy for 1 dys. Rdiogrphs were tken nd mesurement of overll impednce between fixtion pins ws performed weekly fter distrction. At weeks 2, 4, 6, nd 8 fter distrction (n = 6 ech), resistivity of the bone to electricl conductivity ws mesured before killing. Crosssectionl re of the conduction pthwy in cllus nd mximum bending stress were mesured fter excision of the tibi. Results The overll impednce incresed sttisticlly significntly from 1 to 6 weeks fter completion of distrction. The resistivity of bone decresed over 4 weeks nd the cross-sectionl re of cllus decresed significntly over 6 weeks, while the mximum bending stress incresed significntly over the sme time. We observed negtive correltion between the cross-sectionl re of cllus nd mximum bending stress. Interprettion The impednce vlues, which re relted to chnges in electricl conductivity nd the conduction pthwy, incresed due to the chnges in the cross-sectionl re of cllus, despite the reduction in bone resistivity. Since the cross-sectionl re of cllus ws correlted with mximum bending stress nd the impednce vlues incresed with the cllus-remodeling process, we suggest tht temporl increses in overll impednce reflect cllus mturtion. Current clinicl evlution of distrction cllus usully depends only on rdiogrphic exmintions, which my be unrelible; cllus frcture or recurrence of deformity my occur fter removl of the externl fixtion (Simpson nd Kenwright 2). Exmples of methods previously reported for evlution of distrction cllus in niml studies include quntittive computed tomogrphy (CT) (Hrp et l. 1994, Reichel et l. 1998), dul-energy X-ry bsorptiometry (DEXA) (Eyres et l. 1993, Reichel et l. 1998, Tselentkis et l. 21), ultrsound (Eyres et l. 1993, Bil et l. 22), nd bone stiffness testing (Dwyer et l. 1996, Windhgen et l. 2, Arnes et l. 26). CT (prticulrly with multiple exmintions) is ssocited with dditionl rdition nd is costly, nd both CT nd DEXA my be ssocited with rtifcts ttributble to externl fixtion. Ultrsound hs problems with the ccurcy nd imge qulity of coustic conduction. A bone-heling meter nd bending testing still hve vrious problems ssocited with the complexity of instlling the mesuring instruments nd with the evlution method. Mesurement of impednce is esy nd useful for evlution of conductive substnces bsed on their electricl chrcteristics (Ohmine et l. 24), nd it is frequently used for evlution of the physicl properties nd structure of substnces in industry. It is lso used for biologicl systems: for exmple, it hs been used for mesuring percentge of body ft nd muscle volume of the extremities (Miytni et l. 21), s impednce increses with increse in body ft nd muscle volume. Previous investigtions of bone electrophysiology hve shown the electricl nd dielectric behvior of bone (Sh nd Willims 1995), nd the mechnicl properties of humn trbeculr bone hve been evluted by electricl mesurements (Sierpowsk et l. 25). We hve previously reported increses in the electricl impednce of bone during frcture heling (Yoshid et l. 29), focusing on both evlution of bone nd the frcture heling process. We investigted both bone conductivity nd the conduction pthwy itself, nd we observed close involvement of the conduction pthwy in the increse in impednce. Since steel pins re lredy inserted into long bones during cllus distrction, simply by using these pins s electrodes, the overll impednce between pins cn be mesured without the need for ny dditionl invsive procedures. We pplied this method to distrction cllus, which cn be difficult to evlute cliniclly. We nlyzed the temporl chnges in overll Open Access - This rticle is distributed under the terms of the Cretive Commons Attribution Noncommercil License which permits ny noncommercil use, distribution, nd reproduction in ny medium, provided the source is credited. DOI 1.319/

