Continuous Parathyroid Hormone and Estrogen Administration ... - BPS

JOURNAL OF BONE AND MINERAL RESEARCH Volume 16, Number 7, 2001 © 2001 American Society for Bone and Mineral Research

Continuous Parathyroid Hormone and Estrogen Administration Increases Vertebral Cancellous Bone Volume and Cortical Width in the Estrogen-Deficient Rat H. ZHOU,1 V. SHEN,1–3 D.W. DEMPSTER,1,2 and R. LINDSAY1,4

ABSTRACT Generally, it is believed that intermittent administration of parathyroid hormone (PTH) has an anabolic effect on the skeleton, whereas continuous administration is catabolic. However, there is evidence that continuous exposure to PTH may have an anabolic effect, for example, in patients with mild primary hyperparathyroidism (PHPT). The possibility of delivering PTH continuously may have important implications for the treatment of osteoporosis. Furthermore, estrogen treatment may be useful in the medical management of PHPT. Therefore, we examined the skeletal effects of continuous administration of PTH, with or without estrogen, in the estrogen-deficient rat with established osteopenia. Forty 7-month-old SD rats were divided into four ovariectomy (OVX) groups and one sham-operated group. Eight weeks post-OVX, three groups received subcutaneous implants of Alzet mini pumps loaded with PTH(1-34) (30 ␮g/kg per day), 17␤-estradiol (10 ␮g/kg per day) pellet, or both PTH and 17␤-estradiol separately for 4 weeks. OVX and sham control groups were given the mini pumps loaded with vehicle. Two doses of calcein (10 mg/kg) were given subcutaneously to all rats 2 days and 8 days before death. Histomorphometry was performed on cancellous and cortical bone of the fourth lumbar vertebra. At 3 months, post-OVX rats displayed bone loss with high bone turnover. Estrogen reversed OVX-mediated high turnover without restoring cancellous bone volume (BV/TV). PTH infusion further increased bone turnover and partially restored BV/TV. However, PTH infusion increased cortical porosity. Estrogen inhibited PTH-mediated cancellous bone resorption and substantially increased BV/TV above sham control. The combined treatment was associated with a significant increase in peritrabecular fibrosis and woven bone formation. The combined treatment of PTH infusion and estrogen replacement enhanced cortical width but estrogen did not prevent the PTH-induced cortical tunneling. We conclude that continuous administration of PTH and estrogen increases cortical porosity but has substantial beneficial effects on vertebral cancellous bone volume and cortical width in OVX rats. (J Bone Miner Res 2001;16: 1300 –1307) Key words:

parathyroid hormone, hyperparathyroidism, estrogen, histomorphometry, osteoporosis

INTRODUCTION T GENERALLY is believed that intermittent administration of parathyroid hormone (PTH) has an anabolic effect on the skeleton, whereas continuous administration is cata-

I

bolic. However, a mild anabolic action of continuous exposure to PTH on cancellous bone has been recognized in patients with asymptomatic primary hyperparathyroidism (PHPT)(1–3) as well as in animals treated continuously with exogenous PTH.(4 –7) Cancellous bone, a skeletal component

1

Regional Bone Center, Helen Hayes Hospital, New York State Health Department, West Haverstraw, New York, USA. Department of Pathology, Columbia Presbyterian Medical Center, New York, New York, USA. Present address: Skeletech, Incorporated, Bothell, Washington, USA. 4 Department of Medicine, Columbia Presbyterian Medical Center, New York, New York, USA. 2 3

