Vitamin D metabolites prevent vertebral osteopenia in ovariectomized rats

Calcif Tissue Int (1992) 50:228-236

Calcified Tissue International 9 1992 Springer-Verlag New York Inc.

Vitamin D Metabolites Prevent Vertebral Osteopenia in Ovariectomized Rats Reinhold G. Erben, ~ Harald Weiser, 3 Fred Sinowatz, z Walter A. Rambeck, 1 and Hermann Zucker 1 1Institute of Physiology, PhysiologicalChemistry, and Nutrition Physiology;2Institute of Veterinary Anatomy, Veterinary Faculty, Ludwig-Maximilians-University, Munich, Federal Republic of Germany; and 3Research Department, Hoffmann-LaRoche Ltd, Basel, Switzerland Received July 16, 1990, and in revised form September 25, 1990

Summary. The present study investigated the prophylactic effects of vitamin D metabolites and vitamin D metabolite combinations on static and dynamic, tetracycline-based, histomorphometric parameters in the axial skeleton of ovariectomized rats. Forty-three Fischer-344 rats (10 weeks old, 130 g each body weight, BW) were either bilaterally ovariectomized (OVX) or sham-operated (SHAM). The rats were allocated into the following groups: SHAM; OVX; OVX + 7.5 ng l~,25-dihydroxyvitamin D 3 [1,25(OH)2D3]/rat/day; OVX + 15 ng lct,24R,25-trihydroxyvitamin D 3 [1,24,25(OH)3D3]/rat/day; OVX + 75 ng 24R,25-dihydroxyvitamin D 3 [24,25(OH)2D3]/rat/day; OVX + 7.5 ng 1,25(OH)aD3/rat/ day + 15 ng 1,24,25(OH)3D3/rat/day; OVX + 7.5 ng 1,25(OH)2D3/rat/day + 75 ng 24,25(OH)2D3/rat/day. The vitamin D metabolites were fed orally starting 4 weeks after surgery. Urine and blood samples were collected 12 and 16 weeks postovariectomy, respectively. Sixteen weeks after surgery, all rats were sacrificed, and the first lumbar vertebrae were processed undecalcified for histomorphometric analysis. Ovariectomy induced a highly significant reduction (P < 0.001) of cancellous bone mass in the secondary spongiosa of the lumbar vertebral body. The bone loss in OVX rats was accompanied by a distinct elevation of all histomorphometric parameters of bone formation and resorption. 1,25(OH)2D3 and both vitamin D metabolite combinations significantly raised serum calcium levels and prevented the bone loss by inhibiting the increased bone resorption in OVX rats. In the applied dosage, 1,24,25(OH)3D 3 and 24,25(OH)aD 3 alone were ineffective in preserving the cancellous bone of the lumbar vertebra in OVX rats. We conclude that the oral prophylactic application of low doses of active vitamin D metabolites can effectively prevent the osteopenia induced by ovariectomy in the axial skeleton of the rat. Key words: Ovariectomy - Bone turnover - Quantitative bone histomorphometry - Osteopenia - Vitamin D.

The actions of the most potent naturally occurring vitamin D metabolite, lct,25-dihydroxyvitamin D3 [1,25(OH)2D3], on skeletal tissue are complex. 1,25(OH)2D3 is, probably by providing minerals through augmented intestinal absorption of calcium and phosphate, necessary for normal mineraliza-

Offprint requests to: R. G. Erben, Institute of Animal Physiology, Veterinfirstrasse 13, D-8000 Mtinchen 22, Federal Republic of Germany.