2 Act Orthopedic 21; 81 (5): Figure 1. A. Digrm illustrting the experimentl scheme. When mesuring overll impednce, the electricl device ws connected to the inner two pins (P2 nd D1). The voltge (E, mv) between the electrodes ws mesured with digitl oscilloscope vi biologicl mplifier. The impednce vlues were clculted using the eqution: Z (overll impednce) = E / I. B. Photogrph showing mesurement of overll impednce. impednce vlues over time during the cllus mturtion process nd investigted the reltionship between chnges in impednce vlues nd the mechnicl strength of the cllus. Mteril nd methods This study ws conducted ccording to the regultions of Kyoto Prefecturl University of Medicine regrding niml reserch. Overll experimentl design A mid-diphysel osteotomy t the right tibi ws performed in 24 Jpnese white rbbits nd n externl fixtor ws pplied. Rdiogrphic exmintions nd mesurement of overll impednce between fixtion pins were performed once week fter osteotomy or completion of distrction. To nlyze the electricl properties of distrction cllus, bone resistivity s conductivity nd cllus cross-sectionl re s conduction pthwy were evluted. A 3-point bending tester ws used to mesure the bending stress t the mid-cllus. We exmined temporl chnges in the overll impednce, the electricl properties, nd the mechnicl strength of the distrction cllus. Animls nd surgicl procedures We used 24 five-week-old mle Jpnese white rbbits in this study (men body weight: 1.1 (1. 1.2) kg). Under intrvenous nesthesi with pentobrbitl t 3 mg/niml nd locl nesthesi with lidocine t 5 mg/niml, 1-mm-long longitudinl skin incisions were mde t 2 sites: one t one-third of the distnce from the proximl end nd the other t one-third of the distnce from the distl end on the medil spect of the right tibi. 2 threded, stinless-steel pins (2.-mm dimeter, Kirschner wire with thred nd trochr point, resistivity < 1 Wm; Synthes, Inc, West Chester, PA) were inserted t ech incision site perpendiculr to the bone xis t n intervl of 8 mm, followed by ttchment of n Orthofix M-1 externl fixtor. Since nodic oxide coting ws performed on the surfce of the externl fixtor, the intervl between the fixtion pin nd the clmp ws in n insulted stte. The 4 pins were designted P1, P2, D1, nd D2 in order strting from the proximl end (Figure 1A). A 1-mm skin incision ws mde 5 mm proximl to the distl end pin (D1), the skin ws peeled wy to expose the periosteum, holes were drilled using screw 2 mm in dimeter, nd osteotomy ws performed with bone chisel with width of 1.2 mm. After 7 dys, cllus distrction ws performed t rte of 1 mm per dy for 1 dys. The 24 nimls were ssigned to 4 groups (weeks 2, 4, 6, nd 8 fter distrction; n = 6 for ech group). To nlyze the electricl properties of bone during cllus mturtion, we mesured bone resistivity for ech group under intrvenous nesthesi with pentobrbitl, fter removing the surrounding soft tissue including periosteum. During the removl of soft tissue, the posteromedil neurovsculr bundle ws preserved to mintin intrmedullry blood flow. Hemosttic procedures were used when pproprite, t room temperture (22 ± 2 C) with humidity of 5 ± 1%. For ech niml, tissue removl nd impednce vlue mesurements were completed in bout 2 min. The tibie of the nimls were excised fter killing, followed by mesurement of cllus cross-sectionl re to evlute the conduction pthwy nd by mesurement of mximum bending stress for determintion of the mechnicl properties of the distrction site. Rdiogrphic exmintion Rdiogrphic exmintions were performed under nesthesi fter osteotomy nd t 1-week intervls fter completion of distrction. We obtined nteroposterior nd mediolterl views with n effective voltge of 5 kv, current of 1 ma, focl distnce of 1 cm, nd n exposure time of.4 s.