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CONTINUOUS PTH AND ESTROGEN ADMINISTRATION IN OVX RAT

that is affected predominantly in postmenopausal osteoporosis, was relatively preserved in postmenopausal women with PHPT.(1,2,8) Intermittent administration of PTH by daily injection also has been shown to increase bone density in osteoporotic patients.(9) These observations raise the possibility of continuous delivery of PTH for the treatment of osteoporosis. However, in contrast to the anabolic response of cancellous bone,(1–3,8) a catabolic action on cortical bone is a consistent finding in patients with PHPT.(8,10,11) Moreover, the response of cancellous bone volume to continuous elevation of PTH in patients with PHPT is relatively minor and may not augment bone mass sufficiently to prevent fractures in osteoporotic patients. This is especially so because high turnover is an independent risk factor for fracture. Therefore, in consideration of the therapeutic implications of delivering PTH continuously in the treatment of osteoporosis, it is desirable to explore combined treatment with an agent, such as estrogen, that could prevent cortical bone loss, augment cancellous bone gain, and mitigate the increase in turnover. Estrogen replacement therapy (ERT) has been considered in the treatment of PHPT in postmenopausal women.(10,12,13) ERT reduced serum calcium and biochemical markers of bone turnover and increased bone mineral density (BMD) in the axial and appendicular skeleton.(10) However, the mechanism of these effects at the cellular level has not been investigated in either human or animal studies. Exogenous PTH infusion in rats induces changes in both cortical and cancellous bone and in some serum biochemistry that are similar to those seen in patients with PHPT.(4 –7,14) Therefore, in this study, we examined the effects of continuous administration of PTH in the presence and absence of estrogen, on bone structure, and in cellular activity in ovariectomized (OVX) rats. This work extends a previous publication(15) in which we reported the effects of these treatments on biochemical markers, BMD, and bone biomechanics.

MATERIALS AND METHODS In a previous report,(15) we described changes in BMD, biochemical markers, and bone mechanical strength in the same animals as we studied here. Therefore, the experimental protocol will be described only briefly. Forty virgin female Sprague–Dawley rats at 7-months of age were subjected to either bilateral OVX or sham operation under 12.5:2.5 mg/kg ketamine/xylazine anesthesia. The animals remained untreated for an additional 8 weeks to allow the development of post-OVX bone loss. To prevent the OVXinduced excessive hyperphagia, the rats were fed Laboratory Rodent Chow (Purina Mills, St. Louis, MO, USA), 15 g/day per rat, the mean food intake of the sham-operated animals during the experimental period. At the initiation of treatment, the rats were divided into five groups of eight each. One sham and one OVX control group were implanted subcutaneously with Alzet miniosmotic pumps (model 2002; Alza Corp., Palo Alto, CA, USA) loaded with vehicle (1 mM acetic acid). One OVX group was given pumps loaded with vehicle and an additional subcutaneous 17␤-

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estradiol pellet implant (Innovative Research, Inc., Sarasota, FL, USA) delivering 10 ␮g 17␤-estradiol/kg per day. One OVX group was implanted with PTH(1-34) (Bachem, Inc., Torrance, CA, USA) in Alzet miniosmotic pumps delivering 30 ␮g PTH/kg per day. The other OVX group was implanted with both 17␤-estradiol pellets and PTH-loaded pumps. Miniosmotic pumps were replaced biweekly because of the functional life span of the pump. The implantations were performed under light ether anesthesia. Two doses of calcein (Sigma Chemical Co., St. Louis, MO, USA) at 10 mg/kg body weight were given subcutaneously to all the animals at 2 days and 8 days before death. The animals were killed after 4 weeks of treatment by exsanguination under ketamine/xylazine anesthesia. The fourth lumbar vertebrae were fixed in 70% ethanol.

Bone histomorphometry Excised fourth lumbar vertebrae were cut longitudinally to expose the bone marrow using a low-speed metallurgical saw and dehydrated in graded ethanol, defatted in toluene, and embedded in methylmethacrylate. Four-micrometerand 8-␮m-thick sagittal sections of the central region were cut with a Polycut S microtome (Reichert-Jung, Heidelberg, Germany). The 4-␮m sections were stained with Goldner’s trichrome for analysis of static parameters and the 8-␮m sections were left unstained for dynamic histomorphometric analysis. The two-dimensional parameters, obtained by histomorphometry of cancellous bone, were measured on the secondary spongiosa in an area 0.6 mm from the cranial or caudal growth plate. Parameters of cortical bone were measured on both dorsal and ventral cortices. Trabecular bone area and perimeter, osteoid perimeter and area, woven bone area, cortical width and area, and cortical porosity area and number, as well as single- and double-label perimeters and double-label width were measured using a digitizing image analysis system and a morphometric program (OsteoMeasure; OsteoMetrics, Inc., Atlanta, GA, USA), at a magnification of ⫻100 or ⫻200. Woven bone was identified by the presence of randomly oriented collagen fibers under polarized light; larger, rounder, and less uniformly distributed osteocyte lacunae; and diffuse calcein labeling. Osteoblast, osteoclast, and peritrabecular fibrosis perimeters were measured by the point counting method using a 36-point MerzGrid (Carl Zeiss, Oberkochen, Germany) at a magnification of ⫻200.(16) All parameters were expressed and calculated according to Parfitt.(17) The combined area of cortical canals was defined as cortical porosity, expressed as a percentage of total cortical area. The numbers of canals and labeled canals were defined as cortical canal number and mineralized canal number, respectively, expressed per unit of cortical area. Intracortical bone formation rate was calculated as the product of labeled perimeter (double-labeled perimeter ⫹ 1⁄2 single-labeled perimeter) ⫻ intracortical mineral apposition rate/total canal perimeter. A mineral apposition rate of 0.3 ␮m/day was assigned to samples that had single labels but no double labels.(18)

