Maintenance of cortical bone in human parathyroid hormone(1-84)-treated ovariectomized rats

Bone Vol. 28, No. 3 March 2001:251–260

Maintenance of Cortical Bone in Human Parathyroid Hormone(1-84)-Treated Ovariectomized Rats E. SAMNEGÅRD, U. T. IWANIEC, D. M. CULLEN, D. B. KIMMEL, and R. R. RECKER Osteoporosis Research Center, Creighton University, Omaha, NE, USA

Key Words: 17␤-Estradiol; Risedronate; Adult rats; Densitometry; Histomorphometry.

The purpose of this cross-sectional study was to evaluate the effects of human parathyroid hormone(1-84) (hPTH) followed by maintenance treatment with 17␤-estradiol (E2), risedronate (Ris), or a reduced dose of hPTH (LowPTH) on cortical bone in the ovariectomized (ovx) rat. Eight groups of ovx and one group of intact female rats (3.5 months) were left untreated for 11 weeks. For the following 12 weeks, four groups received subcutaneous injections of hPTH (75 ␮g/kg per day on 3 days/week) and four groups received vehicle. Treatments were then changed to E2 (10 ␮g/kg per day on 2 days/week), Ris (3 ␮g/kg per day on 3 days/week), LowPTH (25 ␮g/kg per day on 3 days/week), or vehicle. Bone tissue was collected at weeks ⴚ11 (baseline), 0 (ovx effect), 12 (hPTH effect), 24, 36, and 48 (maintenance effect). Bone mineral density (BMD) and bone mineral content (BMC) of the diaphyseal femur and total cross-sectional area (Tt.Ar), marrow area (Ma.Ar), cortical area (Ct.Ar), and periosteal and endocortical bone formation of the tibia were measured. Ovariectomy resulted in lower BMD (weeks 0 – 48), unaffected BMC, and greater Tt.Ar (weeks 12 and 36), Ma.Ar (week 48), and Ct.Ar (weeks 0 and 12) compared with intact rats. Endocortical and periosteal bone formation were greater in the ovx than in the intact rats up to 23 weeks postovariectomy. Treatment of ovx rats with hPTH for 12 weeks resulted in greater cortical BMD, BMC, and endocortical bone formation than in intact or ovx controls. In ovx rats pretreated with hPTH and then treated with Ris for 36 weeks, BMD and BMC were greater and Ma.Ar was smaller than in ovx controls. In ovx rats pretreated with hPTH and then treated with LowPTH, BMD, BMC, Ct.Ar, and endocortical bone formation were greater and Ma.Ar was smaller than in ovx controls. Treatment of hPTH-pretreated rats with E2 for 36 weeks did not affect cortical BMD, BMC, and Ct.Ar, although periosteal bone formation was lower in the E2 group compared with the ovx group. Thus, in ovariectomized rats, cortical bone gained by 12 weeks of hPTH treatment was maintained for up to 36 weeks by treatment with risedronate or low-dose hPTH, but not with 17␤-estradiol. (Bone 28:251–260; 2001) © 2001 by Elsevier Science Inc. All rights reserved.

Introduction Osteoporosis associated with fragility fractures is a very common disorder among elderly postmenopausal women.2,10 It is associated with low cancellous and cortical bone density,21,42 and poor trabecular architecture.24,28 Estrogen replacement therapy is well known to prevent bone loss and has been used for three decades in the treatment of osteoporosis.1,7,13 Bisphosphonates represent a more recent treatment regime for osteoporosis.5,19,31 Estrogen and bisphosphonates are both antiresorptive agents that prevent bone loss mainly through the inhibition of osteoclast activity.38,43,46 A more ideal treatment would reduce osteoporotic fracture risk by increasing bone formation, bone mass, and bone strength. Due to its bone anabolic properties, human parathyroid hormone (hPTH) has been suggested to be an effective therapeutic agent in the treatment of osteoporosis in humans.14,17,32,41 In the rat model, intermittent administration of hPTH increases cortical bone mass and strength by increasing periosteal and endocortical bone formation.11,16,20,39,53 Cessation of hPTH, unfortunately, is followed by a rapid loss of bone mass.16 Strategies are needed to maximize bone gain and then maintain the newly gained bone. The purpose of this investigation was to increase cortical bone mass with intermittent hPTH(1-84) administration for 12 weeks and then evaluate the effects of antiresorptive maintenance agents (17␤-estradiol and risedronate) or a reduced maintenance dose of hPTH(1-84) on cortical bone mineral density and content, bone structure, and time course bone formation endpoints in an estrogen-depleted rat model. The data presented are part of a larger study including cancellous bone mass and bone strength. Materials and Methods Animals Two hundred sixty-seven, 3.5-month-old, virgin or intact, ovariectomized female Sprague-Dawley rats, weighing 250 ⫾ 13 g, were obtained from Sasco (Omaha, NE). The rats were given free access to drinking water. Body weights of all rats were recorded at the beginning and every 6 weeks throughout the study. Ovariectomized rats were food restricted throughout the experiment to minimize weight gain associated with ovariectomy and weight match to intact control rats.52 The experiment was conducted according to a protocol approved by the Creighton University Animal Resource Facility, and animals were maintained

Address for correspondence and reprints: Eva Samnegård, M.D., Department of Orthopaedic Surgery, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden. E-mail: [email protected] © 2001 by Elsevier Science Inc. All rights reserved.

