Alfacalcidol prevents age-related bone loss and causes an atypical pattern of bone formation in aged male rats

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1 J Musculoskel Neuron Interct 24; 4(1):22-32 Originl Article Hylonome Alfclcidol prevents ge-relted one loss nd cuses n typicl pttern of one formtion in ged mle rts M. Li 1, D.R. Hely 1, Y. Li 1, H.A. Simmons 1, M. Su 2, W.S.S. Jee 2, V.W. Shen 3, D.D. Thompson 1 1 Osteoporosis Reserch, Deprtment of Crdiovsculr nd Metolic Diseses, Pfizer Glol Reserch nd Development, Groton, CT, 2 Rdioiology Division, University of UT School of Medicine, Slt Lke City, UT, 3 Skeletech, Inc., Bothell, WA, USA Astrct The current study ws designed to investigte the skeletl effects of lfclcidol in ged rts. Eighteen-month-old mle rts were treted with,.1, or.2 Ìg/kg/d of lfclcidol y dily orl gvge, 5 dys/week for 12 weeks. At the eginning of the tretments, one group of rts ws euthnized to serve s seline control. At the end of the study, the second lumr vertere nd the right tiil diphysess were processed for one histomorphometric nlysis. The fourth lumr vertere were sujected to strength testing. The control group of rts t 21 months of ge hd decresed serum testosterone levels nd decresed cncellous one mss ssocited with incresed one turnover on the treculr surfce. The older rts hd incresed one turnover on the endocorticl surfce nd decresed one formtion on the periostel surfce compred with the 18-month group. In contrst, lfclcidol tretment incresed cncellous nd corticl one mss in ged mle rts. Treculr one resorption ws decresed wheres one formtion ws mintined or incresed in the rts treted with lfclcidol. In ddition, endocorticl one formtion ws decresed wheres periostel one formtion ws incresed in the rts treted with lfclcidol compred with vehicle-treted rts. Mrrow treculr one re ws incresed y lfclcidol tretment in tiil diphyses. Furthermore, one strength of the lumr verterl ody ws incresed fter lfclcidol tretment. An typicl pttern of one formtion on endostel one surfces ws seen in the rts treted with lfclcidol. The typicl one formtion is chrcterized y smll, focl pckets of newly formed one on treculr nd endocorticl one surfces. This gve the ppernce of the formtion of "one uds" emnting from treculr surfces. These ony outgrowths were minerlized nd demonstrted significnt fluorochrome lel indicting recent minerliztion. Also, lmelle of the ony uds did not run prllel to those of the treculr plte to which they re ttched. Arrest lines presented in most of the "one uds". In summry, lfclcidol tretment incresed cncellous nd corticl one mss nd improved one strength, resulting in the prevention of ge-relted one loss in ged mle rts. An typicl pttern of one formtion oserved in this study my e result of minimodeling sed one formtion stimulted y lfclcidol tretment. Keywords: Alfclcidol, Bone Formtion, Bone Resorption, Bone Mss, Bone Strength Introduction Authors Li, Hely, Li, Simmons, Thompson hve corporte ppointments with Pfizer, Inc. nd stock ownership with Pfizer, Inc; Author Shen hs corporte ppointment with SkeleTech, Inc; Author Jee hs stock in Pfizer, Inc. nd contrct funding with Pfizer, Inc.; Author Su hs no conflict of interest. Corresponding uthor: Dr. M. Li, Deprtment of Crdiovsculr nd Metolic Diseses, MS8118W-28, Pfizer Glol Reserch nd Development, Groton, CT 634 E-mil: mei_li@groton.pfizer.com Accepted 21 Jnury 24 Vitmin D is n essentil fctor for the development nd mintennce of skeleton. In humns, clcitriol (1,25-dihydroxyvitmin D 3 or 1,25(OH) 2 D 3 ) nd its prodrug, lfclcidol (1 -hydroxyvitmin D 3 or 1 (OH)D 3 ) reduce verterl nd hip frctures in postmenopusl nd senile osteoporosis, s well s in glucocorticoid-induced osteoporosis 1-9. However, the mechnism of ction of the ctive vitmin D metolites on one s it contriutes to the frcture reduction remins uncler. In vitro nd in vivo studies hve shown tht clcitriol hs stimultory effects on osteoclstogenesis nd osteoclst ctivity This effect on osteoclsts is elieved to e medited through osteolstic cells 15,16. However, the effects of clcitriol on one resorption re countercted in vivo y suppression of prthyroid hormone (PTH) secretion through oth direct inhiitory effect on the prthyroid glnds nd n indirect effect vi stimultion of intestinl clcium sorption nd susequent rise in serum clcium 17. In recent yers, there is incresing evidence to support the 22