tion of newly formed bone matrix. It stimulates osteoclastic bone resorption, and regulates osteoblast proliferation and osteoblast production of type I collagen, alkaline phosphatase, and osteocalcin [1]. There is considerable controversy as to whether there are specific effects of the 24-hydroxylated vitamin D metabolites 24R,25-dihydroxyvitamin D 3 [24,25(OH)2D3] and l~,24R,25-trihydroxyvitamin D 3 [1,24,25(OH)3D3] on bone. Experiments using 24,24d i f l u o r o - 2 5 - h y d r o x y v i t a m i n D3, a synthetic compound that cannot be hydroxylated in the 24 position, have provided evidence that 24-hydroxylation is neither necessary for healing of rachitic bone lesions nor for normal modeling and mineralization of bone [2--4]. Numerous other studies, however, have strongly suggested a specific role for 24,25(OH)2D3 in bone mineralization, endochondral bone formation, proliferation and differentiation of growth cartilage chondrocytes, and matrix calcification in growth plate cartilage [5-10]. Very little is known about potential specific effects of 1,24,25(OH)3D 3 on skeletal tissue. In rachitic rats, chicks, and quails it can be shown that the bioactivity of 1,25(OH)2D3 in various vitamin D bioassays is enhanced in an overadditive, synergistic fashion when 24,25(OH)ED3 or 1,24,25(OH)3D 3 are administered simultaneously [11-13]. The reinforcing effects of 24hydroxylated vitamin D metabolites on 1,25(OH)2D3 bioactivity might open the possibility of lowering the dosage of 1,25(OH)2D3 without loosing its therapeutic effect, thus possibly also reducing the risks associated with 1,25(OH)aD3 treatment, i.e., the occurrence of hypercalcemia and hypercalciuria. Studies in healthy women in their early natural menopause have demonstrated that prophylactic administration of 0.25 p~g 1,25(OH)aD3, 0.25 ixg lct-hydroxyvitamin D3 [lct(OH)D3], or 10 ~g 24,25(OH)aD3/day cannot prevent early postmenopausal bone loss [14-16]. On the other hand, there is accumulating evidence that 1,25(OH)zD 3 and let(OH)D 3 have a beneficial effect in preventing bone loss and reducing fracture rate in patients with established postmenopausal osteoporosis [17-19]. In these studies, daily doses of 0.7-1.0 Ixg 1,25(OH)2D3 or let(OH)D 3 were used. The bone loss observed in ovariectomized (OVX) rats bears some resemblance to postmenopausal bone loss, and ovariectomy-induced osteopenia in the cancellous bone of the rat lumbar and caudal vertebral body, accompanied by increased bone turnover, is a well-documented phenomenon [20-22]. Compared with the weight-bearing long bones of the rat appendicular skeleton, the advantages of the vertebral body as a sample site for cancellous bone histomorphometry are (1) comparatively slow longitudinal bone growth [22], (2) less biomechanical loading in quadruped animals, and (3) the

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p r e s e n c e o f t h e s a m e t y p e o f r e m o d e l i n g activity t h a t dominates t h e h u m a n a d u l t s k e l e t o n in t h e s e c o n d a r y s p o n g i o s a of the r a t v e r t e b r a [23]. S e v e r a l studies h a v e s h o w n t h a t t h e v i t a m i n D m e t a b o lites 1,25(OH)2D3, let(OH)D3, 1 , 2 4 R - d i h y d r o x y v i t a m i n D3, a n d t h e c o m b i n a t i o n o f 1,25(OH)2D 3 w i t h 1,24,25(OH)3D 3 c a n b e effective in t h e p r e v e n t i o n a n d t h e r a p y of the ost e o p e n i a i n d u c e s b y o v a r i e c t o m y in t h e r a t [24-30]. T h r e e of t h e s e i n v e s t i g a t i o n s [28-30] e m p l o y e d b o n e h i s t o m o r p h o m etry to a s s e s s t h e effects o f v i t a m i n D m e t a b o l i t e s o n t h e s t r u c t u r e a n d m e t a b o l i s m o f skeletal tissue in O V X rats. In t h e s e h i s t o m o r p h o m e t r i c studies, h o w e v e r , p h a r m a c o l o g i c a l d o s e s of 1,25(OH)2D 3 (50-60 ng/rat/day) w e r e a d m i n i s t e r e d , a n d t h e sole u s e o f static h i s t o m o r p h o m e t r i c p a r a m e t e r s ( w i t h o u t t e t r a c y c l i n e d o u b l e labeling) did n o t p e r m i t t h e accurate determination of the influence of vitamin D metabolites o n b o n e f o r m a t i o n . It is n o t k n o w n w h e t h e r low d o s e s of a c t i v e v i t a m i n D m e t a b o l i t e s c a n p r o t e c t against ovariect o m y - i n d u c e d c a n c e l l o u s b o n e loss in t h e axial s k e l e t o n of the rat. T h e c u r r e n t s t u d y e m p l o y e d static a n d d y n a m i c , tetracycline-based histomorphometry to evaluate the ability o f low, o r a l d o s e s o f 1,25(OH)2D 3, 2 4 , 2 5 ( O H ) 2 D 3, a n d 1,24,25(OH)3D3, a l o n e or in c o m b i n a t i o n , to p r e v e n t t h e dev e l o p m e n t o f o s t e o p e n i a in t h e c a n c e l l o u s b o n e o f the first l u m b a r v e r t e b r a l b o d y o f o v a r i e c t o m i z e d rats.

termined according to a modified micromethod by Stegemann [31]. Fasting urinary excretion of calcium and hydroxyproline was expressed as a ratio to creatinine excretion.