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8756-3282/01/$20.00 PII S8756-3282(00)00446-4

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Table 1. Experimental design showing groups and number of rats at necropsya Group

Week ⫺11

Week 0

Week 12

Week 24

Week 36

Week 48

8

7 8

8 8 8

8 8 8

8 8 8

8 8 7

17␤-estradiol Risedronate Low-dose hPTH

8 8 8

8 8 8

8 9 8

17␤-estradiol Risedronate Low-dose hPTH

8 8 7

8 8 8

8 8 7

Surgery

Pretreatment

Maintenance treatment

Intact Ovx-V Ovx-PTH-V

None Ovx Ovx

None Vehicle PTH

None Vehicle Vehicle

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH

Ovx Ovx Ovx

PTH PTH PTH

Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH

Ovx Ovx Ovx

Vehicle Vehicle Vehicle

See Materials and Methods for explanation of treatment for each group. a Six rats were lost to premature death.

according to the NIH Guide for the Care and Use of Laboratory Animals. Experimental Design The rats were randomized by weight into nine groups, one intact control group (intact) and eight ovariectomized groups (ovx) (Table 1). Bone tissue from the baseline intact control group was collected at week ⫺11. The animals were then left untreated for 11 weeks to allow for the development of changes in bone properties in the ovx groups. Bone tissue from the intact and control ovx (ovx-V) groups was collected at week 0. For weeks 0 –12, four ovx groups were injected subcutaneously with human parathyroid hormone(1-84) (hPTH; 75 ␮g/kg per day, three times per week) and four groups received vehicle (1 mL/kg per day, three times per week). Tissue from the intact, ovx-V, and hPTH-treated ovx (ovx-PTH-V) groups was then collected at week 12. For the remaining 36 weeks, treatments were changed to vehicle (three times per week), 17␤-estradiol (E2; 10 ␮g/kg per day, two times per week), risedronate (Ris; 3 ␮g/kg per day, three times per week), or a low dose of hPTH(1-84) (LowPTH; 25 ␮g/kg per day, three times per week). All injections were given subcutaneously. The intact control group remained untreated throughout the duration of the experiment. Bone tissue was collected from all groups at weeks 24, 36, and 48. Preparation of Treatment Solutions Treatment solutions were prepared to deliver 1 mL/kg body weight. 17␤-estradiol (10 ␮g/mL; Sigma Chemical Co., St. Louis, MO) was dissolved in 98% corn oil and 2% benzyl alcohol. Risedronate (3 ␮g/mL) was dissolved in saline. hPTH(184) (Allelix Biopharmaceuticals, Mississauga, ON, Canada) (25 or 75 ␮g/mL) was dissolved in heat-inactivated 2% rat serum (Allelix). The vehicle (V) treatment was heat-inactivated 2% rat serum. Tissue Collection A double-labeling fluorochrome technique was used to determine active mineralization sites and rates of bone formation. Each animal was injected subcutaneously with calcein (8 mg/kg; Sigma Co.) at 10 and 3 days before killing. Bone tissue was collected between 9:00 A.M. and 12:00 A.M., after an overnight fast. Each rat was anesthetized with ketamine HCl (50 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. Blood was drawn through the aorta bifurcation, causing death by exsangui-

nation. The uterine horns were examined to verify successful ovariectomy. For tissue collection, the right leg was disarticulated at the hip, knee, and ankle. The femur and tibia were cleaned of excess muscle and soft tissue. The femur was placed in 70% ethanol for densitometric analysis. The tibia was cut into three sections, a proximal (1 cm), a central (diaphyseal), and a distal section (5 mm). The diaphyseal segment was processed with the tibiofibular junction (TFJ) intact and placed in 10% phosphate-buffered formalin for 24 h and then transferred to 70% ethanol for histomorphometric processing. Densitometry of Femoral Diaphysis Bone mineral content (BMC) of the diaphyseal region of the right femur was measured with a dual-energy X-ray bone densitometry device (Model XR2600, Norland Corp., Ft. Atkinson, WI).25 The right femur was placed on a 1.5-cm-thick piece of plastic and scanned in a distoproximal direction at a resolution of 0.5 mm and a scanning speed of 2 mm/sec. Bone mineral density (BMD) was calculated as BMC divided by projected area. The distal and the diaphyseal regions were isolated by software during analysis. The distal region started at the first scan line with three or more consecutive points ⱖ0.06 g/cm2 of mineral. The diaphyseal region began 9 mm proximal to this line, extended 19 mm proximally, and was centered over the diaphysis. The diaphyseal region is composed of ⬎98% cortical bone and is taken to represent cortical bone.25 Histomorphometry of Tibial Diaphysis The right tibial diaphysis was processed for histomorphometric analysis of cortical bone. The specimens were Villanueva block stained51 for 72 h and placed in 80% ethanol. During the next 2 weeks, the bones were gradually dehydrated in graded ethanols and acetone, and then embedded in methylmethacrylate.3 The embedded diaphyseal samples were cross sectioned at 75 ␮m between 5 and 7 mm proximal to the distal TFJ on a saw microtome (Model 1600, Leica, Nussloch, Germany). One section was mounted on a glass slide with Permount and assigned a random number. Distances, perimeters, and areas were digitally traced with a light/epifluorescent microscope and a camera lucida with a graphics pad interfaced to an IBM PCAT computer and image-analysis software (BIOQUANT II, R&M Biometrics, Nashville, TN). Standard bone histomorphometry nomenclature was used.40 The following data were collected for each specimen: at ⫻20, total tissue area (Tt.Ar) and marrow area (Ma.Ar)