2 M. Li et l.: Effects of lfclcidol in ged mle rts BV/TV (%) A Bseline c N.Oc/BS (#/mm) Vehicle D 3(.1 Ìg) D 3(.2 Ìg) B Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) C Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) BFR/BS (Ìm 2 /Ìm/d) Figure 1. Cncellous one volume (BV/TV), osteoclst numer (N.OC/BS), nd surfce-sed one formtion rte (BFR/BS) of 2 nd lumr vertere of seline rts nd the rts treted with either vehicle or lfclcidol t.1 or.2 Ìg/kg/d, 5 dys/week, for 12 weeks. Dt re expressed s men ± SEM. p <.5 vs. seline; p <.5 vs. vehicle; c p <.5 vs. low dose lfclcidol. Ec.MS/BS Ec.MAR (Ìm/d) A Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) B Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) C Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) Ec.BFR/BS (Ìm 2 /Ìm/d) Pc.MS/BS Ps.MAR (Ìm/d) D Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) E Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) F Bseline Vehicle D 3(.1 Ìg) D 3(.2 Ìg) Figure 2. Dynmic corticl one histomorphometric vriles of the tiil diphyses of seline rts nd the rts treted with either vehicle or lfclcidol t.1 or.2 Ìg/kg/d, 5 dys/week, for 12 weeks. Ec.MS/BS: endocorticl minerlizing surfce; Ec.MAR: endocorticl minerl pposition rte; Ec.BFR/BS: endocorticl one formtion rte; Ps.MS/BS: periostel minerlizing surfce; Ps.MAR: periostel minerl pposition rte; Ps.BFR/BS: periostel one formtion rte. Dt re expressed s men ± SEM. p <.5 vs. seline; p <.5 vs. vehicle; c p <.5 vs. low dose lfclcidol. c Ps.BFR/BS (Ìm 2 /Ìm/d) c notion tht vitmin D my effectively stimulte osteolstic ctivity nd exert n nolic effect on one. Osteolsts, the cells tht re responsile for one formtion, possess undnt vitmin D receptors 18,19. Studies in vitmin D receptor (VDR) knockout mice reveled tht the 1,25(OH) 2 D 3 -VDR complex ws essentil for one growth nd one formtion through its physiologicl effect on intestinl clcium sorption 2,21. In ddition, in vitro studies showed tht clcitriol stimulted the synthesis of vriety of noncollgenous proteins such s osteoclcin, mtrix Gl protein, osteopontin, fironectin, s well s lkline phosphtse ctivity in osteolst model systems In intct rts, high doses of clcitriol up-regulted tiil osteoclcin messenger RNA levels 31 nd incresed the numer of osteolst precursor cells in one mrrow of tii s well s stimulted cncellous one formtion in lumr verter 32. Furthermore, clcitriol nd lfclcidol hve een reported to stimulte one formtion nd increse one mss nd strength in ovriectomized (OVX) rts These results suggest tht ctive vitmin D stimultes osteolstic ctivity nd one formtion. However, the skeletl effects of lfclcidol hve not een ssessed in ged niml models, which my e more relevnt to elderly popultion. Therefore, the im of this study ws to investigte the effects of lfclcidol on one in ged mle rts to further understnd the reltionship etween chnges in one formtion nd one mss s well s one strength. 23

3 M. Li et l.: Effects of lfclcidol in ged mle rts Mterils nd methods Animls nd mterils Eighteen-month-old mle Sprgue-Dwley rts (Hrln, Indinpolis, Indin, USA) weighing n verge of 54g were used in this study. The nimls were housed t 24ÆC with 12h light/12h drk cycle. They were llowed free ccess to wter nd were restricted to 28 grms/dy of commercil diet (Purin Lortory Rodent Chow 51, Purin- Mills, St. Louis, MO) contining.95% clcium,.67% phosphorus, nd 4.5 IU/g vitmin D 3. The experiments were conducted ccording to Pfizer niml cre-pproved protocols, nd nimls were mintined in ccordnce with the ILAR (Institute of Lortory Animl Reserch) Guide for the Cre nd Use of Lortory Animls. Forty rts were rndomly divided into 4 groups with 1 per group. One group of rts ws euthnized t the eginning of the experiment to serve s seline. The remining rts were treted with vehicle lone (cottonseed oil) or lfclcidol t.1 or.2 Ìg/kg ody weight/d y dily orl gvge, 5 dys/week for 12 weeks. The doses nd dosing regimen used in this study were sed on their efficcy shown in rts previously Alfclcidol ws purchsed from Cliochem, L Joll, CA. Cottonseed oil, nd clcein were purchsed from Sigm Chemicl Co., St. Louis, MO. The stock solution of lfclcidol ws mde y dissolving lfclcidol in 1% ethnol t concentrtion of.1 mg/ml, protected from light, nd stored t 4ÆC. The dosing solutions were prepred weekly y diluting the stock solution with cottonseed oil vehicle to the concentrtion of.1 