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TABLE 1. HISTOMORPHOMETRIC VARIABLES Parameters

Sham

Cancellous bone volume (%) Woven bone volume (%) Trabecular number (no./mm2) Trabecular width (␮m) Trabecular separation (␮m) Osteoblast perimeter (%) Osteoclast perimeter (%) Osteoid perimeter (%) Fibrosis perimeter (%) Mineralized perimeter (%) Mineral apposition rate (␮m/day) Bone formation rate (␮m3/␮m2 per day) Cortical width (␮m) Cortical porosity (%) Cortical canal number (no./mm2) Mineralized canal number (no./mm2) Cortical-mineral apposition rate (␮m/day) Cortical-bone formation rate (␮m3/␮m2/per day)

26.3 ⫾ 1.6 0 4.2 ⫾ 0.1 63.1 ⫾ 3.3 179.0 ⫾ 8.5 14.8 ⫾ 1.6 2.0 ⫾ 0.3 7.4 ⫾ 1.7 0 1.3 ⫾ 0.4 0.3 ⫾ 0.1

OF

CANCELLOUS OVX

21.1 ⫾ 1.2* 0 3.7 ⫾ 0.1* 57.0 ⫾ 2.7 213.9 ⫾ 6.8* 18.7 ⫾ 1.7* 3.8 ⫾ 0.7* 7.8 ⫾ 2.3 0 6.4 ⫾ 1.2* 1.0 ⫾ 0.2*

0.004 ⫾ 0.001 0.07 ⫾ 0.02* 175.5 ⫾ 5.6 178.9 ⫾ 11.7 0.4 ⫾ 0.1 0.2 ⫾ 0.2 1.7 ⫾ 0.2 1.0 ⫾ 0.44

AND

CORTICAL BONE

IN

LUMBAR VERTEBRA

Estrogen

PTH

Estrogen ⫹ PTH

21.0 ⫾ 1.6* 0 3.6 ⫾ 0.2* 59.1 ⫾ 3.3 225.7 ⫾ 14.5* 8.9 ⫾ 2.6† 0.7 ⫾ 0.3*,† 0.8 ⫾ 0.3*,† 0 1.4 ⫾ 0.5† 0.4 ⫾ 0.1†

24.0 ⫾ 2.4 0.1 ⫾ 0.1 3.9 ⫾ 0.1 61.6 ⫾ 4.4 200.3 ⫾ 13.5 25.9 ⫾ 2.9*,‡ 13.7 ⫾ 2.4*,†,‡ 22.9 ⫾ 4.4*,†,‡ 0.4 ⫾ 0.4 17.0 ⫾ 2.0*,†,‡ 1.2 ⫾ 0.1*,†,‡

49.5 ⫾ 4.5*,†,‡,§ 10.6 ⫾ 3.3*,†,‡,§ 5.1 ⫾ 0.6‡ 101.8 ⫾ 11.0*,†,‡,§ 108.1 ⫾ 14.3*,†,‡,§ 38.9 ⫾ 3.8*,†,‡,§ 3.0 ⫾ 1.4§ 25.1 ⫾ 10.9*,†,‡ 7.7 ⫾ 4.1*,†,‡,§ 29.9 ⫾ 4.5*,†,‡,§ 2.0 ⫾ 0.2*,†,‡,§