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and periosteal and endocortical bone perimeter (B.Pm); at ⫻200, double (dL.Pm) and single (sL.Pm) labeled perimeter; and at ⫻400, interlabel width (Ir.L.Wi) at sites of double labeling. The following calculations were made for each section: cortical area (Ct.Ar ⫽ Tt.Ar ⫺ Ma.Ar); mineralizing surface [MS ⫽ 100ⴱ(dL.Pm ⫹ 0.5sL.Pm)/B.Pm]; and mineral apposition rate (MAR ⫽ Ir.L.Wi/interlabel time period). When double labels were missing in tibial sections of the same surface, but single labels were present, MAR was set to the biological lower limit of 0.3 ␮m/day.15 If both single and double label were missing on the same surface, MAR was treated as a missing value. Statistics Values in tables are reported as mean ⫾ SD and in diagrams as mean ⫾ SE or as means only. Longitudinal body weight data in animals surviving to week 48 were analyzed using a split-plot two-factor analysis of variance (ANOVA) for differences between groups at each week and differences within groups over time. The Bonferroni post hoc test was used to identify differences among treatment groups at each collection period (weeks 0, 12, 24, 36, and 48). Differences were considered significant at p ⬍ 0.05 for the ANOVA test and p ⬍ 0.02 for the Bonferroni post hoc test. The Kruskal–Wallis test was used to analyze bone data for differences among treatment groups at each timepoint and differences within time.8 If the Kruskal–Wallis test was significant (p ⬍ 0.05), a nonparametric post hoc test (t-statistic adjusted for number of groups and comparisons) was used to identify groups different from intact, ovx-V, ovx-PTH-V, and the group with same maintenance treatment pretreated with vehicle. Statistical significance for the post hoc comparisons was set at p ⬍ 0.02. All statistical analyses were performed using CRUNCH software (Crunch Software Corp., Oakland, CA). Results The survival rate for the rats over the 59 weeks of study was remarkably high; that is, only 6 of 267 were lost to premature death. Atrophied uterine horns confirmed successful ovariectomy in all operated rats. A longitudinal assessment of body weight in animals surviving to week 48 is presented in Figure 1. There were no differences in body weight among these groups at the start of the study (249 ⫾ 13 g). At week 0, all ovx groups were heavier than intact controls, despite restricted food intake. The daily food ration was further lowered for all ovx rats at this time. At week 12, only the ovx-V, ovx-V-E2, ovx-V-Ris, and ovx-PTH-Ris groups were heavier than intact controls. By week 24, the weight differences between the intact and ovx groups were not significant. Body weight increased in all groups up to week 12 (age 9 months). In some ovx groups, animals continued to gain weight up to week 36 (age 14.5 months). Femoral Diaphyseal Densitometry Means and significant treatment differences in bone densitometry and histomorphometry results are presented in Figures 2– 4. Tables 2– 4 contain more detailed presentation of the data. Femoral diaphyseal BMD and BMC were greater in older than in younger rats in both intact and ovx-V groups and followed the age-related weight increase (Figure 2 and Table 2). BMD was lower in the ovx-V rats than in intact controls 11 weeks postovariectomy (week 0) and remained lower in these groups for the duration of the study. Treatment of ovx rats with hPTH (ovx-PTH-V) for 12 weeks resulted in greater BMD than in either intact or ovx-V control groups. Cessation of hPTH

Figure 1. Changes in body weight in rats that survived to week 48. Values are shown as mean ⫾ SE. aDifferent from intact group at same week. Analysis of variance (ANOVA), p ⬍ 0.02.

resulted in BMD values that were intermediate, and not significantly different from either ovx-V or intact rats at weeks 36 and 48. Treatment of hPTH pretreated rats with 17␤-estradiol (ovxPTH-E2) resulted in BMD values not different from ovx-V at week 48. No differences in BMD were detected between animals treated only with 17␤-estradiol (ovx-V-E2) and those treated with vehicle (ovx-V) at weeks 24 – 48. Both ovx-PTH-E2 and ovx-V-E2 groups had lower BMD than intact controls at the end of the study and did not differ from each other. Administration of risedronate, regardless of previous hPTH pretreatment (ovxPTH-Ris and ovx-V-Ris), resulted in BMD values higher than ovx-V controls and not different from intact controls at the end of the study. BMD in ovx-PTH-Ris rats was also higher than in ovx-PTH-V at the end of the study. BMD in ovx-PTH-Ris was higher than in ovx-V-Ris at weeks 24 and 48. Administration of low-dose hPTH, regardless of previous hPTH treatment (ovxPTH-LowPTH and ovx-V-LowPTH) resulted in higher BMD values in comparison to ovx-V, intact controls and ovx-PTH-V at the end of the study. In contrast to BMD, BMC was not different between ovx-V and intact groups at any timepoint (Figure 2A, Table 2). Treatment of ovx rats with hPTH (ovx-PTH-V) for 12 weeks resulted in greater BMC than intact and ovx-V control groups. Cessation of hPTH resulted in BMC values not different from either intact or ovx-V groups at the end of the study. Treatment with 17␤estradiol for 36 weeks, regardless of previous hPTH pretreatment (ovx-PTH-E2 and ovx-V-E2), resulted in BMC values not different from either intact, ovx-V, or ovx-PTH-V at the end of the study. Administration of risedronate to hPTH-pretreated rats (ovx-PTH-Ris) resulted in BMC values higher than in intact and ovx-V controls. Risedronate-treated rats with no pretreatment (ovx-V-Ris) did not differ from either intact, ovx-V, or ovxPTH-V groups at the end of the study. Low-dose hPTH treatment, regardless of previous hPTH treatment (ovx-PTH-Low-

