Presented at the 19th Annual Meeting of the American Society of Bone and Mineral Research. J Bone Miner Res 1997;12(Suppl 1):S232 (abstract).
The Influence of Combined Parathyroid Hormone and Growth Hormone Treatment on Cortical Bone in Aged Ovariectomized Rats†
Article first published online: 1 NOV 2000
Copyright © 2000 ASBMR
Journal of Bone and Mineral Research
Volume 15, Issue 11, pages 2266–2275, November 2000
How to Cite
Andreassen, T. T. and Oxlund, H. (2000), The Influence of Combined Parathyroid Hormone and Growth Hormone Treatment on Cortical Bone in Aged Ovariectomized Rats. J Bone Miner Res, 15: 2266–2275. doi: 10.1359/jbmr.2000.15.11.2266
- Issue published online: 2 DEC 2009
- Article first published online: 1 NOV 2000
- Manuscript Accepted: 29 JUN 2000
- Manuscript Revised: 8 JUN 2000
- Manuscript Received: 18 JAN 2000
- parathyroid hormone;
- growth hormone;
- cortical bone;
- bone formation;
- mechanical strength;
The influence of combined parathyroid hormone (PTH) and growth hormone (GH) treatment on bone formation and mechanical strength was investigated in femoral middiaphysial cortical bone from 20-month-old ovariectomized (OVX) rats. The animals were OVX at 10 months of age, and at 18 months they were treated daily for 56 days with PTH(1-34) alone (60 μg/kg), recombinant human GH (rhGH) alone (2.7 mg/kg), or a combination of PTH(1-34) plus rhGH. Vehicle was given to OVX control rats. All animals were labeled at day 28 (calcein) and at day 49 (tetracycline) of the treatment period. PTH(1-34) alone gave rise to formation of a new zone of bone at the endocortical surface. rhGH alone caused substantial bone deposition at the periosteal surface without influencing the endocortical surface. Combined PTH(1-34) plus rhGH administration enhanced bone deposition at the periosteal surface to the same extent as that of rhGH alone. However, the combined treatment resulted in a more pronounced formation of new bone at the endocortical surface than was induced by PTH(1-34) alone. Both PTH(1-34) alone and rhGH alone increased the mechanical strength of the femoral diaphysis, and further increase in mechanical strength resulted from combined PTH(1-34) plus rhGH treatment. OVX by itself induced the characteristic increase in medullary cavity cross-sectional area and a minor decrease in the mechanical quality of the osseous tissue.
THE ADMINISTRATION of both parathyroid hormone (PTH) and growth hormone (GH) in rats increases diaphysial bone mass and enhances the mechanical strength of the bone.(1–15) Labeling with fluorochromes during the PTH or GH treatment has shown that the PTH administration induces substantial bone formation at the endocortical surface of the diaphysis and limited bone formation at the periosteal surface.(2,8,16) GH injections, on the other hand, induce pronounced periosteal bone formation without influencing the endocortical surface.(10,11,13,14)
In the current experiment, we treated aged ovariectomized (OVX) rats with PTH and GH, either alone or in combination and, using histomorphometric methods, investigated the changes in femoral diaphysial bone mass and the localizations of the new bone formed during treatment. We also investigated changes in the mechanical strength of the femoral diaphysial bone in relation to the different treatments.
In previous studies undertaken on OVX rats in our laboratory we found no differences in the mechanical strength or quality of femoral diaphysial bone using compression tests.(17,18) Using a torsion test, Aerssens et al. found OVX did not affect torsion stiffness, whereas OVX enhanced torsion strength.(19) However, using bending tests, other laboratories have reported minor decreases in the mechanical strength and the mechanical quality of diaphysial bone from OVX rats.(5,7,20) We have therefore evaluated the influence of OVX on the mechanical properties of diaphysial cortical bone by using bending test procedures.
MATERIALS AND METHODS
Ninety-six 10-month-old female Sprague-Dawley rats (Iffa-Credo, L'Arbresle Cédex, France) were OVX or sham-operated (Sham). Daily injection with PTH, GH, or PTH plus GH was commenced when the OVX rats were 18 months old. Synthetic human PTH fragment (1-34) [PTH(1-34); Bachem, Inc., Torrance, CA, U.S.A.] was dissolved in vehicle (0.15 M saline with 2% heat-inactivated rat serum, pH 5) and recombinant human GH (rhGH; Norditropin, Novo Nordisk, Gentofte, Denmark; specificity, 1 mg = 3 IU) was dissolved in distilled water. Duration of hormone administration was 56 days (20 months old when killed). PTH(1-34) was injected subcutaneously once daily in a dose of 60 μg/kg body weight, and rhGH was injected subcutaneously in a dose of 2.7 mg/kg body weight (divided into two daily doses). Vehicles [vehicle-PTH(1-34) plus vehicle-rhGH] were given to control animals. The rats were weighed once a week, and the dose of hormones was adjusted in accordance with the actual body weight. The experiment included the following eight groups (age at killing [k] and number of animals [n] are given in parentheses): (1) intact animals (k = 10 months; n = 10); (2) Sham (k = 18 months; n = 12); (3) OVX (k = 18 months; n = 10); (4) Sham vehicle-PTH(1-34) plus vehicle-rhGH (k = 20 months; n = 11); (5) OVX vehicle-PTH(1-34) plus vehicle-rhGH (k = 20 months; n = 12); (6) OVX PTH(1-34) plus vehicle-rhGH (k = 20 months; n = 11); (7) OVX rhGH plus vehicle-PTH(1-34) (k = 20 months; n = 11); and (8) OVX PTH(1-34) plus rhGH (k = 20 months; n = 12). The influence of the different hormone administrations on OVX animals was determined by comparing groups 5, 6, 7, and 8. The influence of OVX on diaphysial bone was evaluated by comparing groups 2, 3, 4, and 5. The rats were caged with 12 h light (6:00 a.m. to 6:00 p.m.) and 12 h darkness and had free access to tap water and pellet food (Altromin diet 1324 containing 0.9% calcium and 0.7% phosphorous; Chr. Pedersen Ltd., Ringsted, Denmark). All animals in the experiment were labeled with calcein (Sigma, St. Louis, MO, U.S.A.; 15 mg/kg intraperitoneally [ip]) 4 weeks before killing and tetracycline (Sigma; 20 mg/kg ip) 1 week before killing. The rats were anesthetized with pentobarbital (SA, Copenhagen, Denmark; 50 mg/kg ip) and killed by exsanguination. The hind legs were exarticulated in the hip joints and stored at −80°C until examination.