3 63 Act Orthopedic 21; 81 (5): Figure 2. A. Photogrphs demonstrting mesurement of bone resistivity between the P2 nd D1 regions. Resistivity ws clculted by mesuring trnsverse dimeter nd nteroposterior dimeter t the P2 region, the distrction site, nd the D1 region, nd bone tissue ws ssumed to be in the shpe of n ovl column bsed on verging of these vlues. B. Digrms illustrting mesurement of men cross-sectionl re (A men ) nd cllus crosssectionl re (CS-A). C. Digrm illustrting mesurement of bone resistivity (ρ). Mesurement of overll impednce The overll impednce (Z (kw)) between the P2 nd D1 pins ws mesured using n lternting current (AC) device (MES Co. Ltd., Tokyo, Jpn) (Kwmoto et l. 25). The frequency ws set t 2 ±.4 Hz to limit the rectnce element of AC current to negligible rnge during mesurement. The impednce vlues were mesured for ech group under intrvenous nesthesi before distrction nd once week fter completion of distrction (Figure 1B). AC ws pplied using the device with constnt current output of 3 ± 6 μa nd loding resistnce of 6 kw. A 1-kW resistor ws connected to the AC device. The output voltge (E (mv)) of the AC device ws mesured with digitl oscilloscope vi biologicl mplifier, nd the constnt current (I (μa)) ws mesured using the eqution: I = E /R s (Figure 1A), where R s is the stndrd resistnce (1 kw). The voltge (E (mv)) between the electrodes ws mesured with digitl oscilloscope vi biologicl mplifier. The impednce vlues were clculted using the eqution: Z (overll impednce) = E/I. Bone resistivity between P2 nd D1 The bone nd distrction cllus in the region from P2 to D1 were ssumed to resemble cylindricl conductor, nd the bone resistivity (ρ (Wm)) of ech group ws clculted s follows. The trnsverse dimeter (-vlues) nd nteroposterior dimeter (b-vlues) of the P2 region, proximl region of the distrction cllus, middle of the distrction cllus, distl region of the distrction cllus, nd D1 region were designted 1, 2, 3, 4, nd 5 nd b 1, b 2, b 3, b 4, nd b 5, respectively, nd were mesured using clipers. Their cross-sectionl res (A men (m 2 )) were clculted using the eqution: A men = p [ ( ) / 5] [(b 1 + b 2 + b 3 + b 4 + b 5 ) / 5] / 4 (Figure 2A nd B). The length between P2 nd D1 (L (mm)) ws constnt t 35 mm for ll nimls fter completion of distrction. The bone resistivity ws clculted using the formul: ρ (bone resistivity) = (bone impednce) A men / L (Figure 2C) using the bone impednce obtined in the stte in which soft tissues, including periosteum, hd been removed. Cross-sectionl re of cllus The trnsverse dimeter nd nteroposterior dimeter of the middle of the distrction cllus of the excised bone tissue, which ws the re enclosed by the cllus envelope presented, were designted 3 nd b 3, nd the cross section of the cllus ws pproximted to resemble n ovl shpe, followed by clcultion of the cllus cross-sectionl re (CS-A (mm 2 )) using the formul: CS-A (cllus cross-sectionl re) = p 3 /2 b 3 /2 (Figure 2B). Mximum bending stress The mechnicl properties of the distrction cllus for ech group were mesured using sttic 3-point bending tester (Instron 55R; Instron Corp, Cnton, MA; lod cell, 5 N; rupturing speed, 1 mm/min). In clculting the mximum bending stress (B mx (N/mm 2 )) of the distrction cllus, we used formul bsed on the ssumption tht the cross section of the distrction cllus ws ovl. The distnce between the fulcr t both ends ws 4 mm. The bending stress ws clculted from the formul: B mx (mximum bending stress) = M y/i (Shrp 23), where M is the moment, Force (N) 2 (mm); y is the mximum distnce from neutrl xis, b 3 /2 (mm); nd I is the inerti of the cross section, p 3 b 3 3 /64 (mm 4 ). Thus, B mx = [Force 2 (b 3 /2)] / [p 3 b 3 3 /64] Sttisticl nlysis ws performed by testing mong multiple groups using the one-wy nd repeted-mesures ANOVA. Testing for the presence of correltion ws performed using Spermn s rnk correltion coefficient.

