Effect of dihydrotachysterol on bone induction in ovariectomized rats

359

Effect cjf dihydrotachysterol on bone induction in ovariectomized rats Chikage Tabuchi’, David J. Simmons2,Aurora Fausto’, lzhak Bindermad and Louis V. Avioli’

Summary we havedemonstratedvia marr”v atromatCEH Eld1”TC6 and the aaeoinductiverclpanmIOdeminera. lizedbone@tr (DSMj i.*, ,ilUcvr,icaibu.. &r&f in ,hc ovrrirtamized (CWX),a.tMueeLspostop) ispdmarilydue

,oimpaircdo~,eopraS~ni,orcellproliiera,ion.

andthatdihydmtachy~tsrol

(DHT)

men, can be protective. In cultured marrow stromal cells from DVX rats. short-lem~ DHT-R,

mar-

uagger-

a,ed the already subnormal pattern 01 ma,,ow stromal cell proliferation. However. in DBM grafts. DHT lrealmen, benefited the time-course of mcxnchymal cell DNAsyntheris asmsasurcd by triliated Ihymid,ne incorporation z!+ weagenic cell ma,u,a,irn as measured by alkaline phmphatase mncen,m,ian. and established a r”SSes,ive ,rcnd D ward normalizado” of bone forma,io~minera,iza,ion (24 h %a incorporation). The dam from this animal model infer that DHT could moderate the bone (OS normally ~fcn in ovarieclomized mavia an activation of the arfeapmgenitorcell

population.

Key wwdm Osreoinduaion; Bone morphogenetie protein; Deminerakzed bone; Dihydmrachywsrol; Alkaline phospharau; Ovaris,amy

Introduction Although the ovariectomized rat is an inexact model for the postmenopausal osteoporotic subject, the animal has been used extensively to study the biological mechanisms involved in this metabolic disorder which has significant medical problems and social implications [1.17,21,22,28,29.32,34.38,39]. Importantly, like certain populations of women who show elevated #terns of bone turnover after the menopause [2.31,33], they and ovariectomized rats rapidly lose both trabecular and cortical bone mass [I ,32.39]. Correspondence 10: David I. Simmons, Ph.D., University of Texas Medical Branch, Depanmen, Surgery, Division Orthopedic Surgery, Galveston. TX77550, USA.

0169&WS91$03.S0~

198.9Ekvier

Science Publishers B.V. (Biomedical Divirion)

of

360 Protracted

vitamin D administration

with its active skeletal metabolite,

(12-16

months [11,15,23])

1.25dihydroxyvitamin

and treatment

D, for 4-30 months [24]

have been used as therapeutic modalities to improve calcium balance in postmenopausal patients. But the effects have been controversial

since the improvement

in

calcium balance has not always been matched by an increase in bone formation and bone density. It is not certain whether the variable skeletal effects of vitamin D or 1,25(OH),D,

therapy per se is due to the age-related decrease in renal l-hydroxy-

lase activity [W] which would impair the metabolism of the vitamin to the active 1,25(OH),D,

compound, or to the concomitant estrogen lack. While estrogen lack

is not thought to influence the metabolism of vitamin D [28]. this effect is probably expressed in terms of a failure to suppress bone resorption interest

to pursue the question of whether

1,25(OH),D,

[S,11,30].

It is clearly of

or other structurally

re-

lated substances will prove to benefit this patient population. In this paper we report hydrotachysterol

(DHT)

the results

lized bone implant (DBM) DHT

of treating ovariectomized

rats with di-

and its effects on bone induction using the Urist deminerasystem. DHT

is metabolized by the liver to 25(OH)-

which has been shown to be an analogue of 1,25-dihydroxyvitamin

terms of its affinity

for 1,25(OH),D,

logical potency [6,16.20]. in viva DHT

D, in

receptors in target tissues and its similar bio-

We have previously shown in the ovariectomized rat that

treatment stimulates (a) the proliferation

of ntesenchymal osteoproge-

nitnr cells in cultures of their hone marrow and (6) bone formation induced by allogeneic DMB

implants with and without the addition of autologous bone marrow in

viva [32]. However,

the design of the in viva studies did not permit us to determine

whether the major effect of DHT osteoprogenitor function.