Ìg/ml nd.2 Ìg/ml for the.1 Ìg/kg/d nd.2 Ìg/kg/d group, respectively. All rts were injected sucutneously with clcein on -12 nd -2 dys prior to necropsy t dose of 1 mg/kg. This regimen resulted in deposition of single or doule fluorochrome lel t one surfces tht were ctively minerlizing t the time of the injections. At the conclusion of 12 weeks of tretment with lfclcidol, the nimls were fsted 16 hours prior to necropsy. At the dy of necropsy, whole lood smples were collected y crdic puncture for iochemicl ssessments immeditely fter euthnsi y CO 2 sphyxition. Blood smples were centrifuged to otin the ser, which were stored t 2ÆC until ssy. The 2 nd lumr vertere (LV 2 ) nd the right tiil diphyses were hrvested nd prepred for one histomorphometric nlysis. The 4 th lumr vertere (LV 4 ) were hrvested nd prepred for iomechnicl test. Serum iochemistry Serum clcium (C) nd phosphte (P i ) concentrtions were mesured with the Cos Fr 2 nlyzer (Roche Diognostic System, Hoffmn-L Roche Inc. Indinpolis, IN). Totl serum testosterone levels were mesured with Cot-- Count kit-tktt1 (Dignostic Prodocts Corp., Los Angeles, CA). The limit of detection is.4 ng/ml. The cross rectivity is less thn 5% for dihydrotestosterone nd less thn 1% for other steroids, including ll mjor ndrogens nd estrogens. Cncellous one histomorphometry of lumr vertere The LV 2 were fixed in 1% formldehyde for 24 hours nd then dehydrted in grded concentrtion of ethnol nd emedded undeclcified in methyl methcrylte 41. Longitudinl frontl sections of lumr verter were cut t 4- nd 1-Ìm thickness using Reichert-Jung Polycut S microtome (Leic Corp., Deerfield, IL, Heidelerg, Germny). One set of the 4-Ìm sections ws stined with modified Msson s Trichrome stin for sttic histomorphometric mesurements. Another set of the 4-Ìm sections ws stined for cement lines y using surfce-stining technique descried y Eren 42. The 1-Ìm sections remined unstined for the mesurements of fluorochrome-sed indices of one formtion. Histomorphometric mesurements were performed in cncellous one tissue etween.5 mm from the crnil nd cudl growth pltes of the lumr verterl ody using n Imge Anlysis System (Osteomesure, Inc., Altnt, GA). Cncellous one volume s percentge of one tissue re (BV/TV), osteoclst surfces s percentges of totl cncellous perimeter (Oc.S/BS), nd osteoclst numer per one surfce (N.Oc/BS) were mesured in 4-Ìm thick, stined sections. Treculr numer (T.N), thickness (T.Th) nd seprtion (T.Sp) were clculted s descried y Prfitt et l. 43. Fluorochrome-sed indices of one formtion including the percentge of cncellous one surfce with doule fluorochrome lel (minerlizing surfce, MS/BS) nd minerl pposition rte (MAR) were mesured in 1-Ìm thick, unstined sections. In ddition, one formtion rte (one surfce referent) ws clculted y multiplying minerlizing surfce y minerl pposition rte (BFR/BS). Vlues for minerl pposition rte were not corrected for oliquity of the plne of section in cncellous one 44. Corticl one histomorphometry of tiil diphyses The tiil diphyses were fixed in 7% ethnol, stined with Villnuev one stin (Polysciences, Inc., Wrrington, PA) 45, dehydrted in scending concentrtions of ethnol, cetone, nd emedded undeclcified in methyl methcrylte 46. Cross-sections of tiil diphyses (just proximl to the tiiofiulr juction) were swed to 23-Ìm thickness using n isomet low speed sw (Buechler, Lke Bluff, IL) nd then further ground y hnd to out 3 Ìm for corticl histomorphometric mesurements 46. Sttic histomorphometric vriles mesured in tiil diphyses included totl tissue re (T.Ar), percent corticl re (% Ct.Ar), percent mrrow re (% M.Ar), corticl width (Ct.Wi), mrrow treculr one re (M.T.Ar), nd endocorticl percent eroded surfce (ES/BS). Dynmic histomorphometric vriles included the percent minerlizing surfce (Ec.MS/BS), minerl pposition rte (Ec.MAR), nd surfced-sed one formtion rte (Ec.BFR/BS) on the endocorticl surfce nd the percent minerlizing surfce (Ps.MS/BS), minerl pposition rte (Ps.MAR), nd surfced-sed one formtion rte (Ps.BFR/BS) on the periostel surfce. 24