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Figure 2. Bone mineral density (A) and bone mineral content (B) of diaphyseal femur (mg/cm2) after 12 weeks of hPTH or vehicle treatment, followed by vehicle, 17␤-estradiol (E2), risedronate (Ris), or low-dose hPTH (LowPTH) treatment for 36 weeks in ovariectomized rats. Values are shown as means. Differences are shown only for weeks 0, 12, and 48. a Different from intact group at same week; bdifferent from ovx-V group at same week; cdifferent from ovxPTH-V group at same week; ddifferent from same maintenance treatment, pretreated with vehicle, at same week. Kruskal–Wallis post hoc test, p ⬍ 0.02. Mean, SD, and comprehensive statistics are shown in Table 2. Note that values refer to different groups at each timepoint. Lines are provided to simplify identification of treatment groups, not to imply continuity over time. Table 2. Mean values, standard deviations, and comprehensive statistics for diaphyseal femur bone mineral density and bone mineral content Week ⫺11

Week 0

Week 12

Week 24

116 ⫾ 6

134 ⫾ 5 127 ⫾ 5

141 ⫾ 4 133 ⫾ 4a 146 ⫾ 4a,b

153 ⫾ 14 139 ⫾ 4a 150 ⫾ 7b

151 ⫾ 8 142 ⫾ 6a 148 ⫾ 12

146 ⫾ 8 135 ⫾ 6a 140 ⫾ 6

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH

150 ⫾ 6b,d 155 ⫾ 7b,d 152 ⫾ 6b,d

152 ⫾ 11b,d 157 ⫾ 7b 156 ⫾ 8b

136 ⫾ 7a 153 ⫾ 5b,c,d 161 ⫾ 9a,b,c

Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH

135 ⫾ 6a,c 142 ⫾ 7a 145 ⫾ 5

135 ⫾ 4a,c 148 ⫾ 9 159 ⫾ 8b,c

136 ⫾ 7a 143 ⫾ 6b 166 ⫾ 12a,b,c

⬍0.0001

⬍0.0001

Group

Week 36

Week 48

2

Bone mineral density of diaphyseal femur (mg/cm ) Intact Ovx-V Ovx-PTH-V

p value Bone mineral content of diaphyseal femur (mg) Intact Ovx-V Ovx-PTH-V

132 ⫾ 7

⬍0.03

⬍0.0002

⬍0.0001

153 ⫾ 5 154 ⫾ 6

168 ⫾ 7 165 ⫾ 7 178 ⫾ 8a,b

172 ⫾ 26 175 ⫾ 6 192 ⫾ 13b

182 ⫾ 16 181 ⫾ 9 184 ⫾ 19

185 ⫾ 15 186 ⫾ 11 192 ⫾ 9

180 ⫾ 13 192 ⫾ 11b 187 ⫾ 9b

188 ⫾ 16d 194 ⫾ 15 190 ⫾ 13

185 ⫾ 14 204 ⫾ 12a,b 213 ⫾ 12a,b,c

170 ⫾ 11c 179 ⫾ 13 179 ⫾ 8

165 ⫾ 12c 181 ⫾ 22 200 ⫾ 13a,b

182 ⫾ 11 192 ⫾ 11 225 ⫾ 24a,b,c

0.008

0.004

0.0001

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH p value

0.49

0.007

See Materials and Methods for explanation of treatment for each group. Values expressed as mean ⫾ SD. Kruskal–Wallis test, p ⬍ 0.05; post hoc test, p ⬍ 0.02. Comparisons within the same week: adifferent from intact; bdifferent from ovx-V; cdifferent from ovx-PTH-V; ddifferent from same maintenance treatment, pretreated with vehicle.

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Figure 3. Tibial cross-sectional total tissue area (A), marrow area (B), and cortical area (C) (mm2) after 12 weeks of hPTH or vehicle treatment, followed by vehicle, 17␤-estradiol (E2), risedronate (Ris), or low-dose hPTH (LowPTH) treatment for 36 weeks in ovariectomized rats. Values are shown as means. Differences are shown only for weeks 0, 12, and 48. aDifferent from intact group at same week; bdifferent from ovx-V group at same week; cdifferent from ovx-PTH-V group at same week. Kruskal–Wallis post hoc test, p ⬍ 0.02. Mean, SD, and comprehensive statistics are shown in Table 3. Note that values refer to different groups at each timepoint. Lines are provided to simplify identification of treatment groups, not to imply continuity over time.

PTH and ovx-V-LowPTH), resulted in higher BMC values in comparison to intact, ovx-V, and ovx-PTH-V groups at week 48. Tibial Diaphyseal Structure Differences between treatment groups in histomorphometric variables representing bone structure and formation endpoints were noted at the tibial diaphysis (Figures 3 and 4 and Tables 3 and 4). Tibial diaphyseal total cross-sectional area (Tt.Ar, bone ⫹ marrow) was greater in the ovx groups (ovx-V and ovx-PTH-V) compared with intact controls at week 12 (Figure 3A). Although a similar pattern was observed for all ovx groups at the remaining timepoints, the differences were not significant at the end of the study. Marrow area (Ma.Ar) tended to be greater in ovx-V rats than in intact rats, but the differences were significant only at week 48 (Figure 3B). Administration of hPTH (ovx-PTH-V) for 12 weeks did not affect Ma.Ar. After discontinuation of hPTH, Ma.Ar did not differ between the ovx-PTH-V and ovx-V control groups at the end of the study. Treatment with 17␤-estradiol, regardless of previous hPTH treatment, had no effect on Ma.Ar compared with ovx-V rats. However, Ma.Ar was greater in the 17␤-estradioltreated rats than intact controls at week 48. Ma.Ar was smaller in rats treated with risedronate or low-dose-hPTH, regardless of previous hPTH treatment, as compared with ovx-V rats at the end of the study. Ma.Ar was greater in the ovx-V-Ris group compared with the intact group at week 48. Cortical area (Ct.Ar) was greater in ovx-V rats than in intact control rats at weeks 0 and 12, but not subsequently (Figure 3C).