was on cell proliferation

For the effects of DHT

anticipated that DHT

to be consistent with those of 1,2S(OH),D,,

would augment the proliferative

cells in the host bed to the implant’s diffusible and promote osteoblast differentiation performance 1,25(OH),D, [3,4,14],

to increase the size of the

pool, oc on the subsequent steps of osteoblast differentiation

of the functional suppresses

but not necessarily the osteogenic

This

collagen production

issue is ciouded because while in hone organ and cell cultures

it increaoes collagen and non-collagen protein synthesis in MC3T3-Fl

teoblasts [I? .19]. In order to gain some insight about the effect of DHTon rameters, we have evaluated the in vitro effect of DHT stromal cell proliferation, proliferative,

on osteoprogenitor

os-

these pamarrow

as well as the in viva influences of this substance on the

maturational

the osteoinductive

we

response of mesenchymal

bone morphogenetic protein [4,32],

[9,26,37],

osteoblasts.

and

and differentiated

cell responses which are critical to

end point, i.e., newly formed and subsequently calcified carti-

lage and thence bone.

Materials

and Methods

Young adult female rats (Sprague-Dawley were ovariectomized

(OVX)

Strain,

5-6 weeks old, 200 g body wt)

and they and their age-matched sham-operated con-

361 trols were maintained in a viva&m under conditions of ad-lib feeding (Purina rat chow (Ca = l.Ol%, P = 0.6%)) and tap water ad libitum for 5 weeks. Both groups were then subdivided at random so that half of the control and OVX rats could be fed dietary supplements of diiydrotachysterol(2pg/lOO g body wt) three times per week for a total period of 4 weeks: Group Group Group Group

1: sham-op control 2: sham-op treated with oral DHT 3: ovariectomized 4: ovariectomized: treated with oral DHT

On the 6th postoperative week (1 week DHT pretreatment), the animals in each group were lightly anesthetized with ether and they were grafted i.m. (paraspinous muscle) with four to six pieces of allogeneic demineralized cortical bone matrix (DBM) weighing 9-15 mg each.

Graftpreparation

The demineralized bone grafts (DMB) were prepared according to the method of Urist et al. [35] (see review by Harakas [13]). Marrow-free mid-shaft cortical bone segments were obtained from the femurs of donor rats (Sprague Dawley strain). The tissues were washed in cold water (2 h), defatted in 1:l chloroform methanol (1 h), demineralized in 0.6 N HCI at 4 “C (24 h), washed in running water (6 h), lyophiliaed and gas sterilized (3 h). Postgrafting DHT-trearmenr The rats in Groups 2 (controls) and 4 (WX), which had been pretreated with DHT, continued to receive DHT at the same dose level and treatment interval. In viva isotopic techniques Cell metabolic and osteoinductive endpoints in implanted DBM matrices were monitored at 1,2 and 3 weeks postimplantation. At each of these timepoints, six to eight rats from each of the four groups were injected with 5pCi radiocalcium (“Ca: New England Nuclear Corp., Boston, MA) 24 h prior to sacrifice, and with 0.25 @i/g body wt [3H]thymidine (New England Nuclear Corp., Boston, MA) 1 h before killing. The 24-h retention of radiocalcium was used as a measure of newly forming/mineralizing cartilage and bone matrix. The 1.0 h retention of 3H in strttctural DNA served as an index to cell proliferation. Graft cell alkaline phosphatase levels were also assessed (see specific methodologies below). At autopsy Blood. Just prior to the time the grafts were recovered, the animals were lightly anesthetized with ether and bled by cardiac puncture. Serum samples were stored frozen at -80 “C for serum calcium and phosphorus determinations. Serum calcium was determined by fluorometry (Turner fluorometer Model) (81. Serum phospho-

362 rus was determined

by the method of Fisk and Subbarw

Marrow cell /mrvestlculfures. their epiphyses cat awy.

[S].

The femurs and tibias of the rats were resected and

Marrow

‘plugs’ were transferred from the mid-shaft dia-

physeal segments into chilled alpha-MEM

medium, and the samples from each of

the four groups were pooled. Single cell suspensions were prepared by passing the plugs through a fine metal mesh. and cell counts performed with a hemocytometer using the trypan blue exclusion technique to identify damaCed cells. Viable were plated-out

in 25 cm’ T-flasks at a density of 5-10

grown in alpha-MEM clone Laboratories, ture (Whittaker

medium supplemented Logan, UT)

M.A.