4 M. Li et l.: Effects of lfclcidol in ged mle rts Group Vriles Bseline Vehicle 1 -D 3 (.1 Ìg) 1 -D 3 (.2 Ìg) Body Weight (g) 539 ± ± ± ± 15 Testosterone (pg/ml) 82.9 ± ± ± ± 6. Clcium (mg/dl) 12.4 ± ± ± ±.2 c Phosphorus (mg/dl) 7.4 ± ± ± ±.3 Dt re expressed s men±sem. Bseline: rts were euthnized t the eginning of tretments; Vehicle: vehicle-treted controls; 1 -D 3 (.1 Ìg): rts treted with.1 Ìg/kg/d of lfclcidol; 1 -D 3 (.2 Ìg): rts treted with.2 Ìg/kg/d of lfclcidol. p <.5 vs. seline group; p <.5 vs. vehicle-treted controls; c : p <.5 vs. rts treted with.1 Ìg/kg/d of lfclcidol. Tle 1. Body weights nd iochemicl vriles. Group Vriles Bseline Vehicle 1 -D 3 (.1 Ìg) 1 -D 3 (.2 Ìg) T.N (#/mm) 2.9 ± ± ± ±.1 T.Th (Ìm) ± ± ± ± 5.6 c T.Sp (Ìm) ± ± ± ± 6.6 c Oc.S/BS (%) 1.2 ± ±.2 1. ± ±.1 MS/BS (%) 9.3 ± ± ± ± 1.1 MAR (Ìm/d) 1.2 ±. 1.3 ±. 1.3 ± ±. Dt re expressed s men±sem. Bseline: rts euthnized t the eginning of tretments; Vehicle: vehicle-treted controls; 1 -D 3 (.1 Ìg): rts treted with.1 Ìg/kg/d of lfclcidol; 1 -D 3 (.2 Ìg): rts treted with.2 Ìg/kg/d of lfclcidol. T.N: treculr numer; T.Th: treculr thickness; T.Sp: treculr seprtion; Oc.S/BS: osteoclst surfce; MS/BS: minerlizing surfce; MAR: minerl pposition rte. p <.5 vs. seline group; p <.5 vs. vehicle-treted controls; c p <.5 vs. rts treted with.1 Ìg/kg/d of lfclcidol. Tle 2. Cncellous one histomorphometric vriles in lumr vertere (LV 2 ). Biomechnicl properties Mechnicl properties of LV 4 were determined y compression test 47,48 using n Instron Mechnicl Testing Mchine (Instron 4465 retrofitted to 55, Instron Corportion, Cnton, MA). The spinous process nd posterior pedicle rch were first removed from LV 4 using lowspeed sw (Buechler, Lke Bluff, IL). Both crnil nd cudl ends of epiphyses were lso removed with the sme sw to otin verterl ody specimen with two prllel surfces nd height pproximtely equl to 4 mm. Width in the medil-lterl nd nterior-posterior directions t oth the crnil nd cudl ends ws mesured using digitl clipers. Vlues otined from the two ends were recorded nd the verged vlue ws used in the clcultion of crosssectionl re. The height of the verterl ody specimen ws mesured with the clipers. The specimen ws then compressed to filure t displcement rte of 6 mm/min using 5-kN lod cell. The lod nd displcement curve ws recorded y testing mchine softwre (Merlin II, Instron) from ech test. The loctions for mximum lod t filure, stiffness nd energy sored were selected mnully from the lod nd displcement curve nd then clculted y the mchine softwre. The intrinsic properties, stress, elstic modulus nd toughness were clculted from mximum lod, stiffness, energy sored, cross-sectionl re, nd height with the following equtions. Mchine mesurements Mximum lod (unit: N) (F u ) Stiffness (unit: N/mm) (S) Energy sored (unit: mj) (W) 25

5 M. Li et l.: Effects of lfclcidol in ged mle rts Group Vriles Bseline Vehicle 1 -D 3 (.1 Ìg) 1 -D 3 (.2 Ìg) T.Ar (mm 2 ) 6.42 ± ± ± ±.1 %Ct.Ar (%) 81.2 ± ± ± ±.8 %M.Ar (%) 18.8 ± ± ± ±.8 Ct.Wi (Ìm) 89.1 ± ± ± ± 21.2 M.T.Ar (Ìm 2 ). ±. 1.5 ± ± ±.2 ES/BS (%) 2.4 ± ± ± ±.1 Dt re expressed s men±sem. Bseline: rts were euthnized t the eginning of tretments; Vehicle: vehicle-treted controls; 1 -D 3 (.1 Ìg): rts treted with.1 Ìg/kg/d of lfclcidol; 1 -D 3 (.2 Ìg): rts treted with.2 Ìg/kg/d of lfclcidol. T.Ar: totl tissue re; %Ct.Ar: percent corticl re; %M.Ar: percent mrrow re; Ct.Wi: corticl width; M.T.Ar: mrrow treculr one re; ES/BS: percent eroded surfce. p <.5 vs. seline group; p <.5 vs. vehicle-treted controls; c p <.5 vs. rts treted with.1 Ìg/kg/d of lfclcidol. Tle 3. Sttic histomorphometric vriles in the tiil diphyses. Cliper mesurements Width in the nterior-posterior direction (unit: mm) () Width in the medil-lterl direction (unit: mm) () Height of the verterl ody in the crnio-cudl direction (unit: mm) (h) Derived prmeters Cross-sectionl re (unit: mm 2 ) (CSA) Formul CSA = p * * /4 Stress (unit: N/mm) (Û) Formul Û = F u /CSA Elstic modulus (unit: MP) (E) Formul E = S/(CSA/h) Toughness (unit: MJ/m 3 ) (T) Formul T = W/(CSA*h) Sttisticl nlysis Dt re expressed s the men ± SEM for ech group. Sttistics were clculted using SttView 4. pckges (Acus Concepts., Inc., Berkeley, CA). Sttisticl differences etween groups were evluted with ANOVA followed y the Fisher PLSD test for multiple comprisons. Proilities (p) less thn.5 were considered significnt. Results Body weights nd serum iochemistry. The men ody weights of ech group remined constnt throughout the experimentl period nd were not significntly different from ech other (Tle 1). Serum testosterone ws significntly lower in the rts t 21 months of ge thn t 18 months of ge (Tle 1). This prmeter ws not ltered y lfclcidol tretments. The rts treted with lfclcidol