4 Act Orthopedic 21; 81 (5): Figure 3. A. Anteroposterior nd lterl rdiogrphs reveling the formtion of tubulr structures of distrction cllus t 4 weeks. Thickening of corticl bone nd progression of the remodeling process were observed from 4 to 8 weeks. Overll impednce (kω) Completion of distrction (n=24) (n=18) (n=12) (n=6) Mesurement of the overll impednce of distrction cllus llows ny chnges in electricl properties to be evluted during the cllus mturtion process. Advntges of this method include the bsence of dditionl invsive procedures, since the pins lredy inserted for fixtion re used s electrodes nd mesurements cn be performed esily nd fre- Osteotomy 1 p <.1 (by ANOVA) B. Grph showing sequentil chnges in overll impednce. The impednce vlues incresed considerbly, ccompnying increses in the distnce between fixtion pins due to the 1-mm distrction. Although slight decresing trend ws subsequently observed until 1 week, the impednce vlues incresed over 6 weeks. Reltive chnge in impednce 1.2 p <.1 (by ANOVA) p <.1 vs week 1 (by ANOVA) Completion.1 of distrction (n=24) (n=18) (n=12) (n=6) C. Grph showing the rte of chnge bsed on the vlues t completion of distrction. Although slight decresing trend ws observed over 1 week, the rte of chnge of impednce vlues incresed substntilly over 6 weeks nd remined nerly constnt t tht time. Results Rdiogrphic findings After completion of distrction, cllus ws observed ner the proximl nd distl ends of the osteotomized tibi, while rdiolucent regions were observed in the center of distrction cllus. These minerliztion bnds progressed towrd the center nd fused t 2 weeks. Corticliztion nd medullriztion begn from both the proximl end of the cllus nd the distl end, nd then tubulr formtion by new corticl bone ws observed t 4 weeks. Thickening of corticl bone nd progression of the remodeling process were observed from 4 to 8 weeks (Figure 3A). Overll impednce The overll impednce incresed fter completion of distrction, ccompnied by n increse in length of 1 mm resulting from cllus distrction. Although the vlues slightly decresed in week 1 fter distrction, they incresed with time fter tht nd showed mximum vlues t weeks 6 8 (p <.1) (Figure 3B). Although the rte of chnge bsed on the impednce vlues t completion of distrction demonstrted slight decresing trend through week 1, it incresed substntilly from 1 6 weeks nd remined nerly constnt t tht time (p <.1). Differences were observed between 1 week nd 4, 5, 6, 7, nd 8 weeks (p <.1) (Figure 3C). There ws positive correltion between the vlues of overll impednce nd bone impednce obtined fter removl of soft tissues including periosteum (p <.5, correltion coefficient.43). Bone resistivity Although the vlues of bone resistivity decresed from weeks 2 to 4 fter completion of distrction, they subsequently remined nerly constnt over 8 weeks. No sttisticlly significnt differences were observed (Figure 4A). Cross-sectionl re of cllus The cllus cross-sectionl re decresed over time, ccompnying mturtion of the distrction cllus nd reching minimum t 6 weeks fter completion of distrction. Differences were observed between 2 weeks nd 4, 6, nd 8 weeks (p <.5, p <.1, nd p <.1, respectively) (Figure 4B). Mximum bending stress The mximum bending stress incresed over time, ccompnying mturtion of the distrction cllus. After hving become nerly constnt strting t 6 weeks, there were no differences observed between 6 nd 8 weeks. However, differences were observed between 2 weeks nd 4, 6, nd 8 weeks (p <.1) nd lso between 4 weeks nd 6 nd 8 weeks (p <.5) (Figure 4C). There ws negtive correltion between cllus sectionl re nd mximum bending stress (p <.1; correltion coefficient.79) (Figure 4D). Discussion