37 ‘C in a tissue culture incubator was usually changed completely of the marrow

with 15% fetal calf scrm (FCS: Hy-

and a 1% penicillin-streptomycin_Fungizone

Bioproducts,

Walkersvilla,

MD).

mix-

Cells were incubated at

under an atmosphere of 5% CO,. The medium

after the first 24 h and every 2-3 days thereafter.

The clonal

growth

ter IO-12

days of culture (fibroblast

mesenchymal-like

stromal

colony formation

these counts were used to index the proliferative teoprogenitor

cells

x 10” cells. The cells were

cells was measured af-

units (FCFlJs)/flask),

potential

cells [32]. The flasks also contained macrophages,

conditions obviated the survival of hematopoietic

and

of these putative osbut the culture

elements [27].

Bone implunts. The bony implants were recovered. stripped of soft tissues, fmzen in liquid N, and stored at -80 “C. The wet wt of the tissue was determined

after

thawing. Graft alkaline phosphatase (Alk.Pase) a commercially

Table

available

levels were measured after sonication by

kit (Sigma Corp.

St. Louis,

MO).

The data were ex-

I

Effect of dihydmtachysterol muscular demineralized

on serum calcium and phosphorus in rats bearing intra-

bone matrix grafts

(iroup”

Phasphorus(mg%)

Sha#lI-“p Shsm-op+ DH7 nvx ovx + “HI 2

Sham-ap ShamoP + DHl ovx cwx + DH, Shim-np Sbm.oP + DHT OVX ovx + DHT

6.92 * 0.41 i4j s.99 f ,1.26,4) 7.116f 0.27 (5)

363 pressed in terms of the preimplantation

weight of the demineralized

bone matrix:

this index is known to provide data which is as reliable as that reported in terms of other denominators which reflect cell and matrix composition (DNA, This preparative

procedure did not result in the leaching of DNA

tracted in 1 N NaOH acid-soluble DNA

and precipitated

in trichloroacetic

protein) [37]. which was ex-

acid (TCA)

to eliminate

pools. The implants were then dissolved in ft.6 N HCI and heated

at 110 ‘C for 18 h. The radioactivity

in aliquots of these solutions (after background

subtraction) were used to determine the amount of “H and 4*Ca incorporated

in the

cells and mineral. All data were expressed in terms of units per milligram of original implant weight.

Himlogy. Three grafts fmm each group of rats were fixed in 10% neutral formalin, decalcified in 10% ethylenediaminetetraacetic

acid (EDTA,

pH 7.4). and em-

bedded in paraffin by routine procedures. The tissues were serially sectiwted at 5 pm parallel to their long axes, and the sections were stained by hematoxylin,

eosin

and Azure 11.

Smfisrical evnlrrariort. All data are reported as mean f SE. The statistical significance of differences between groups was established by the Student’s r-test when the variances were equal. and by a non-parametric

P-test when the variances were

unequal. A 5% level of confidence was considered to be statistically significant.

.Se,nm calciwn ~miphosphorrrs (Table I) Serum calcium values remained within the normal range in ovariectomized in the intact and OVX mal in OVX

rats treated with DHT.

rats and OVX+DHT

z -

.

rats and

Serum phosphorus values were nor-

groups throughout

the study, but serum phos-

50. o+

2 WEEKS

3

Fig. 1 Graph showing()Hlthymidinc incarporulionin implantsin sham.shim

+ DHTrafsat 1. Zand 3 wceksnncrimplantarioo.

+DHT.

OVX andOVX

364 phorus tended to be elevated in the DHT-treated intact control animals at the end of the first and second weeks.