Materials and Methods

Animal Procedures Forty-three female 10-week-old Fischer-344 rats (Institute for Biological and Medical Research, Switzerland) weighing about 130 g each, were used for this experiment. Thirty-five rats were bilaterally ovariectomized by dorsal approach under xylazine/ketamine anesthesia, and the remaining 8 rats were sham-operated (SHAM). The animals were kept in individual cages at 24~ with a 12 hour/12 hour light/dark cycle, and were fed a standard laboratory diet (Altromin, FRG) containing 0.9% calcium, 0.75% phosphorus, and 600 IU/kg vitamin D 3. Food and tap water were available ad libitum to all rats. The rats were allocated by weight in the following, weight-matched groups: group 1 SHAM (n = 8); group 2 0 V X (n = 6); Group 3 OVX + 7.5 ng 1,25(OH)2D3/rat/day (n = 6); group 4 0 V X + 15 ng 1,24,25(OH)3D3/rat/day (n = 6); group 5 0 V X + 7.5 ng 1,25(OH)zD3/rat/day + 15 ng 1,24,25(OH)3D3/rat/day (n = 6); group 6 0 V X + 75 ng 24,25(OH)2D3/rat/day (n = 6); group 7 0 V X + 7.5 ng 1,25(OH)2D3/rat/day + 75 ng 24,25(OH)2D3/rat/day (n = 5). The vitamin D metabolites were dissolved in ethanol/1,2 propandiol (t:10) and added to the diet, starting 4 weeks after surgery. During the experimental period, the rats were weighed every 2 weeks. Urine was collected in metabolic cages 4 weeks prior to the end of the experiment. Blood samples were obtained by orbital sinus puncture under ether anesthesia at the end of the trial. The serum and urine samples were stored at - 40~ until assayed. Oxytetracycline (TerravenOs, Pfizer Co., FRG) at a dose of 20 mg/kg BW was injected intraperitoneally on the 9th and 4th day before sacrifice. Sixteen weeks postovariectomy, all rats were sacrificed by an ether overdose. Success of ovariectomy was confirmed by failure to detect ovarian tissue and observation of marked atrophy of the uterine horns.

Blood and Urine Analysis Total calcium in serum and urine was determined by flame photometry (ELEX 6361, Eppendorf Co., FRG). Serum inorganic phosphate and serum alkaline phosphatase activity were measured with commercially available testkits (Boehringer Co., FRG and Sigma Chemical Co., FRG, respectively). Urinary hydroxyproline was de-

Histology At autopsy, the first lumbar vertebrae were carefully defleshed and fixed immediately in 40% ethanol at 4~ for 48 hours on a magnetic stirrer [32]. After fixation the bones were embedded undecalcified in methylmethacrylate, as described previously [29]. Five and 10 ~m thick, undecalcified, median sections were prepared with a Jung Polycut E sledge microtome (Reichert-Jung, FRG). The 5 Ixm thick sections were stained with von Kossaltoluidine blue [29], toluidine blue at acid pH [32], and, after "edging" with formic acid and methanol, with toluidine blue for demonstration of cement lines [33].