0.005 ⫾ 0.001† 189.6 ⫾ 11.5 0.2 ⫾ 0.1 0.7 ⫾ 0.3*

0.2 ⫾ 0.03*,†,‡ 186.1 ⫾ 15.0 2.9 ⫾ 0.6*,†,‡ 6.1 ⫾ 1.2*,†,‡

0.6 ⫾ 0.1*,†,‡,§ 237.1 ⫾ 12.5*,†,‡,§ 2.9 ⫾ 0.5*,†,‡ 7.0 ⫾ 0.8*,†,‡

0.001 ⫾ 0.02a

0.01 ⫾ 0.01b

0

0.2 ⫾ 0.1*,†,‡

0.3 ⫾ 0.1*,†,‡

0.08 ⫾ 0.05

0.04 ⫾ 0.04

0

0.7 ⫾ 0.2*,†,‡

1.2 ⫾ 0.3*,†,‡

0.01 ⫾ 0.01

0.004 ⫾ 0.01

0

0.1 ⫾ 0.04*,†,‡

0.3 ⫾ 0.1*,†,‡,§

The data are expressed as mean ⫾ SEM. a Two out of eight sham had mineralized canals. b One out of eight OVX had mineralized canals. * p ⬍ 0.05 versus sham; †p ⬍ 0.05 versus OVX; ‡p ⬍ 0.05 versus estrogen-treated group; §p ⬍ 0.05 versus PTH-treated group.

Statistical analysis All values are expressed as the mean ⫾ SEM. Analysis of variance (ANOVA) with Duncan’s multiple-range test provided by SAS software (SAS Institute, Inc., Cary, NC, USA) was performed for the comparison of differences among treatments. A value of p ⬍ 0.05 was considered significant.

RESULTS Mean values for each treatment group are given in Table 1; Figs. 1-3 show percent changes versus sham control. As expected, 3 months of estrogen deficiency decreased trabecular bone volume and trabecular number and increased trabecular separation (Fig. 1). No change occurred in cortical width or cortical porosity (Fig. 2). High bone turnover was indicated by increases in indices of bone formation and an increase in osteoclast perimeter (Fig. 3).

Effects of estrogen treatment alone Estrogen treatment reversed OVX-stimulated trabecular osteoclast perimeter to values lower than the sham control (Fig. 3). Cancellous bone formation also was reduced, as indicated by decreases in osteoblast perimeter, osteoid perimeter, mineralized surface, mineral apposition rate, and bone formation rate (Fig. 3). The lowered bone turnover did not result in restoration of cancellous bone volume and

FIG. 1. Changes in trabecular bone structure. All data are graphed as the percentage change from the sham control value. OVX, ovariectomized group; OE, OVX ⫹ 17␤-estradiol treatment group; OP, OVX ⫹ PTH(1-34) infusion group; OPE, OVX ⫹ PTH(1-34) infusion ⫹ 17␤estradiol treatment group. ap ⬍ 0.05 versus sham; bp ⬍ 0.05 versus OVX; cp ⬍ 0.05 versus OE; dp ⬍ 0.05 versus OP.


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FIG. 2. Changes in cortical bone structure and turnover. All data are graphed as the percentage change from the sham control value. OVX, ovariectomized group; OE, OVX ⫹ 17␤estradiol treatment group; OP, OVX ⫹ PTH(134) infusion group; OPE, OVX ⫹ PTH(1-34) infusion ⫹ 17␤-estradiol treatment group. a p ⬍ 0.05 versus sham; bp ⬍ 0.05 versus OVX; cp ⬍ 0.05 versus OE; dp ⬍ 0.05 versus OP.

FIG. 3. Changes in trabecular bone surface and turnover. All data are graphed as the percentage change from the sham control value. OVX, ovariectomized group; OE, OVX ⫹ 17␤estradiol treatment group; OP, OVX ⫹ PTH(134) infusion group; OPE, OVX ⫹ PTH(1-34) infusion ⫹ 17␤-estradiol treatment group. ap ⬍ 0.05 versus sham; bp ⬍ 0.05 versus OVX; cp ⬍ 0.05 versus OE; dp ⬍ 0.05 versus OP.

architecture in the OVX rats, but no further bone loss occurred (Fig. 1). Estrogen treatment did not change cortical width and porosity compared with sham and OVX but decreased cortical canal number by 60% compared with sham (Fig. 2).