Hisfology. All grafts, irrespective of host type, showed cartilage formation within vascular channels and surface crevices at the end of the first week. During the second week, bone formation and/or osteochondroid formed on the external and internal surfaces of the implants. Ossification was progressive into the third week. Rodiorhymidineincorporation (Fig. 1). In animals not treated with DHT, graft DNA synthesis increased from the first to the second week and then declined at the end of the third week. DHT-treatment significantly increased implant DNA synthesis in both the sham-op and OVX groups (P < 0.01) during the first 2 weeks, but the effect occurred at a slower rate in the OVX rats. The level of DNA synthesis had declined in all groups by the end of the third week. Alkaline phospharase (AP) (Fig. 2). Post-implant AP-levels in the untreated and DHT-treated sham-operated control rats were virtually identical throughout the course of the study. The initial values were unchanged during the first 2 weeks, but enzyme levels increased 2- to 3-fold at the end of the third week. In the OVX rats, the 1 week implant AP levels were subnormal irrespective of DHT-treatment, but DHT treatment (OVX+DHT) was effective in normaliziog implant AP-levels at the end of the third week. The implants io the OVX-DHT group had shown a temporary recovery at 2 weeks, but again exhibited subnormal enzyme levels at the end of the study. Irrespective of the individual group time-course, it was of interest that the peak implant alkaline phosphatase values were on the order of 200 f 20% of their 1 week baselines. The greatest relative change occurred in the OVX rats treated with DHT (+275%).

Fig. 2. Graphshowing alkaline phovphatase activityin implants in rhum, sham + + DHTretsat I, land 3 weeksafterimplamatian.

DHT.

CJYX and OVX

365

Radiocalcium incorporation (Figs. 3,4). Figure 3 shows that the grafts in sham-op rats incorporated little “Ca at the end of the first week when the vascular channels and crevices were filled with cartilage and a loose connective tissue stroma. Cartilage calcification and newly formed bone mineralization was progressive during the second and third weeks. There were no differences in %a incorporation by the im-

Fig. I(. Graph showingthe relativechangeof “Co incorporationin implantsat econd and third week lram the fira week.

366 Table

2

Effect of dihydroxytachysterol

treatment

(started 1 week prior to grafting) on the

clonal growth of stromal cells from the marrow of rats implanted

with deminera-

lized bone grafts Fibrablastcolony6xminp units(IO&q incubation)

Group

Implanthawesrinrervals(weeks)

1 .____--

Sham-q Sham-op+ DHT OVX OVX + DHT St”dent’s

2

29.6 2 3.3 (5) 28.6*3.7(s) 19.3 f I.7 (5)yl 13.6 + 1.3 ~41’“’

3

27.5 t 76.8 * 15.2 * 7.6 *

7.5 (5) 7.0 (S)b’ 2.8(5) 1.3 (SP’“2

25.8 f 3.4 (5) 23.0 + 3.5 (4) 6.6 + 0.5 (3)” 3.9 * 1.3 (5)““i

I-tes,s

vs. Sham-q.

VS.OVX a2 b2 c2

Sl bl cl

Pvaluc 40.05 40.01
plants in ovariectomized grafts in ovariectomized

rats and control rats at the end of the first week, but the rats showed a statistically significant reduction in minerali-

zation at the end of the third week. In neither the sham-op nor OVX DHT-treatment

affect these parameters.

fust week showed that “Ca incorporation DHT-treatment)

by the grafts in the intact sham-op rats (k

was nearly twice that of the grafts in the OVX

rats (2 DHT-treat-

ment) (Fig. 4). There was no clear indication that DHT-treatment direct index ufosteoblast Host mmvw

performance

stromol cdl clotdgrowth

In intact control rats, DHT colony formatiun formation

improved this in-

in either the intact sham-op or OVX

rats,

(Table 2)

treatment provided a transitory stimulus to stromal cell

2 weeks after implantation.

In the untreated

was always subnormal and decreased significantly

postgraft time. DHT

hosts did

A plot of the relative changes from the

had a negative impact in OVX

OVX

rats, colony

with the postsurgical/

rats, further compromising

by

50% marrow stromal cell proliferation.