Administration of hPTH for 12 weeks resulted in greater Ct.Ar in ovx-PTH-V rats than in intact controls. However, Ct.Ar in ovx-PTH-V rats did not differ from ovx-V controls. After withdrawal of hPTH, Ct.Ar was greater in the ovx-PTH-V rats than in the intact controls at weeks 24 and 36, but not at the end of the study. At the end of the study, only groups that had received maintenance treatment with low-dose hPTH (ovx-PTH-LowPTH and ovx-V-LowPTH) had greater Ct.Ar than ovx-V and intact controls. Tibial Diaphyseal Histomorphometry Periosteal and endocortical mineralizing surface (MS/BS) was lower at each sequential timepoint up to week 24 in all groups (except for the ovx-V-LowPTH group between weeks 12 and 24) due to aging (Figure 4A,B and Table 4). On the tibial periosteum, a transient, threefold increase in MS/BS was observed in the ovx-V group at week 12 compared with intact controls (Figure 4A). Differences between groups were not detected at subsequent times. Treatment with hPTH for 12 weeks had no effect on periosteal MS/BS compared with either intact or ovx-V groups. Treatment of rats with estrogen, regardless of previous hPTH treatment, resulted in lower periosteal MS/BS compared with ovx-V rats at week 48. Administration of low-dose hPTH in hPTH-pretreated rats also resulted in a lower periosteal MS/BS compared with ovx-V rats at the end of the study. Endocortical mineralizing surface (MS/BS) tended to be higher in the ovx-V group relative to the intact group at week 0, but did not differ at subsequent timepoints (Figure 4B). Treat-

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Figure 4. Periosteal mineralizing surface (A) and endocortical mineralizing surface (B) after 12 weeks of hPTH or vehicle treatment followed by vehicle, 17␤-estradiol (E2), risedronate (Ris), or low-dose hPTH (LowPTH) treatment for 36 weeks in ovariectomized rats. Values are shown as means. Differences are shown only for weeks 0, 12, and 48. aDifferent from intact group at same week; bdifferent from ovx-V group at same week; cdifferent from ovx-PTH-V group at same week. Kruskal–Wallis post hoc test, p ⬍ 0.02. Mean, SD, and comprehensive statistics are shown in Table 4. Note that values refer to different groups at each timepoint. Lines are provided to simplify identification of treatment groups, not to imply continuity over time.

ment with hPTH (ovx-PTH-V) for 12 weeks, resulted in higher endocortical MS/BS compared with both ovx-V and intact controls at week 12. Cessation of hPTH resulted in MS/BS values not different from intact controls at the end of the study. Administration of low-dose hPTH, regardless of prior hPTH treatment, resulted in higher MS/BS in comparison to ovx-V and intact rats. Although double labels were absent in sections of the cortical tibia at 60% of the measured surfaces (periosteal and endocortical), only 7% (36 of 522 surfaces) were missing both single and double labels. The latter were treated as missing values and were excluded from calculated MAR data. Forty-two percent of the animals had no double label in either surface (periosteal or endocortical) of the tibia. When double labels were evaluated in diaphyseal and metaphyseal tibiae, all rats had double labels in at least one measured site. No individual MAR measurement was as low as the finite lower biological limit of 0.3 ␮m/day (range 0.45–5.08). Periosteal mineral apposition rate (MAR) was not different between groups at any timepoint, but was lower due to aging in all rats up to week 12 (age 9 months) compared with week ⫺11 (week ⫺11 2.30 ⫾ 0.20 ␮m, week 12 0.77 ⫾ 0.08 ␮m, week 48 0.52 ⫾ 0.03 ␮m, data not shown). Endocortical MAR also did not vary among groups at weeks 0 and 12, but was lower due to aging in all rats up to week 12 (age 9 months) compared with week ⫺11 (week ⫺11 2.58 ⫾ 0.36 ␮m, week 12 0.79 ⫾ 0.08 ␮m). hPTH treatment for 12 weeks and withdrawal of hPTH resulted in lower MAR compared with ovx-V and intact controls at week 48 only. Estrogen and risedronate treatment, regardless of previous hPTH treatment, resulted in lower MAR compared with ovx-V controls. Ovx-PTH-E2 and ovx-PTH-Ris groups had lower MAR than intact controls at the end of the study. In the groups treated with low-dose hPTH, regardless of previous hPTH