demonstrted significntly incresed serum clcium in dose responsive mnner when compred to those treted with vehicle (Tle 1). Serum phosphorous levels were lso significntly incresed in dose responsive mnner in rts treted with lfclcidol s compred to those treted with vehicle (Tle 1). Lumr vertere (LV 2 ). Bone histomorphometric dt from LV 2 re shown in Figure 1 nd Tle 2. Bone volume in the 21-month control rts ws significntly decresed wheres treculr seprtion, osteoclst surfce, osteoclst numer, minerlizing surfce, minerl pposition rte, nd one formtion rte were incresed compred with 18- month-old rts. A trend towrd decresed treculr numer nd treculr thickness ws lso seen in 21-month-old rts reltive to 18-month-old rts. In contrst, one volume of the rts treted with.1 or.2 Ìg/kg/d of lfclcidol ws incresed y 31% or 45%, respectively, compred with tht of vehicle-treted rts. Treculr numer nd treculr thickness were significntly incresed wheres treculr seprtion ws significntly decresed in the rts treted with lfclcidol compred with seline nd vehicle-treted controls. When compring the two lfclcidol-treted groups, the rts treted with the higher dose of lfclcidol hd higher vlues for one volume nd treculr thickness ut lower vlues for treculr seprtion. Osteoclst surfce nd osteoclst numer were significntly decresed fter lfclcidol tretments reltive to vehicle tretments with the osteoclst numer decresing elow seline level. Both minerlizing surfce nd one formtion rte in the rts treted with lfclcidol were significntly lower thn those of rts treted with vehicle ut higher thn those of seline controls. The minerl pposition rte for the rts treted with lfclcidol t oth doses ws not different from those treted with vehicle ut it ws significntly higher for the rts treted with the higher dose of lfclcidol compred with seline controls. Tiil diphyses. Bone histomorphometric dt from tiil diphyses re listed in Tle 3 nd shown in Figure 2. When compred with the 18-month-old rts, the 21-month- 26

6 M. Li et l.: Effects of lfclcidol in ged mle rts Group Vriles Bseline Vehicle 1 -D 3 (.1 Ìg) 1 -D 3 (.2 Ìg) Mximum Lod (N) 258 ± ± ± ± 19 Stiffness (N/mm) 1385 ± ± ± ± 134 Energy Asored (mj) 4.8 ± ± ± ± 5.2 Ultimte Strength (N/mm 2 ) 2.4 ± ± ± ± 2. Elstic Modulus (MP) ± ± ± ± 85.3 Toughness (mj/mm 2 ).77 ±.7.61 ±.6.92 ±.9.94 ±.8 c Dt re expressed s men±sem. Bseline: rts euthnized t the eginning of tretments; Vehicle: vehicle-treted controls; 1 -D 3 (.1 Ìg): rts treted with.1 Ìg/kg/d of lfclcidol; 1 -D 3 (.2 Ìg): rts treted with.2 Ìg/kg/d of lfclcidol. p <.5 vs. seline group; p <.5 vs. vehicle-treted controls; c p <.5 vs. rts treted with.1 mg/kg/d of lfclcidol. Tle 4. Biomechnicl vriles ssessed y compression test in lumr vertere (LV 4 ). old rts hd significnt increse in percent mrrow re, endocorticl eroded surfce, endocorticl minerlizing surfce, endocorticl minerl pposition rte, nd endocorticl one formtion rte nd significnt decrese in percent corticl re, periostel minerlizing surfce, periostel minerl pposition rte, nd periostel one formtion rte. The men vlues of totl tissue re nd corticl width in the 21- month-old rts were not significntly different from those of 18-month-old rts. In contrst, the rts treted with lfclcidol t oth doses exhiited significntly incresed totl tissue re, percent corticl re, corticl width nd decresed percent mrrow re compred with those treted with vehicle. There ws no treculr one oserved in the mrrow cvity of seline rts. Four out of ten of vehicle-treted rts presented one or two miniscule treculr spicules, which contriuted to the verge of 1.5-Ìm 2 mrrow treculr one re. However, treculr one ws seen in the mrrow cvity of eight out of ten rts treted with.1 Ìg/kg/d of lfclcidol nd the verge mrrow treculr one re ws incresed to 39-Ìm 2. All rts treted with.2 Ìg/kg/d of lfclcidol hd significnt mount of treculr one in their mrrow cvity (Figure 3), which hd n verge re t 58- Ìm 2. Endocorticl eroded surfce of the rts treted with lfclcidol ws significntly decresed reltive to those of oth seline nd vehicle-treted rts. Interestingly, the men vlues for the endocorticl one formtion prmeters including endocorticl minerlizing surfce, endocorticl minerl pposition rte (except higher dose) nd endocorticl one formtion rte were significntly lower wheres the men vlues for the periostel one formtion prmeters including periostel minerlizing surfce, periostel minerl