The results of the present study indicate that there are at least two factors which may be responsible at the organ level for the development chronically ability

ovariectomized

to maintain

adequate

these osteoprogenitor

populations

of osteoprogenitor

cells appear unable to either differentiate

teoblasts. or to subserve a paracrine factors/mediators

of osteopenia in the

rat. The most significant contributing

which

support

function by producing

the proliferation

and/or

clement is the

in-

cells. Secondarily, into functional osa cytokine or other

differentiation

of os-

teogenic stem cells [lo] on or adjacent to endosteal bone surfaces. The ovaries, then, exert the protective effect that would be anticipated from the recent demonstration that osteoprogenitor cells and osteoblasts have estrogen receptors and that estrogen stimulates the proliferation and functional activity (Type 1 collagen mRNA) of osteoblast-like cells in vitro [7]. The data which support our thesis are all indirect, pertaining to the ovariectomized rat’s subnormal marrow stromal cell clonal growth in vitro and the slower time-course of radiothymidine incorporation and attainment of normal alkaline phosphatase values in viva. The impairment of marrow stromal cell growth in OVX rats is consistent with our earlier observations [32] which demonstrated that only protracted periods of oral DHT treatment (2&100 g body wt. for 3.5-7 months) could benefit marrow stromal cell proliferation in vitro and in a composite bone graft model (marrow cell + DBM), and also the connective tissue mesenchymal cell osteoinductive response to DBM grafts. A shorter 2-week course of DHT treatment did not appear to affect these end points. The present studies confirm those findings; but suggest as well that short-term DHT treatment could have some limited benefit in terms of promoting the early proliferative rem sponse of muscle mesenchymal cells to the DBM-diffusible bone morphogenetic protein and enhancing alkaline phosphatase production. The effects of DHT treatment observed in this study were then not entirely unlike the responses of osteoblast-like cells exposed in vitro to BMP and a 1,2S(OH),D, challenge [4,9,26], or to the responses to DBM grafts in viva in animals treated in viva with 1,25(OH),D1 1371. Our data is most closely approximated by Vukicevic’s experiences in intact male rats [37]. While i.p. injections of 1,2S(OH)zD3 (50 @day for 35 days) impaired mesenchymal cell ingrowth into DBM implant and bone growth during the first 2 weeks postgrafting, implant alkaline phosphatase values significantly increased above normal at 24l-21 days and bone formation normalized at 35 days. Vitamin D treatment did not affect the normal pattern of decline in implant alkaline phosphatase levels after 21 days. Although DHTlike 1,25(OH),D,-did not appear to enhance the osteogenic end-point in the present study on OVX female rats, the trends in the alkaline phosphatase activity and “Ca incorporation data suggest that this effect might have been expressed if the DHT-treatment time been extended. Benefit was reported in grafts of DBM + autologous bone marrow implanted in OVX rats treated with DHT for 6 months (321. Thus, the importance of the postovariectomy time course on the basal rate of bone turnover in rats must be considered in the interpretation of these experiments. Wronski et al. [40] has shown that the period of most active bone turnover (histomorphometry) occurs within 14 to 70 days post-OVX and that it rapidly diminishes thereafter. This was the period within which Turner et al. 1341reported, by histomorphometriccriteria, stimulation of osteoinduction by particulate implants of demineralized bone matrix in OVX rats treated with 1,25(OH),D,. Our studies, on the other hand, involved DHTtreatment at later post-OVX times (approximately 84-105 days) when the rapid acceleratory response had begun to wane. In this situation, the moderate enhancements of DNA synthesis and alkaline phosphatsse activity were more adequate indices to the osteoinductive stimulus than “Ca incorporation.

368 Since the effects of dihydrotachysterol on the osteoinductive response to DBM matrix appear to be like those of 1.2%dihydroxyvitamin D,, it is likely that these agents operate via common mechanisms. The liver metabolite of DHT, 25(OH)DHT, binds to the 1,25(OH),D, receptor in target tissues and appears to have a similar biological potency on stimulating bone resorption [16,20]. Vitamin D appears essential to the activation and differenti:.tion of elements of the immune system (e.g., the inflammatory response) which are critical to osteoclast formation and to the critical ‘coupling’ response which OCEUISbetween bone resorption and formation [36]. These are considerations which point to an important action of DHT in promoting the early stages of osteoprogenitor cell proliferation and differentiation. From the standpoint of the (quasi-postmenopausal) ovariectomized rat model, the rationale for 1,2S(OH),D, or DHT therapy lies in its ability to maintain an adequate population of osteoprogenitor cells.

This work was supported by a Winthrop Corporation fellowship awarded to Dr Chikage Tabuchi.

opeinpostmenopausal 3 Gnatis

osteoporosis. J Clin Endocrinol Mctab 1987:M:954-959.