Effects of PTH treatment alone PTH infusion partially restored cancellous bone volume and architecture in the OVX rats (Fig. 1). A small amount of woven bone appeared in cancellous bone (Table 1). PTH infusion dramatically stimulated trabecular osteoclast perimeter (Fig. 3). Bone formation and turnover also were increased, as shown by increases of osteoblast and osteoid perimeters, mineralized surface, mineral apposition rate, and bone formation rate (Fig. 3). Peritrabecular fibrosis was present on 0.4% of the trabecular surface (Table 1). PTH infusion did not change cortical width but increased cortical remodeling as evidenced by increases in cortical porosity, cortical canal number, mineralized canal number, cortical

mineral apposition rate, and bone formation rate compared with sham and OVX controls (Fig. 2).

Effects of PTH plus estrogen Combined treatment with PTH and estrogen completely restored the OVX-induced loss of cancellous bone and architecture and further enhanced bone volume and trabecular thickness to values substantially higher than sham controls (Fig. 1). A significant amount (10.6%) of woven bone was present. Static and dynamic indices revealed a dramatically increased bone formation rate (Fig. 3). On the other hand, osteoclast perimeter was significantly reduced compared with PTH alone and was similar to sham and OVX controls (Fig. 3). Fibrosis was observed on 7.7% of trabecular surface (Table 1). This is in contrast to PTH treatment alone, in which only a small amount (0.4%) was present, and estrogen treatment alone, in which there was none. In the cortices, the combined treatment enhanced cortical width compared with sham and OVX groups (Fig.

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2). However, cortical porosity and bone formation variables were elevated (Fig. 2).

Effects of PTH plus estrogen versus estrogen alone Compared with estrogen treatment alone, the combined treatment resulted in a gain in cancellous bone volume with marked woven bone formation, increases in trabecular number and thickness, and a decrease in trabecular separation (Fig. 1). Peritrabecular fibrosis was observed only in the combination treatment (Table 1). Osteoclast perimeter was 4-fold higher than in the group treated with estrogen alone, although this difference was not statistically significant (Fig. 3). However, bone formation indices were all significantly higher than with estrogen treatment alone (Fig. 3). Furthermore, the combined treatment enhanced cortical width. Cortical porosity and bone formation variables also were increased compared with estrogen treatment alone (Fig. 2).

some histomorphometric studies(2,8) and densitometric studies have indicated greater bone loss at sites rich in cortical bone.(8,10,20) ERT has been shown to have beneficial effects on bone density and turnover in postmenopausal women with PHPT.(12,13) Consistent with the changes in biochemical markers of resorption in humans(13,21) and rats,(15) addition of estrogen to the PTH-treated rats significantly reduced osteoclast surface. However, unlike the situation in humans, histomorphometric indices of formation were not reduced by estrogen in the PTH-treated rats. Consequently, the increment in bone density was greater than that seen after estrogen administration in patients with PHPT. This will be discussed further below. Although the changes in bone histomorphometry seen in the PTH-infused rats were similar in direction to those reported in patients with mild PHPT, they were greater in magnitude. As we have previously reported in these animals,(15) PTH infusion resulted in increases in serum calcium and PTH that were well beyond the normal range. Further investigation of the potential of the PTH-infused rat as a model of PHPT in humans will require the use of lower doses of PTH.

DISCUSSION The present study is in good agreement with our previous report(15) of changes in BMD and mechanical strength in these animals and provides additional insight into the underlying cellular mechanism. The following are the principal findings of this study: (1) combined continuous elevation of PTH and estrogen is a potent stimulator of bone formation, resulting in marked increases of cancellous bone volume and cortical width in the lumbar vertebral body of OVX rats; (2) estrogen, at a dose of 10 ␮g/kg per day, is capable of preventing PTH-mediated increments in osteoclast recruitment and activation in cancellous bone but is not able to prevent PTH-mediated tunneling in cortical bone; and (3) continuous administration of PTH(1-34) at a dose of 30 ␮g/kg per day provides a model of PHPT in OVX rats. This model is characterized by increased osteoclast recruitment, marrow fibrosis, increased bone formation rate, preserved trabecular bone volume and structure, and increased cortical tunneling.