treatment, MAR was higher than in ovx-V and intact controls at weeks 24 and 36, but was not different at the end of the study. Discussion In this cross-sectional study of ovariectomized rats, cortical bone mineral density and content were greater in the group treated with hPTH for 12 weeks compared with the untreated group. This difference was also present after 36 weeks in the hPTHpretreated groups that had received maintenance treatment with risedronate or low-dose hPTH, but not in groups that had received 17␤-estradiol as maintenance treatment. This indicates that risedronate and low-dose hPTH may have the capacity to maintain increased bone mass gained with hPTH. As seen in other studies of estrogen-depleted rats, we found slightly increased cortical total tissue and marrow area following ovariectomy.4,44,49,50 We also found a transient increase of cortical area, but no change was seen beyond 23 weeks postovariectomy. Earlier studies have reported that cortical area does not change4,44,50 or decreases49 after ovariectomy. However, there are major differences between these studies in rat age, breed, restricted feeding, and follow-up period at 12, 6, 8, and 1 months, respectively. In accordance with other studies, femoral cortical BMD decreased after ovariectomy.26,44 However, the low BMD values were due to the increase in total bone area measured by densitometry, because cortical BMC was not affected by ovariectomy. Several studies where ovariectomized rats were followed for a maximum of 15 weeks have shown an increase in cortical bone turnover.50,53,54 Such an increase was seen in our study during the initial 23 week period, but not subsequently. This supports the previous finding that cortical bone turnover is no longer affected 1 year after ovariectomy.4

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Table 3. Mean values, standard deviations, and comprehensive statistics of diaphyseal tibia total tissue area, marrow area, and cortical area Group Total tissue area (bone and marrow) of the tibial diaphysis (mm2) Intact Ovx-V Ovx-PTH-V

Week ⫺11

Week 0

Week 12

5.1 ⫾ 0.5

5.8 ⫾ 0.6 6.3 ⫾ 0.4

5.5 ⫾ 0.4 6.4 ⫾ 0.5a 6.5 ⫾ 0.5a

Week 24

Week 36

Week 48

6.0 ⫾ 0.5 6.5 ⫾ 0.2 6.8 ⫾ 0.6

5.8 ⫾ 0.5 6.6 ⫾ 0.3a 6.6 ⫾ 0.8a

6.1 ⫾ 0.5 7.0 ⫾ 0.5 6.9 ⫾ 0.4

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH

6.3 ⫾ 0.7 6.7 ⫾ 0.6 6.6 ⫾ 0.5

6.6 ⫾ 0.6a 6.6 ⫾ 0.5a 6.6 ⫾ 0.3a

6.7 ⫾ 0.4 6.7 ⫾ 0.6 6.7 ⫾ 0.4

Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH

6.4 ⫾ 0.4 6.7 ⫾ 0.5 6.5 ⫾ 0.5

6.1 ⫾ 0.6 6.5 ⫾ 0.7a 6.7 ⫾ 0.4a

6.7 ⫾ 0.6 6.6 ⫾ 0.7 7.1 ⫾ 0.9

⬍0.07

⬍0.002

⬍0.14

⬍0.04

1.7 ⫾ 0.3 1.8 ⫾ 0.4

1.3 ⫾ 0.2 1.6 ⫾ 0.3 1.6 ⫾ 0.2

1.6 ⫾ 0.3 1.8 ⫾ 0.2 1.7 ⫾ 0.2

1.4 ⫾ 0.4 1.9 ⫾ 0.2 1.7 ⫾ 0.3

1.3 ⫾ 0.3 2.1 ⫾ 0.4a 1.9 ⫾ 0.3a

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH

1.5 ⫾ 0.3 1.6 ⫾ 0.3 1.6 ⫾ 0.3

1.7 ⫾ 0.3 1.5 ⫾ 0.2 1.5 ⫾ 0.3

1.8 ⫾ 0.2a 1.5 ⫾ 0.3b,c 1.3 ⫾ 0.3b,c

Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH

1.8 ⫾ 0.4 1.8 ⫾ 0.3 1.8 ⫾ 0.3

1.5 ⫾ 0.3 1.7 ⫾ 0.3 1.6 ⫾ 0.2

2.0 ⫾ 0.4a 1.6 ⫾ 0.3a,b 1.5 ⫾ 0.4b,c

p value Marrow area of the tibial diaphysis (mm2) Intact Ovx-V Ovx-PTH-V

1.5 ⫾ 0.4

p value Cortical area of the tibial diaphysis (mm2) Intact Ovx-V Ovx-PTH-V

3.6 ⫾ 0.2

⬍0.42

⬍0.07

⬍0.31

⬍0.10

4.1 ⫾ 0.3 4.5 ⫾ 0.3a

4.1 ⫾ 0.3 4.8 ⫾ 0.2a 4.9 ⫾ 0.3a

4.4 ⫾ 0.3 4.6 ⫾ 0.1 5.1 ⫾ 0.5a,b

4.3 ⫾ 0.3 4.7 ⫾ 0.2 4.9 ⫾ 0.6a

4.8 ⫾ 0.3 4.9 ⫾ 0.3 5.0 ⫾ 0.4

4.8 ⫾ 0.4 5.0 ⫾ 0.4a 5.0 ⫾ 0.4a

4.9 ⫾ 0.4a 5.1 ⫾ 0.4a 5.0 ⫾ 0.3a

4.9 ⫾ 0.3 5.2 ⫾ 0.4 5.3 ⫾ 0.3a,b

4.6 ⫾ 0.3c 4.9 ⫾ 0.3a 4.7 ⫾ 0.3 ⬍0.006

4.6 ⫾ 0.4 4.8 ⫾ 0.5 5.1 ⫾ 0.3a,b ⬍0.005

4.8 ⫾ 0.3 5.0 ⫾ 0.5 5.6 ⫾ 0.7a,b ⬍0.006

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH p value

⬍0.09

⬍0.008

⬍0.0006

⬍0.0001

See Materials and Methods for explanation of treatment for each group. Values expressed as mean ⫾ SD. Kruskal–Wallis test, p ⬍ 0.05; post hoc test, p ⬍ 0.02. Comparisons within the same week: adifferent from intact; bdifferent from ovx-V; cdifferent from ovx-PTH-V.