Histomorphometry All measurements were performed on the secondary spongiosa of the cancellous bone of the first lumbar vertebra. With a semiautomatic system (Videoplan, C. Zeiss, FRG), 30 fields (4.4 mm 2) were evaluated in each section at • magnification (• plan objective). The area within 0.8 mm from the cranial and caudal growth plates was excluded from the measurements [34]. Static histomorphometric parameters were measured in 5 ~m thick sections stained with toluidine blue at acid pH (generally 1 section per animal), and dynamic, tetracycline-based parameters were measured in unstained, 10 ~m thick sections (generally 2 sections per animal). The following parameters were determined: total tissue area (Tt.T.Ar), total bone area (Tt.B.Ar), total b o n e p e r i m e t e r (Tt.B.Pm), total osteoid area (Tt.O.Ar), total osteoid perimeter (Tt.O.Pm), total osteoblast perimeter (Tt.Ob.Pm), number of osteoclasts (N.Oc), number of bone marrow mast cells (N.Mc), total single-labeled tetracycline perimeter (Tt.sL.Pm), and total doublelabeled tetracycline perimeter (Tt.dL.Pm). Osteoblasts were defined as mononuclear, basophilic, cuboidal cells with a prominent Golgi apparatus and in contact with osteoid. Flat, terminal osteoblasts apposed to osteoid surface were not counted as "osteoblasts" [35, 36]. Osteoclasts were defined as large, irregularly shaped cells with a foamy, slightly metachromatic cytoplasma containing one or more nuclei, and residing within Howship's lacunae. Typical osteoclast profiles without a nucleus were also regarded as "osteoclasts." The distance between tetracycline double labels was measured at • magnification. The mineral apposition rate (MAR) was given by the arithmetic mean of at least 50 such distance measurements per animal (equally weighed sample [37]), divided by the marker interval (5 days). The wall width (W.Wi) of completed cancellous bone structural units was measured in sections stained with toluidine blue for demonstration of cement lines at • magnification. Each completed remodeling site (at least 25 per animal) was sampled at three to four equidistant points. Values for MAR and W.Wi were not corrected for obliquity of the plane of section. From these data, the following parameters were calculated [36]: Bone area (B.Ar/T.Ar) Bone perimeter (B.Pm/T.Ar) Trabecular width (Tb.Wi) Osteoid area (O.Ar/B.Ar) Osteoid perimeter (O.Pm/B.Pm) Osteoblast perimeter (Ob.Pm/B.Pm) Osteoid width (O.Wi) Osteoclast number (N.Oc/Md.Pm) Mast cell number (N.Mc/Ma.Ar) Single-labeled perimeter (sL.Pm/B.Pm)

= = = =

Tt.B.Ar/Tt.T.Ar * 100 (%) Tt.B.Pm/Tt.T.Ar (mm/mm 2) Tt.B.Ar/Tt.B.Pm * 2000 (p~m) Tt.O.Ar/Tt.B.Ar * 100 (%)

= Tt.O.Pm/Tt.B.Pm * 100 (%)

(1) (2) (3) (4) (5)

= Tt.Ob.Pm/Tt.B.Pm * 100 (%) (6) = Tt.O.Ar/Tt.O.Pm * 1000 (~m) (7) = N.Oc/Tt.Md.Pm (#/mm)

(8)

= N.Mc/Tt.Ma.Ar (#/mm 2)

(9)

= Tt.sL.Pm/Tt.B.Pm * 100 (%) (10)

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R.G. Erben et al,: Vitamin D Metabolites in Ovariectomized Rats Results

Body Weight (Table 1) Upon completion of the experiment, the body weights of sham-operated and OVX rats did not differ significantly. However, the rats in Gp 4 (OXV + 1,24,25(OH)3D3) and Gp 6 (OVX + 24,25(OH)2D3) weighed significantly more than the SHAM rats (P < 0.005). Compared with untreated OVX rats, the application of vitamin D metabolites did not result in a reduced weight gain in any treated group.

Serum and Urine Biochemical Findings (Table 1)

Fig. 1. Representative, undecalcified, median sections (5 ~m thick) of the first lumbar vertebral body of a sham-operated rat (A), an OVX rat (B), and an OVX rat treated with 7.5 ng 1,25(OH)2D3/rat/ day (C). Note the pronounced loss of cancellous bone mass and the increased discontinuity of the bone trabeculae in the vertebral body of the OVX rat (B). Also note that these changes were completely prevented by prophylactic treatment of OVX rats with 1,25(OH)zD3 (C). Von KossaJtoluidine blue stain. • 11.

OVX rats exhibited significantly higher levels of total serum calcium that S H A M animals, 16 weeks postovariectomy (P < 0.01). There was only a nonsignificant trend towards increased values for serum alkaline phosphatase and urinary hydroxyprotine/creatinine excretion in OVX rats relative to S H A M controls. In OVX rats, the application of 7.5 ng 1,25(OH)zD3/rat/day alone (Gp 3) or in combination with 1,24,25(OH)3D 3 (Gp 5) or 24,25(OH)zD3 (Gp 7) caused a highly significant rise in serum calcium (P < 0.005 versus OVX), a rise in serum phosphate (P < 0.05 for Gps 3 and 5 versus OVX), and about a 5- to 6-fold increase in urinary calcium/creatinine excretion (P < 0.01 versus OVX). At the same time, urinary hydroxyproline/creatinine excretion was slightly (NS) decreased in OVX rats receiving 1,25(OH)zD3 (Gps 3, 5, and 7 versus OVX). Relative to untreated OVX rats, serum alkaline phosphatase activity was significantly (P < 0.005 versus OVX) reduced in Gp 7 (OVX + 1,25(OH)zD3 + 24,25(OH)zD3). Treatment of OVX rats with 1,24,25(OH)3D 3 (Gp 4) and 24,25(OH)zD 3 alone (Gp 6) did not result in any significant effects on biochemical parameters in serum and urine.