A rodent model of PHPT After PTH infusion at a dose of 30 ␮g/kg per day for 4 weeks, the lumbar vertebral body of estrogen-depleted rats exhibited histomorphometric characteristics that mimic the changes in the axial skeleton of postmenopausal women with PHPT.(1–3) PTH infusion increased osteoclast surface in cancellous bone, which presumably contributed to the sustained elevation of serum calcium previously reported.(15) The increments in osteoblast, osteoid, and mineralizing perimeters confirmed that bone formation was coupled to resorption. These findings are consistent with the preservation of cancellous bone mass and architecture in the presence of enhanced bone turnover seen in the postmenopausal women with PHPT.(1–3) PTH infusion did not change cortical width compared with sham and OVX controls. This is consistent with a previous histomorphometric study,(19) in which no change was noted in cortical width in patients with PHPT. However, reductions in cortical width have been noted in

Continuous PTH or continuous PTH and estrogen administration in the treatment of estrogen-deficiency bone loss Intermittent administration of PTH has a significant anabolic action on the skeleton of both humans and rodents.(9,22–25) Continuous infusion of PTH, on the other hand, has been shown to reduce bone mass in rodents(14) and to change bone markers in favor of a catabolic action in humans.(26) This has led to the current axiom that intermittent administration of PTH has an anabolic effect on the skeleton, whereas continuous administration is catabolic. However, our data present a challenge to that view in that we saw no evidence of a deleterious effect of continuous PTH treatment on cancellous bone volume or cortical width. The only negative effect on bone mass was the increase of cortical porosity. Although PTH treatment did increase osteoclast surface, this was compensated for by an increase in the indices of bone formation. Our finding that cancellous bone volume and cortical width were maintained in the face of continuous PTH infusion of 3 ␮g of PTH(1-34)/100 g body weight (b.w.) per day is consistent with several previous studies in dogs.(27,28) Hock and Gera have reported that infusion of 4 ␮g/100 g b.w. per day of PTH(1-34) in intact rats increased total bone mass to the same extent as a dose of 8 ␮g/100 g b.w. per day given intermittently.(5) The authors also showed that both models of administration were capable of stimulating bone formation. More recently, Watson et al.(29) reported that continuous infusion of 5 ␮g/100 g b.w. per day PTH(1-84) in rats produced dramatic osteoblast development. However, in that study, the authors found decreased expression of insulin-like growth factor I (IGF-I) and increased expression of IGF binding proteins in osteoblasts. They speculated that this might lead to a negative bone balance. Combining estrogen with intermittent PTH treatment has long been promoted as a stratagem to prevent or mitigate the


potentially deleterious effect of PTH on cortical bone and perhaps to enhance its effects on cancellous bone.(22,24,30) In this study, we found estrogen and PTH had a synergistic effect on the parameters of bone formation. In addition, estrogen effectively suppressed cancellous osteoclast surface. Consequently, there was a dramatic increase in cancellous bone volume and cortical width in combination treatment compared with estrogen treatment alone. Recently, Gowen and colleagues(31) have reported that continuously elevated, endogenous PTH has an anabolic effect on bone in OVX rats. This effect was achieved only when estrogen was administered concomitantly. Although our findings of a marked anabolic effect of combined estrogen and continuous PTH treatment are encouraging, they must be tempered by the fact that serum calcium was elevated(15) and significant amounts of peritrabecular fibrosis and woven bone formation was seen. Nevertheless, our results indicate that further investigation of this mode of PTH administration is warranted. In the combination treatment, the augmentation of cancellous bone volume primarily was caused by an increase of trabecular thickness, with no significant change in trabecular number. A more prolonged treatment may have resulted in an increase in trabecular number. It has been shown that the initial response to PTH treatment is trabecular thickening and that this then is followed by tunneling resorption within trabeculae, which increases trabecular number.(32) The anabolic action of intermittent PTH treatment on bone is thought to be mediated by enhanced osteoblastic expression of IGF-I.(33) As noted previously, continuous infusion at a dose similar to that used here dramatically enhanced osteoblast development but the osteoblasts apparently did not express IGF-I.(29) Because estrogen stimulates IGF-I expression in rat and human osteoblasts,(34) it is possible that in the presence of estrogen, the proposed inhibitory effect of continuous PTH infusion on IGF-I expression(29) is overridden and that the osteoblasts have normal functional capacity. In an analogous fashion, Jagger et al.(35) showed that estrogen enhanced the bone formation response to mechanical stimulation, a response that is thought to be mediated by enhanced IGF-I expression in osteocytes.(36) After continuous PTH infusion or in PHPT, a negative effect on cortical bone, manifested by a diminution of cortical width and/or an increase in cortical porosity, generally has been observed in animal and human studies.(4,8,10,11) Cortical bone plays an important role in mechanical load bearing even in the vertebra, a site where both cortical and cancellous bone share this function.(37) Therefore, to protect cortical bone from a catabolic effect and, preferably, to increase cortical thickness and mass is a highly desirable feature in any treatment regimen for osteoporosis. Our finding that coadministration of estrogen and PTH infusion increased the cortical width in the lumbar vertebral body in the current report and BMD of the femoral midshaft in our previous study(15) is encouraging. Furthermore, an increase in cortical thickness in the ilium recently has been reported in postmenopausal woman treated with combined PTH and estrogen and in men treated with PTH.(38) Our study did not include an assessment of peri-