Consistent with previous studies, treatment of ovariectomized rats with hPTH(1-84) (225 ␮g/kg per week) for 12 weeks, increased cortical bone mineral density and content to above the level of intact rats.27,36,37,44 The elevated BMD and BMC were associated primarily with endocortical bone formation. Others have shown similar bone formation effects,48 or that hPTH stimulated both endocortical and periosteal bone formation.4,29,44 The differences in response on the periosteal surface could be due to differences in animal age and the start and duration of treatment after ovariectomy. Although BMD and BMC increased after 12 weeks of hPTH treatment in ovx rats, statistically significant effects on cortical area were not seen until week 48 after an additional 36 weeks of low-dose hPTH administration. Due to increased endocortical bone formation resulting in smaller marrow area, the cortical area was significantly larger than both intact and ovx-V rats at the end of the study. This late bone response contrasts with other studies reporting areal cortical bone effects after 12–36 weeks of hPTH(1-34) or other PTH

analogs.4,36,37,44,49 It has been shown that the increase in cancellous16,33,45 and cortical bone density or bone mass16 is lost after cessation of hPTH. In our study, 24 weeks after termination of hPTH, cortical BMD values were intermediate and BMC was not different from intact and ovx-V rats, indicating a relative decrease in BMD and BMC over time. The same pattern for BMD was seen in a longitudinal study of ovariectomized rats treated with hPTH for 24 weeks, followed by 16 weeks of hPTH withdrawal, where femoral diaphyseal BMD slowly decreased and was not different from either sham- or vehicle-treated ovariectomized rats at the end of the 16 week period.27 In our study, after 24 weeks of hPTH withdrawal, cortical BMD, BMC, and bone formation were not different compared with ovx-V animals. This suggests that the anabolic effect of hPTH does not persist in cortical bone after cessation of treatment. We used a dose of 225 ␮g of hPTH(1-84) per kilogram per week, administered intermittently as three 75 ␮g/kg doses per week. The anabolic data for this dose of hPTH are consistent

258

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Bone Vol. 28, No. 3 March 2001:251–260

Table 4. Mean values, standard deviations, and comprehensive statistics of diaphyseal tibia periosteal mineralizing surface and endocortical mineralizing surface Group

Week ⫺11

Week 0

Week 12

Week 24

Periosteal mineralizing surface of the tibial diaphysis (%) Intact Ovx-V Ovx-PTH-V

33.7 ⫾ 13.9

25.7 ⫾ 10.8 33.5 ⫾ 16.6

8.2 ⫾ 16.5 25.2 ⫾ 13.9a 18.9 ⫾ 17.1

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH p value Endocortical mineralizing surface of the tibial diaphysis (%) Intact Ovx-V Ovx-PTH-V

59.9 ⫾ 16.0

Week 48

4.1 ⫾ 7.6 7.9 ⫾ 13.5 5.2 ⫾ 9.0

9.2 ⫾ 12.0 13.9 ⫾ 15.1 10.6 ⫾ 10.0

15.6 ⫾ 23.3 18.6 ⫾ 11.6 13.8 ⫾ 19.0

0.8 ⫾ 1.3 0.3 ⫾ 0.3 3.3 ⫾ 6.4

3.4 ⫾ 5.2 2.7 ⫾ 4.8 6.9 ⫾ 8.8

2.2 ⫾ 2.7b 12.3 ⫾ 13.1 6.0 ⫾ 7.6b

2.1 ⫾ 2.5 8.7 ⫾ 9.0 9.1 ⫾ 10.3 ⬍0.31

2.4 ⫾ 3.8b 15.7 ⫾ 10.3 12.6 ⫾ 12.3 ⬍0.005

7.9 ⫾ 4.9 8.3 ⫾ 4.2 7.8 ⫾ 2.7

⬍0.30

⬍0.04

1.1 ⫾ 1.3 7.5 ⫾ 12.9 7.6 ⫾ 8.4 ⬍0.12

24.4 ⫾ 15.7 41.2 ⫾ 13.0

5.6 ⫾ 4.9 7.2 ⫾ 7.5 25.6 ⫾ 14.4a,b

3.8 ⫾ 2.9 7.6 ⫾ 3.3 7.1 ⫾ 2.9

4.9 ⫾ 2.7 6.1 ⫾ 1.6 9.1 ⫾ 3.9a

5.9 ⫾ 4.0 6.1 ⫾ 4.1 11.2 ⫾ 4.6a

5.9 ⫾ 2.5d 3.8 ⫾ 1.3c 19.8 ⫾ 5.2a,b,c

7.4 ⫾ 3.9 5.1 ⫾ 2.9 22.3 ⫾ 10.1a,b,c

6.7 ⫾ 3.7 5.2 ⫾ 1.9 16.2 ⫾ 7.1a ⬍0.006

3.5 ⫾ 2.8b,c 4.2 ⫾ 2.3c 29.8 ⫾ 13.6a,b,c ⬍0.0001

9.5 ⫾ 5.0 8.3 ⫾ 3.2 33.8 ⫾ 13.3a,b,c ⬍0.0001

Ovx-PTH-E2 Ovx-PTH-Ris Ovx-PTH-LowPTH Ovx-V-E2 Ovx-V-Ris Ovx-V-LowPTH p value

Week 36

⬍0.04

⬍0.03

See Materials and Methods for explanation of treatment for each group. Values expressed as mean ⫾ SD. Kruskal–Wallis test, p ⬍ 0.05; post hoc test, p ⬍ 0.02. Comparisons within the same week: adifferent from intact; bdifferent from ovx-V; cdifferent from ovx-PTH-V.