E. Eff~fccrsofhormanesandgrowth

tbesisinculturcd

ratcalvariae.

Metabolism

factorron alkaline phorphalane activity andcollagen syn. 1983;32:14-20.

4 Canalis E. Ccntrella M. Urirt MR. Effect of pardelly purified bone morpho~enelic pmtein on DNA synthesis and cell replication in colvari~l cst&dlast and fibrablast ;uftures. Clin Okthop 191(5;198:289-296. 5 Caniggia A, Nuti R, Lore F. Vnuimo A. The hormonal form of vitamin D in the pathopbyriology and rbcrapy ofpmtmcnopausd

oatsoporosia. J Endocrinol lnvcst 1984:7:373-378.

6 ChenTL, Hint MA, Feldman D. A receptor-like binding macmmolecrde for la,25-dihydroxycholccalciferol in cultured mouse bone cells. 1 Biol Chcm 197%254:749l-7494. 7 Emrr M. Schmid Ch, Fraesch ER. Enhanced osreoblasr proliferatio 1 and collagen gene expression by estradiol. Proc Natl Acad Sci USA 1988,85:2307-2310. 8 Fiske CH. Subbnrow. Y. The colorbnerric determination of phosphorus. J Biol Chem 1!325;66: 37s-4w. 9 FritschJ,Gmse

BN. Liberhcrr M, Balsan S. 1.25-Dihydroxyvitamin

pendem expression of alkaline phwphatase

D,isrequired

in cultured rat o~teoblasts. C&if

fargrowrb-inde-

Tissue lnt 1985;37:

639-645. 10 Gwit D. Shteyer A. Bnb I. Marrow-derived factors regulating bane cell proliferation and activity. Bone 1%76;7:3R9. Dnrmbnnchcr MA. Guncaen J. LauffcnbureerT. Lammic BB. Olah J. 1,25(OHI, Vita-

I1 Haas HO.

min D, in &oporosis: n pilot study. l&n Mclab Rer1979: II: 168-171. T. Kuiriharn N. lkedn K, Kumceawa M. lu-2J.Dihvdroxvvitamin

12Hirncii

_.

D, and an~lowcs of vita-

369 B&hem

3983;‘34:1127-1132

13 HarakasNK.

Demineralized bone matrix inducedostsogeneris. ClinOnbop

W84;t88:z39-251.

14 Hock JM, Kream BE, Raisz LG. Autoradiographic study of the effect of 1.25.dihydroxyvitamin I), on bone matrix synthesis in vitamin D replete rats. Caldf Tissue Int 1982:34:347-351. 15 Jensen GF, Christiansen C, Tmnsbol I. Tmatmem of postmenopausal ortcoporosis. A controlled therapeutic trial comparing oestrogen, gestation, 1,25~dibydraxyvitamin D, and calcium. Clin Endo. ainol lY82;16:515-524. 16 Johannesson AJ, Raisz LG. Effects of low medium magnesium concentration on bone resorption in response to psmthymid hormone and 1.25.dihydroxyvitamin D in organ culture. Endocrinology 19B3;113:22Y4-22Y8. 17 Kalu DN. Hardin RR, Cockerham R. Evaluation of the pathogen& tomizrdmta.

of skelettd changes in ovariec-

Endocrinology 1984;115:507-512.

IS Kepner BL. Hercules DM.

Fluorometric determination of calcium in blood ~emm. Anal Chem

,963;35:1238-1240. 19 Kuribam N, lkeda K, Hakcda Y, Tunai

M, Maeda M, Kumegawa M. Effect of L,25-dihydraryvita-

min Dj on alkaline phosphatasc activity and collagen synthesis in osteoblastic cc,,s, Clone MC3T3El.Biochem

BiopbysResCammuo

lY84:119:767-771.

20 Lemer U. Relationship between bone resorption and lyso~omal enzyme release as demonstrated by the effect of

la-hydroxy-vitamin

D-3

in an organ culture system. Biochim Biophys Acta

198$632:204-213. 21 Lindgren U, DeLwa

HF. Role of pamthyroid hormone and 1,25.dihydmxyvitamin D, in the dewl-

opmentofosteocaia inovarieetamizedrats. CalcifTissue Int L982:34:510-514. 22 &dgren JC, Lhdbolm TS. Effect of I-alpha-hydmuyvitamin D, on osteoporosis in rats induced by oopborectamy. Calrif Tissue Int 1979:27:Lhl-164. 23 Lindholm TS. Sevastikogbw IA.