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osteal versus endosteal bone formation, but recent studies in animals and humans suggest a significant proportion of the cortical bone gain under the influence of PTH may occur on the endosteal surface.(38 – 41) However, it should be noted in the present study that estrogen failed to protect the cortices from PTH infusion– mediated remodeling, as evidenced by the lack of reduction of cortical porosity and labeled canals in the combined treatment compared with PTH infusion alone. This implies that osteoclast recruitment in intracortical bone may not be as sensitive to estrogen as that in cancellous bone. The reason for this is unclear. However, it is important to note that despite the increase in cortical porosity, the mechanical properties of cortical bone in the midshaft of the femur were improved by the combined treatment.(15) It seems reasonable to assume that this effect was caused by an increase in cortical thickness and/or diameter and that this overcompensated for any deleterious effects associated with enhanced porosity. Further work is needed on the anabolic response of “pure” cortical, for example, diaphysis of long bones compared with that of cortical bone that surrounds cancellous bone, for example, spine, proximal femur, and ilium. Although there have been many investigations of the anabolic action of PTH on the skeleton, we still know remarkably little about the conditions, that is, the magnitude and duration of the elevation in circulating PTH, that result in an anabolic response. As previously reported,(15) the circulating concentrations on the PTH-infused animals studied here were approximately double those in controls (PTHtreated, 100 –120 pg/ml; control, 40 – 60 pg/ml). Therefore, we may conclude that a continuous 2-fold elevation of circulating PTH has a mild anabolic action on cancellous bone of estrogen-deficient animals and, in the presence of estrogen treatment, exerts strong anabolic action on both cancellous and cortical bone. Dobnig and Turner(7) studied the effect of programmed administration of PTH(1-34) over various intervals to intact rats. Infusion of the hormone for 1 h/day at a rate that resulted in a 7-fold elevation of the mean circulating intact PTH concentration had an anabolic effect. However, this anabolic effect was lost if the infusion was extended to 2 h. Continuous infusion for 6 days at a rate that increased the circulating intact PTH concentration by a factor of 60 did not result in a decrease in cancellous bone area, but there was an increase in osteoclast perimeter and marrow fibrosis. With the standard regimen used to treat osteoporosis in humans [400 U PTH(1-34)/day by subcutaneous injection], circulating concentrations of PTH(1-34) reached an average of 10 times normal within 30 minutes after the injection and then declined rapidly. However, at 4 h the PTH(1-34) concentration was still twice normal. Clearly, we need more information on the pharmacokinetics underlying the anabolic action of PTH. In conclusion, the data presented here suggest that continuous infusion of PTH to rats may provide a reasonable model of PHPT in humans. In addition, they lend further support for the combined use of estrogen and PTH in the treatment of postmenopausal osteoporosis. Finally, in view of the anabolic action of continuously elevated PTH, further

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investigation of this mode of PTH administration is warranted. 15.

ACKNOWLEDGMENTS The authors are grateful to R. Birchman, D.D. Wu, and M. Schnitzer for their technical assistance. This study was supported by grants from the National Institutes of Health (AR 39191) and the Bristol Myers Squibb Co.

16.

17.

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Address reprint requests to: David W Dempster, Ph.D. Regional Bone Center Helen Hayes Hospital Route 9 West West Haverstraw, NY 10993-1195, USA Received in original form August 14, 2000; in revised form December 7, 2000; accepted January 31, 2001.