Vertebral Cancellous Bone Histomorphometry (Table 2) Mineralizing perimeter (M.Pm/B.Pm) = Tt.dL.Pm/Tt.B.Pm * 100 (%) Bone formation rate (BFR/B.Pm) = MAR * Tt.dLPm/Tt.B.Pm (~mZ/~zrn/day) Formation period (FP) = W.Wi/MAR (day) Osteoid maturation time (Omt) = O.Wi/MAR (day)

Effects of Ovariectomy (11) (12) (13) (14)

Because the structural elements in the cancellous bone of the rat vertebral body show a markedly anisotropic distribution (Fig. 1), we used only two-dimensional terms. Osteoclasts usually avoid osteoid, and therefore the parameter N.Oc/Md.Pm was related to the mineralized bone perimeter (Md.Pm) with Tt.Md.Pm = Tt.B.Pm Tt.O.Pm. The number of mast cells (N.Mc) was related to the marrow area (Ma.Ar) with Tt.Ma.Ar = Tt.T.Ar - Tt.B.Ar.

Statistical analysis The data were analyzed using the nonparametric Kruskal-Wallis H-test. When the Kruskal-Wallis H-test performed over all groups indicated a significant (P < 0.05) difference among the groups, statistical differences between two groups were evaluated with the twotailed Wilcoxon-Mann-Whitney U-test. P values of less than 0.05 were considered significant. The data are presented as the mean ~+-SD.

When c o m p a r e d with the S H A M group, O V X rats showed about a 30% reduction in B.Ar/T.Ar (P < 0.001, Fig. 1), and also significantly decreased values for B.Pm/T.Ar and Tb.Wi (P < 0.01). Values for all static and dynamic parameters of bone formation (O.Ar/B.Ar, O.Pm/B.Pm, Ob.Pm/B.Pm, MAR, M.Pm/B.Pm, and BFR/B.Pm), as well as for the bone resorption parameter N.Oc/Md.Pm, were distinctly elevated in OVX rats. No significant differences between the S H A M and the OVX group were observed for the parameters O.Wi, N.Mc/Ma.Ar, W.Wi, sL.Pm/B.Pm, FP, and Omt.

Effects of Treatment with Vitamin D Metabolites Treatment of OVX rats with 1,25(OH)2D 3 alone (Gp 3) or in combination with 1,24,25(OH)3D 3 (Gp 5) or 24,25(OH)2D 3 (Gp 7) proved highly effective in preserving the cancellous bone of the first lumbar vertebral body (Fig. 1). In these groups, the prophylactic application of vitamin D metabolites resulted in significantly decreased values for static and dynamic parameters of bone formation and bone resorption relative to untreated OVX animals. 1,25(OH)2D 3 alone (Gp 3) or in combination with 1,24,25(OH)3D3 (Gp 5) did not suppress BFR/B.Pm in OVX rats to a level significantly below that of the S H A M controls. Statistically significant dif-

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Table 1. Body weight, serum, and urine biochemical parameters in SHAM rats, OVX rats, and OVX rats orally treated with vitamin D metabolites, 16 weeks postovariectomy (12 weeks postovariectomy for urine parameters) Treatment

Variable

Group 1 (n = 8) SHAM

Group 2 (n = 6) OVX

Group 3 (n = 6) OVX + 1,25(OH)2D

Group 5 Group 4 (n = 6) (n = 6) OVX + OVX + 1,25(OH)2D + 1,24,25(OH)3D 1,24,25(OH)3D