Lindgren U. Treatment of patients with bin&

and carticosteroid-induced osteopnrmir with la-hydmxyvitamin

pest-mcnopawd

D, and calcium: short. and long-

rermeffects. Clin Endocrinol,977;7(Suppl.):183S-189S. 24 Lindholm TS. Scvastikoglou JA, Lindgren tJ. Interim report on treatment of orteoporotic patients with la-hydroxpitamin D,and calcium. Clin Grthop lY78:335:232-240. 25 Lore F. Nuti R. Vmtimo A. Caniggin A. Vitamin D metabalites in postmenopausal osteoporosis. Harm MetabRcs ,Yt14:,6% 26 Man&gas

SC, Burton DW. Deftos LG. 1,25-Dihydmryvitsmin

D, stimulates the alkaline phospha.

vaseactivity of osteoblast-like cells. 3 Biol Chem 1981;256:7115-7117. 27 Owen M. Lineage of osteogenic cells and their relationship to the stmma, system, In: Peck WA, ed. Bone and mincml research, Vo,. 3, Amsterdam, New York. &ford:

Elsevier, lYS5;,-25.

28 Pavlovitch H. Clemens TL. Laouari D. O’Riordan JLH. Balsan S. Lack of effect of ovatiectomy on the membolism of vitamin D and intestinal calcium binding protein in female rats. J Endocrinol ,980:86:419-4&t. 29 Saville PD. Changes in skeletal mass and fragility with castrmion in the mt: a mode, of osteoporosis. J Am GcriatrSoc

196r1;17:lSS-166.

.

30 Simmons DJ. Comparative phwiotow of bone. In: Bourne GH. ed. The biochemistry and phvsiolo. gyofbonc.

New York: Academic?&

1976:445-516.

31 Stepan JJ. Prcsl J, Broulik P. Paeovsky V. Serum osleocalcin levels and bone alkaline phosphatare isoennyme nftcr oophorectomy and in primary hyperparatbyroidirm. 1 Clin Endocrinol Metab 1987:6431079-tOR22. 32 Tabuchi C, Simmons DJ, Rusto A. Russell JE, Binderman I, Avioli LV The bone deficit in ovariec. tomized rats: the funeriona, amttibution of the mormw stmmat cell populntion and the effect of oral dihydrotxhyslerol

treatment. J Clin lnvcst lY86;78:637-642.

33 Thomsen K. Riir 8. Christiansen C. Effect of ertragenigertngen and 24R,25-dihydmxyvitamin

D,

thcraov on bone formation in oostmenooausa, women. J Bone Min Rcs 1986:1:503-507. 34 T&RT, Vtmdertccnbove~ JJ. Bc8~l-l. Tbc cffectsofovariectomyand ,7!%cstmdio, oncortical bone histomorphometry in growing mm. J Bone Min Rcr 1987.2~1t&,22. 35 Uris.1 MR. Silverman BF. Buring K. Dubuc FL, Rosenberg HM. The bone induction principle. Clin Orthap 1967;53:243-283.

370

37 Vukicwic S. Slavijenic A. Bagi C. Gumicic G. Kracun 1. Winter 1. la-Dihydroxyvitamin

D, stimu-

l~,es alkaline phosphatasc ecrivity and inhibits salt-tissue protileradon in implants of bone malrix. Clin Onhop ,985;,9h:ZR5-29,. 38 Wranski Tj, Lowry Pi.. Walsh CC. Ir_waszcwki LA. Skcleta, alterations in ovariectomized rats. CalcilTissuc In, IPRS;37:324-328. 39 Wronski TJ. Walsh CC. Ignoszcwski LA.

Hir,ologic evidence for orteopenia and increased bone

tunmver in ovariectomired rats. Bone 19P&7:1 i9-123. 4, Wronski TJ, C;n,ron M. Dann LM. Tcmwral Bone Min Res 19S7:2(Suppl. l):Ahsma&72.

variations in hone turnover in ovaricctomized ra,s. .,