pposition rte, nd periostel one formtion rte were significntly higher in the rts treted with lfclcidol thn those treted with vehicle. The periostel minerl pposition rte nd periostel one formtion rte for the rts treted with higher dose of lfclcidol were significntly higher thn tht those treted with lower dose of the compound. The men vlue of periostel one formtion rte for the rts treted with higher dose of lfclcidol ws lso significntly greter thn tht of seline controls. Bone iomechnicl strength. Mechnicl properties of LV 4 s determined y compression testing re shown in Tle 4. A trend towrd decresed mximum lod (-13%), stiffness (-6%), energy (-23%), ultimte strength (-12%), elstic modulus (-6%), nd toughness (-21%) ws oserved in the rts t 21 months of ge compred with the rts t 18 months of ge. However, the mximum lod of LV 4 in the rts treted with.1 or.2 Ìg/kg/d of lfclcidol ws significntly incresed y 32% or 43%, respectively, compred with vehicle-treted rts. The men vlue of stiffness ws not significntly chnged fter tretment with either vehicle or lfclcidol. Energy sorption, ultimte strength, toughness, nd elstic modulus were significntly incresed y lfclcidol tretments compred with vehicle tretments. The men vlues for mximum lod, ultimte strength nd toughness in the rts treted with the higher dose of lfclcidol were lso significntly higher thn those of seline controls. "Bone Buds". An unusul pttern of one formtion on endostel one surfces (treculr nd endocorticl surfces) ws oserved in the rts treted with lfclcidol (Figure 4). In cncellous one, n typicl one formtion pttern ws oserved, chrcterized y smll, focl pckets of newly formed one on treculr one surfces. This gve the microscopic ppernce of the formtion of "one uds" emnting from treculr surfces. The "one uds" ppered to e rndomly formed long the one surfces. Also they were minerlized nd demonstrted significnt fluorochrome lel indicting recent minerliztion. Under polrized light, it ws oserved tht the lmelle of the "one ud" did not run uniformly prllel to those of the treculr plte to which they re ttched. Arrest lines were seen in most of the "one uds", indicting tht the formtion of "one uds" ws initit- 27

7 M. Li et l.: Effects of lfclcidol in ged mle rts Figure 3. Microscopic oservtion of the spicules produced y lfclcidol tretments in the mrrow cvity of tiil diphysis. Note very smll spicule (rrow) in rt treted with vehicle (A) nd two lrger size spicules (rrow) in the rts treted with higher dose of lfclcidol (B). Villnuev one stined. M: mrrow; Cor: cortex. Originl mgnifiction: X25. ed on quiescent surfce. Some "one uds" hd single rrest line t the order where they connected with the ttched treculr plte. Some "one uds" hd severl rrest lines independent of their size. Similr "one uds" were oserved on the endocorticl surfce. These "one uds" were oserved in the rts treted with higher nd lower doses of lfccidol with more pronounced in the rts treted with higher dose of the compound. No evidence of osteomlci ws oserved in these nimls treted with lfclcidol. Discussion The current study demonstrted tht lfclcidol incresed oth cncellous nd corticl one mss s well s one strength, resulting in the prevention of ge-relted one loss in ged mle rts. The loss of verterl cncellous one seen in the rts t 21 months of ge reltive to those t 18 months of ge ws ssocited with incresed one turnover s evident y incresed osteoclst surfce, minerlizing surfce, minerl pposition rte, nd one formtion rte in these nimls. Similr findings were seen t the proximl tiil metphyses of the sme rts in this study (dt not shown). In corticl one of tiil diphyses, high one turnover ws evident on the endocorticl surfce wheres decresed one formtion ws seen on the periostel surfce, resulting in decrese in percent corticl one re nd n increse in percent mrrow re. These chnges in the 21-month-old mle rts were similr to those reported in the ndrogen deficient rts induced y orchidectomy Therefore, the significntly decresed testosterone level towrd hypogondl stte oserved in this study is likely cuse of one loss in these 21 month-old mle rts. Consistent with previous findings in reltively young rts treted with ctive vitmin D metolites 33-35,37-4,52, our dt showed tht lfclcidol dose-dependently incresed cncellous one mss nd improved treculr structure resulting in n increse of one strength in the lumr verterl odies of ged mle rts. In