Group 7 Group 6 (n = 5) (n = 6) OVX + OVX + 1,25(OH)zD + 24,25(OH)2D 24,25(OH)2D

Body wt (g) 181 - 11 189 -+ 13 192 -+ 13 207f -+ 10 187 + 8 199f + 6 187 -+ 10 Serum calcium (retool/liter) 2.42b-+ 0.09 2.55 + 0 . 0 5 2.79~'f-+ 0 . 0 6 2.55a-+ 0 . 1 2 2.84c'g --+ 0.13 2.49 + 0.07 2.91~'f-+ 0.16 Serum phosphate (mmoFliter) 2.05 -+- 0.62 1.79-+ 0.53 2.49~ -+ 0 . 4 6 1.98 -+ 0 . 3 6 2.60a'a - 0 . 2 4 1.80 + 0 . 2 5 2.I5 -+ 0.33 Serum alkaline phosphatase (U/liter) 134 -+ 13 158 + 31 147 --- 23 157 - 27 129 +- 30 158* +- 17 115~ -+ 16 Urinary calcium/ creatinine (mmol/mmol) 0.47 - 0 . 1 8 0 . 4 2+- 0 . 1 5 1.99c'f-+ 0 . 5 2 0.57 -+ 0.15 2.17c'g -+ 0 . 6 6 0.28a-+ 0 . 1 0 2.58bx+ 0.79 Urinary hydroxyproline/ creatinine (txmol/mmol) 20.1 - 2.9 23.5 -+ 6.3 I9.9 -+ 7.4 25.8 -+ 2.3 19.1 -+ 3.8 20.4 --+ 4.3 18.0 -+ 6.1 All values are means -+ SD a p < 0.05; bp < 0.01; cp < 0.005 vs. Gp 2 (OVX); dp < 0.05; ep < 0.01; fP < 0.005; gP < 0.001 vs. Gp 1 (SHAM)

ferences (P < 0.05) between Gps, 3, 5, and 7 and the SHAM animals (Gp 1) were only observed for the parameters B.Pm/ T.Ar (Gps 3 and 5 versus Gp 1), N.Oc/Md.Pm (Gps 3 and 7 versus Gp 1), M.Pm/B.Pm (Gp 7 versus Gp 1), and BFR/ B.Pm (Gp 7 versus Gp 1). Compared with the SHAM group, the values for these p a r a m e t e r s were lowered in the 1,25(OH)2D3-treated Gps 3, 5, and 7. Significant differences between the treatment with 1,25(OH)/D3 alone (Gp 3) and the treatment with the metabolite combinations (Gps 5 and 7) were not demonstrable. However, relative to the treatment w i t h 1,25(OH)2D3 a l o n e ( G p 3), t h e i n c l u s i o n o f 24,25(OH)zD 3 with 1,25(OH)zD 3 (Gp 7) tended to enhance (NS) the suppressive effect of 1,25(OH)zD 3 on the dynamic bone formation parameters M.Pm/B.Pm and BFR/B.Pm. The parameters O.Wi, N.Mc/Ma.Ar, W.Wi, MAR, sL.Pm/ B.Pm, FP, and t r o t did not show significant changes under treatment of OVX rats with vitamin D metabolites. There were no statistically significant differences in any parameter between the untreated OVX group and the OVX Gps 4 and 6 receiving 1,24,25(OH)3D3 and 24,25(OH)zD3 alone, respectively. A strong inverse relationship was found between the parameters N.Oc/Md.Pm and B.Ar/T.Ar (Fig. 2), and between the parameters BFR/B.Pm and B.Ar/T.Ar (Fig. 3). The regression equations were (linear regression analysis, least square method) N.Oc/Md.Pm = 5.16 - 0,175 * B.Ar/T.Ar (r = - 0 . 7 4 ; P < 0.001; n = 43; 95% confidence interval (CI) for r: 0.57 ~< Irl -< 0.82; Fig. 2), and BFR/B.Pm = 0.83 0.0232 * B.Ar/T.Ar (r = - 0 . 7 0 ; P < 0.001; n = 43; CI: 0.51 ~< Ir] ~< 0.83; Fig. 3), respectively. All other static and dynamic parameters of bone formation were also significantly negatively (P < 0.001) correlated with B.Ar/T.Ar (data not shown). F u r t h e r m o r e , the resorption parameter N.Oe/ Md.Pm was found to increase in a highly significant positive (P < 0.001) correlation with static and dynamic bone formation parameters, for example, N.Oc/Md.Pm = 2.68 9 BFR/ B.Pm + 0.38 (r = 0.68; P < 0.001; n = 43; CI: 0.48