with earlier studies that have shown bone effects after 30 days of hPTH(1-84) treatment with doses of 218 ␮g/kg per week (23.1 nmol/kg per week at 3.3 nmol/kg per day), whereas doses of 72 ␮g/kg per week (7.7 nmol/kg per week at 1.1 nmol/kg per day) have shown no effects.34,39 In contrast, we found that the low dose of hPTH (75 ␮g/kg per week), started 23 weeks after ovariectomy, resulted in greater cortical BMD and BMC, cortical area, and endocortical mineralizing surface than in untreated ovariectomized rats. The lower dose of hPTH was as effective as the higher dose in increasing these endpoints, but a longer treatment period (24 vs. 12 weeks) was necessary. We also found that, after maintenance treatment with low-dose hPTH was started, regardless of previous hPTH pretreatment, there was a continuous increase in BMD and BMC, cortical area, and endocortical mineralizing surface with time. This suggests that a hPTH dose of 75 ␮g/kg per week may be sufficient to increase bone mass in ovariectomized rats. In several studies of ovariectomized rats, hPTH treatment was used in combination with antiresorptive agents, such as estrogen, bisphosphonate, and calcitonin, to increase cortical bone mass.4,36,47 The rationale for using antiresorptive agents as cotherapy to hPTH is to stimulate osteoblast activity by hPTH and at the same time suppress hPTH-mediated bone resorption,9,22,23,38 resulting in an optimal increase in bone mass. However, it has been shown in several rat studies that treatment with hPTH alone is as effective as cotreatment therapy with these antiresorptive agents on both cancellous and cortical bone.30,35,36,47,53 In humans, hPTH treatment and sequential calcitonin treatment did not have any greater anabolic effect on the spine than hPTH treatment alone.18 Because antiresorptive

cotherapy seems unable to fortify hPTH bone effects, and withdrawal of hPTH results in loss of gained bone, other treatment regimens are needed. Treatment with hPTH for a limited time period, followed by maintenance treatment with antiresorptive agents or a lower dose of hPTH (as in this study), seems to be a reasonable approach to the problem. In this study, where ovariectomized rats were given hPTH for 12 weeks followed by maintenance treatment with risedronate for 36 weeks, cortical BMD was higher than in ovx-V and ovx-PTH-V and BMC was higher than in intact and ovx-V, indicating that risedronate is fully capable of maintaining BMD and BMC gained by previous hPTH treatment. This is in accordance with other studies where intact12 and ovariectomized37 rats were pretreated with hPTH followed by risedronate. The same pattern has been reported for the bisphosphonate, icadonate disodium, in ovx rats.48 Although risedronate alone was able to increase BMD slightly, pretreatment with hPTH followed by risedronate was much more efficient at increasing and maintaining cortical BMD. As in other studies, risedronate did not affect bone formation at either the periosteal or the endocortical surface, regardless of pretreatment with hPTH.4,36,53 Risedronate treatment resulted in a smaller marrow area than vehicle-treated ovx rats, suggesting an inhibition of osteoclasts, thereby preventing bone resorption on the endocortical surface. The maintenance of endocortical bone by risedronate has been described previously in severely osteopenic rats,4 in old male rats,12 and for the trabecular endeosteum in intact rats with immobilization-induced osteopenia.33 The response to 17␤-estradiol treatment after ovariectomy was similar to that seen by other investigators, where 17␤estradiol decreased cortical bone formation.4,50,54 The response

Bone Vol. 28, No. 3 March 2001:251–260

was similar regardless of pretreatment with hPTH. However, in our study, the cortical BMD and BMC gained by previous hPTH was lost during 17␤-estradiol maintenance treatment and, after 36 weeks, the BMD and BMC levels were similar to those of untreated ovx-V rats. A possible explanation for the poor antiresorptive effects of estrogen could be that higher doses of estrogen are needed to maintain new bone gained by hPTH.6 Another explanation could be that animals respond to cellregulating hormones differently at an older age. In conclusion, in ovariectomized rats pretreated with hPTH(184) for 12 weeks, risedronate and low-dose hPTH were capable of maintaining the hPTH-related gain in cortical bone mass for up to 36 weeks. Low-dose hPTH not only maintained but also increased bone mass above the level of intact rats. Both risedronate and hPTH acted mainly on the endocortical surface with risedronate preventing bone resorption and hPTH increasing bone formation. In contrast, 17␤-estradiol did not maintain cortical bone mass over time. Consequently, treatments with hPTH followed by risedronate or prolonged low-dose hPTH appear to be most effective for gaining and maintaining bone. This supports previous evidence that hPTH is of potential interest for osteoporosis treatment. Our study indicates that the relationship between bone mass restoration and maintenance with different hPTH dosages and different antiresorptive drugs is an important area for further research.

Acknowledgments: The authors acknowledge Allelix Biopharmaceuticals, Ltd., who supplied the hPTH(1-84) used in this experiment. We also acknowledge Toni Howard, Susan Bare, Dwayne Belognia, Jennye Matula, Julia Westrich, Nina Veccio, and Michelle Hunke-Daffer for injecting and pair-feeding the experimental animals and sectioning of their cortical bones. This work was supported by USNIH Grant AR39221-09 and by a grant from the Fo¨renade Liv Mutual Group Life Insurance Co., Stockholm, Sweden.

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Date Received: July 3, 2000 Date Revised: October 10, 2000 Date Accepted: October 27, 2000