ddition, lfclcidol incresed corticl one mss y inhiiting the incresed one resorption nd turnover on the endocorticl surfce nd y stimulting one formtion on the periostel surfce in ged rts. These effects of lfclcidol on one completely prevented the gerelted cncellous one loss nd corticl one chnges nd dded dditionl one to the skeleton of ged rts. In concert with previous studies in younger rts, the current study demonstrtes tht the skeletl effects of lfclcidol re independent of ge in rts. The suppressed one resorption on the treculr nd endocorticl surfces y lfclcidol tretment in ged mle rts in the current study is consistent with the previous findings in reltively young rts 33-35,37,39,4,52. Similr effects hve lso een documented in OVX dogs 53,54 nd postmenopusl women treted with clcitriol 5. The inhiitory effect of lfclcidol on one resorption ws ccompnied with dosedependent increse in serum clcium levels in ged rts. The oserved elevtion of serum clcium is proly due to incresed intestinl sorption of clcium y lfclcidol tretments. Although serum PTH levels were not determined in this study, previous studies hve clerly shown decresed serum PTH levels fter lfclcidol tretments in rts 34,4. Since PTH is n importnt physiologicl regultor of one resorption nd one turnover, the suppression of one resorption y lfclcidol in the current study is considered to e lrgely consequence of diminished PTH ctivity. The effects of lfclcidol tretment on one formtion in ged mle rts were vried on different one surfces. In cncellous one of lumr verter, lthough one formtion rte in ged rts treted with lfclcidol ws lower thn tht of ge-mtched controls, ut treculr one volume of lfclcidol-treted rts ws significntly incresed ove seline controls. These dt indicted tht lfclcidol tretment, if not incresed, t lest mintined the overll one 28

8 M. Li et l.: Effects of lfclcidol in ged mle rts Figure 4. Microscopic oservtion of the "one uds" produced y lfclcidol tretments. Lumr verterl cncellous one tissue from vehicle-treted rt (A) nd lfclcidol treted rts (B-F). Note "one uds" (rrows) pper on the treculr nd endocorticl surfces in the rt treted with higher dose of lfclcidol (B-F). The "one uds" hve significnt fluorochrome lel (B) nd possess single or multiple smooth cement lines (rrow heds, C nd D). The lmelle of "one uds" do not run prllel to those of the treculr plte or cortex to which they re ttched (E nd F, viewed under polrized light). Unstined (A nd B), toluidine lue surfce stin (C nd D), nd modified msson s trichrom stin (E nd F). T: treculr one; M: mrrow; Cor: cortex. Originl mgnifiction: X25. formtion in the ged mle rts. In other words, during the course of the lfclcidol tretment period, the comintion of mintined one formtion nd decresed one resorption on cncellous one surfces of ged rts produced positive one lnce etween one formtion nd resorption resulting in thickening of treculr one nd net one gin in these ged nimls. In corticl one, our dt showed tht lfclcidol tretment prevented the incresed one resorption nd mintined one formtion on the endocorticl one surfce in ged rts with decresed testosterone levels. These comintion effects resulted in net one gin on this one surfce nd ccounted for decrese in mrrow cvity 29

9 M. Li et l.: Effects of lfclcidol in ged mle rts re. On the periostel surfce, lfclcidol not only prevented the decresed one formtion ut lso mintined (t lower dose) or even incresed (t higher dose) one formtion in ged rts. Interestingly, mrrow treculr one of tiil diphyses ws significntly incresed in ged rts fter 12 weeks of lfclcidol tretment in the current study. In considering the lck of treculr one spicules in the seline rts, this finding my suggest tht lfccidol tretments lso stimulted one formtion without pre-existing one surfce nd creted new treculr spicules in mrrow cvity in ged rts. The disprity etween the existence of few spicules of one in the 21 month-old vehicle group s compred to the 18 month-old seline group cn not e explined. It is noted tht this is rre occurrence nd only very smll mount of one is oserved. Since one or two very smll treculr spicules were oserved t the tiil diphyses in four out of ten vehicle-treted rts, we cnnot rule out the possiility tht lfclcidol tretment enlrged previously existing spicules within one mrrow. Although lfclcidol tretment t the doses used in the current study dose-dependently incresed one