5 632 Act Orthopedic 21; 81 (5): Bone resistivity (ρ) (Ωm) Figure 4. A. Grph showing chnges in bone resistivity vlues. The vlues decresed substntilly from 2 to 4 weeks fter completion of distrction nd subsequently remined nerly constnt. Mximum bending stress (B mx ) (N/mm 2 ) p <.1 vs week 2, b p <.5 vs week 4 (by ANOVA),b Cllus cross-sectionl re (CS-A) (mm 2 ) quently. Kwmoto et l. (25) investigted enhncement of mturtion of distrction cllus nd mesured the impednce of excised bone. Hirshim et l. (29) mesured overll impednce in ptients with distl end frcture of the rdius nd they reported tht impednce vlues incresed concomitnt with bone union. We lso investigted the increses in overll impednce ccording to the frcture heling process nd found tht the vlues peked t the time when mechnicl strength reched plteu (Yoshid et l. 29). However, there hve been no studies nlyzing the chnges in impednce vlues of distrction cllus, nd the resons for the temporl increses in impednce vlues of bone with intrmedullry,b C. Grph showing chnges in mximum bending stress vlues. The vlues incresed over time, nd differences were observed between 2 weeks nd 4, 6, nd 8 weeks, nd lso between 4 weeks nd 6 nd 8 weeks p <.5, b p <.1 vs week 2 (by ANOVA) B. Grph showing chnges in cllus cross-sectionl re vlues. The vlues decresed over time during cllus mturtion, reching minimum t 6 weeks. Differences were observed between 2 weeks nd 4, 6, nd 8 weeks. Cllus cross-sectionl re (mm 2 ) Mximum bending stress (B mx ) (N/mm 2 ) b y = -1.2x r = -.79, p <.1 D. Grph showing correltion between cllus cross-sectionl re nd mximum bending stress. b blood flow still remin unknown. West nd Astle (1981) mesured the resistnce rte in representtive tissues to be 3, Wm in dry bone nd 3 Wm in corticl bone moistened with physiologicl sline. Thus, the intrmedullry blood flow is importnt for evlution of the electricl conductivity of the cllus. One of the objectives of our study ws to identify the fctor elevting the overll impednce with cllus mturtion. The electricl properties of substnce re defined in terms of the distnce between the substnce, the electricl conductivity of the mteril, nd the conduction pthwy. Blck nd Mttson (1982) nd Kosterich et l. (1984) hve evluted increses in electricl conductivity of bone ssocited with chnges in bone mturtion, such s corticliztion nd medullriztion, in vitro. We investigted corticliztion nd medullriztion s chnges in bone resistivity in vivo. We lso evluted the current conduction pthwy by investigting osteomorphologicl chnges in the crosssectionl re of the distrcted region. The mesurement distnce between P2 nd D1 remined constnt t 35 mm fter completion of distrction, while the bone resistivity nd the cross-sectionl re of the distrction cllus fluctuted during cllus mturtion. The bone resistivity decresed until 4 weeks fter the completion of distrction, which influenced the bone by reducing the impednce. As fctor elevting the impednce, reduction of cllus cross-sectionl re, i.e. reduction of the conduction pthwy, ws substntilly involved, s previously observed in rbbit frcture model (Yoshid et l. 29), nd strong negtive correltion ws noted between the cllus sectionl re nd mximum bending stress. This shows tht the reduction of cllus cross-sectionl re influences the elevtion of impednce s the mechnicl strength increses. We lso considered tht increses in the mechnicl strength nd impednce re involved through chnges in the cross-sectionl re of cllus. We found tht increses in the impednce dispper s chnges in the cllus cross-sectionl re dispper. The process reches bone mturtion t this time point, which ws lso supported by the mechnicl test findings. However, the impednce vlues would be influenced by the pthwy of the current, nd mximum bending stress is influenced by the trbeculr structure, such s corticliztion. The vlues of over-