on ll one surfces in ged mle rts, its ction on different surfces ws vried. On the treculr or endocorticl surfces, where one resorption nd turnover were incresed, lfclcidol tretment inhiited osteoclst one resorption nd decresed one turnover, nd t the sme time it stimulted focl one formtion nd formed "one uds" (discussed next). On the periostel surfces, where one formtion ws decresed, lfclcidol tretment stimulted one formtion. However, whether the effects of lfclcidol nd other ctive vitmin D nlogs on one formtion re direct or indirect due to the incresed intestinl clcium sorption remins uncler. The dt from studies with Ro , vitmin D nlog reported to e selective in osteolst cells compred with intestinl cells support direct effect of ctive vitmin D on one formtion. This nlog up-regultes osteolst-specific gene products such s osteoclcin nd osteopontin s well s TGF- 1 nd - 2 mrna 55. It lso increses osteolst numer nd cncellous one mss t nonclcemic dose in OVX rts. Another vitmin D nlog, ED-71, provided similr profile 56,57 nd hs een shown to increse lumr verterl one minerl density in osteoporotic ptients 58. However, highly selective or one specific nlogs with significntly reduced or eliminted hyperclcemic effects re needed for fully understnding the direct ction of ctive vitmin D on one. Another interesting finding of this study ws n typicl pttern of one formtion, "one uds," oserved in the rts treted with lfclcidol for 3 months. This finding hs not een previously reported lthough similr experiments hve een crried out in reltively younger rts, which were treted with the sme dose nd durtion of lfclcidol 37. However, similr oservtion ws reported y Eren et l. in dult rts hving received phrmcologicl doses of clcitriol 59,6. Bsed on their microntomicl feture, the "one uds" re likely result of the ccumultion of micro pcket one formtion on one surfce stimulted y lfclcidol tretment minly through minimodeling processing, where one formtion occurs on quiescent one surfces without preceding osteoclst one resorption 61. Evidence of minimodeling ws found in tiil nd verterl cncellous one in rts 42 nd more importntly, it hs lso recently een reported in the trnsilic iopsy specimens from the ptients who underwent totl hip rthroplsty 62. The ppernce of the foclly formed one t the minimodeling site in these humn one specimens is similr to some of the "one uds" oserved in the rts treted with lfclcidol in the current study. Another interesting feture of the "one uds" is the successive lyers of rrest lines. Some "one uds" hve severl lyer of rrest lines suggesting tht they hve formed through multiple formtion periods or cycles. On the other hnd, some "one uds" hve only single rrest line tht is locted t the se of the "one uds" suggesting tht they my hve formed y single continuous formtion period. The mount of one formed in the "one uds" within single rrest line suggests tht lfclcidol tretment my hve ugmented the ility of osteolst to synthesize nd minerlize mtrix. Chnges in the microenvironment in the mrrow cvity, significntly incresed serum clcium levels, nd the durtion of the tretment my contriute to the initition of this microscopic pttern of loclly destined one formtion. In summry, lfclcidol tretment incresed cncellous nd corticl one mss nd improved one strength in ged mle rts, resulting in the prevention of ge-relted one loss. A unique pttern of one formtion oserved in this study my e result of minimodeling sed one formtion stimulted y lfclcidol tretment. References 1. Aloi, JF, Vswni A, Yeh JF. Clcitriol in the tretment of postmenopusl osteoporosis. Am J Med 1988; 84: Gllgher JC, Riggs BL, Recker RR, Goldgr D. The effect of clcitriol on ptients with postmenopusl osteoporosis with specil reference to frcture frequency. Pro Soc Exp Biol Med 1989; 191: Gllgher JC, Goldgr D. Tretment of postmenopusl osteoporosis with high doses of synthetic clcitriol. A rndomized controlled study. Ann Intern Med 199; 113: Tilyrd MW, Spers GF, Thomson J, Dovey S. Tretment of postmenopusl osteoporosis with clcitriol or clcium. N Engl J Med 1992; 326: Sirnen S, Kärkkäinen M, Tähtelä R, Litinen K, Mäkelä P, Lmerg-Allrdt C, Välimäki MJ. Bone mss nd mrkers of one nd clcium metolism in postmenopusl women treted with 1,25- Dihydroxyvitmin D (Clcitriol) for four yers. Clcif Tissue Int 2; 67: Shirki M, Ito H, Orimo H. 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