6 Act Orthopedic 21; 81 (5): ll impednce re chnged by ll the fctors ssocited with chnges in bone structure nd morphology during bone mturtion. Regrding the limittions of this study, detiled verifiction of the bone mturtion, structure, nd morphology by histologicl investigtion nd μct remins to be performed, nd we re plnning to do this in the future. We consider tht bone mturtion should be evluted in individul cses bsed on the time course of chnges in overll impednce, nd not the bsolute vlue. Our dt suggest tht mesurement of the temporl chnges in impednce vlues over time my be useful for evlution of cllus mturtion. TY, WCK, nd TK designed the study. TY, YO, nd NY performed the experiments, nd collected nd nlyzed the dt. TY wrote the drft mnuscript. TY, WCK, nd TK revised it. This study ws supported by reserch grnts from Nktni Foundtion of Electronic Mesuring Technology Advncement, Tokyo, Jpn. No competing interests declred. Arnes G T, Steen H, Kristinsen L P, Festo E, Ludvigsen P. Optimum loding mode for xil stiffness testing in limb lengthening. J Orthop Res 26; 24: Bil H J, Kolbeck S, Krummrey G, Weiler A, Windhgen H J, Hennies K, Run K, Rschke MJ. Ultrsound cn predict regenerte stiffness in distrction osteogenesis. Clin Orthop 22; (357): Blck J, Mttson R U. Reltionship between porosity nd minerliztion in the hversin osteon. Clcif Tissue Int 1982; 34: Dwyer J S, Owen P J, Evns G A, Kuiper J H, Reichrdson J B. Stiffness mesurements to ssess heling during leg lengthening: preliminry report. J Bone Joint Surg (Br) 1996; 78: Eyres K S, Bell M J, Knis J A. Methods of ssessing new bone formtion during limb lengthening: ultrsonogrphy, dul energy X-ry bsorptiometry nd rdiogrphy compred. J Bone Joint Surg (Br) 1993; 75: Hrp J H, Aronson J, Hollis M. Noninvsive determintion of bone stiffness for distrction osteogenesis by quntittive computed tomogrphy scns. Clin Orthop 1994; (31): Hirshim T, Kim W C, Kwmoto K, Tsuchid Y, Ok Y, Hosokw M, Yoshid T, Tsuji Y, Kudo T. Evluting of bone union of distl rdius frcture by mesuring impednce vlues. Orthopedics 29; Kwmoto K, Kim W C, Tsuchid Y, Tsuji Y, Fujiok M, Horii M, Mikmi Y, Tokung D, Kubo T. Effects of lternting current electricl stimultion on lengthening cllus. J Peditr Orthop B 25; 14: Kosterich J D, Foster K R, Pollck S R. Dielectric properties of fluid sturted bone the effect of vrition in conductivity of immersion fluid. IEEE Trns Biomed Eng 1984; 31: Miytni M, Knehis H, Msuo Y, Ito M, Fukung T. Vlidity of estimting limb muscle volume by bioelectricl impednce. J Appl Physiol 21; 91: Ohmine Y, Morimoto T, Kinouchi Y, Iritni T, Tkeuchi M, Hku M, Nishitni H. Bsic study of new dignostic modlity ccording to non-invsive mesurement of the electricl conductivity of tissues. J Med Invest 24; 51: Reichel H, Lebek S, Alter C, Hein W. Biomechnicl nd densitometric bone properties fter cllus distrction in sheep. Clin Orthop 1998; (357): Sh S, Willims P A. Comprison of the electricl nd dielectric behvior of wet humn corticl nd cncellous bone tissue from the distl tibi. J Orthop Res 1995; 13: Shrp A. Bicycles & Tricycles: A Clssic Tretise on Their Design nd Construction. Mineol, NY: Dover; 23. Sierpowsk J, Hkulinen M A, Töyräs J, Dy J S, Weinns H, Jurvelin J S, Lpplinen R. Prediction of mechnicl properties of humn trbeculr bone by electricl mesurements. Physiol Mes 25; 26: Simpson A H, Kenwright J. Frcture fter distrction osteogenesis. J Bone Joint Surg (Br) 2; 82: Tselentkis G, Owen P J, Richrdson J B, Kuiper J H, Hddwy M J, Dwyer J S, Evns G A. Frcture stiffness in cllotsis determined by dul-energy x-ry bsorptiometry scnning. J Peditr Orthop B. 21; 1: West R C, Astle M J. CRC hndbook of chemistry nd physics. 62nd edn. Boc Rton, FL: CRC Press; 1981: Windhgen H, Kolbeck S, Bil H, Schmeling A, Rschke M. Quntittive ssessment of in vivo bone regenertion consolidtion in distrction osteogenesis. J Orthop Res 2; 18: Yoshid T, Kim W C, Kwmoto K, Hirshim T, Ok Y, Kubo T. Mesurement of bone electricl impednce in frcture heling. J Orthop Sci 29; 14: 32-9.