The right femur was dissected free and the mechanical competence of the femoral diaphysis was measured using a standardized three-point bending procedure (distance between bars, 15 mm; loading point, 55% of total femoral length from the top of the caput; deflection speed, 2 mm/minute). Load-deflection curves were recorded continuously, and ultimate load (maximum load) and ultimate stiffness (equal to the maximum slope of the load-deflection curve) were calculated. The mechanical quality of the bone tissue was analyzed by normalizing the mechanical data to the external and internal dimensions (ultimate stress from ultimate load, elastic modulus [Young's modulus] from ultimate stiffness.(10)
The left femur was dissected free, and by means of a precision bone saw (Exakt-Apparatebau; Otto Herrmann, Norderstedt, Germany), a transverse section approximately 200 μm in thickness was cut out of the diaphysis at the location corresponding to the load insertion point of the right femoral diaphysis. The sections were embedded in alkylacrylate (Entellan; Merck, Darmstadt, Germany) on glass slides. The slides were placed in an epifluorescence microscope (Leitz DMRBE; Leica, Wetzlar, Germany) at a magnification of ×200. A translucent star-shaped grid, with the center point of the star placed in the midpoint of the marrow cavity, was used to measure periosteal and endocortical mineralizing surfaces to total surfaces (MS/TS) and mineral apposition rate (MAR).(10) However, there was a very pronounced response at the endocortical surface in the group given the combined PTH(1-34) plus rhGH treatment. Therefore, the MAR had to be measured from the endocortical boundary line induced at the start of treatment to the calcein labeling line induced at day 28 of treatment. The same procedure was performed on the group given PTH(1-34) alone. The sections were then imaged in full on a screen by a microscope (Model SJ; Carl Zeiss, Oberkochen, Germany) at magnification ×20, and the periosteal and endocortical boundaries were outlined. Because femoral diaphysial bone of old female rats can be divided into an outer zone and an inner zone,(17) the boundary between these two zones was outlined. PTH(1-34) administration induced a new zone of bone from the endocortical surface, and the boundary of this zone also was outlined. The figures were read by the graphic tablet into a computer, and the following cross-sectional areas were calculated: total bone, outer zone, inner zone, endocortical PTH(1-34)-induced zone, and medulla. The lengths of the following circumferences were measured: periosteal, inner zone, cavernae, and endocortical. Mineralized bone formation rate (MBFR) at the periosteal and the endocortical surfaces was calculated as [MAR] × [length of respective circumference]/1 mm sectional height. Finally, the external and internal diameters, corresponding to the load direction and perpendicular to the load direction, were measured and used for calculation of the mechanical quality parameters of the bone.
During the 8-month period after OVX/sham operation (from month 10 to month 18), 6 rats died (1 Sham and 5 OVX). During the following 8-week treatment period, 1 rat from the PTH(1-34) group died. The experiment was approved by the Danish Animal Experiment Inspectorate.
The data were tested for normal distribution and homogeneity of variances, and when these conditions were fulfilled, parametric analyses were applied or else nonparametric analyses were used.
The effects of the different hormone treatments were evaluated using the following groups: OVX-vehicle, OVX-PTH(1-34), OVX-rhGH, and OVX-PTH(1-34) plus rhGH. Differences between these groups were tested by one-way analysis of variance or Kruskal-Wallis test. In cases in which differences occurred, all pairwise multiple comparison procedures were applied (Student-Newman-Keul's method or Dunn's method).
The changes due to age and OVX were evaluated using the following groups: baseline control, Sham 18 months old, OVX 18 months old, Sham 20 months old, and OVX 20 months old. Differences between these groups were tested by one-way analysis of variance or Kruskal-Wallis test. In cases in which differences occurred, all pairwise multiple comparison procedures were applied (Student-Newman-Keul's method or Dunn's method). The influence of OVX (18 months old and 20 months old) versus Sham (18 months old and 20 months old) was further examined by stepwise multiple regression analysis. The value of p < 0.05 (two-tailed) was considered statistically significant.
Changes in femoral middiaphysial cross-sectional bone areas caused by hormone administration are given in Table 1 and depicted in Fig. 1. Compared with the OVX vehicle-injected group, the PTH(1-34) treatment induced a new zone of bone at the endocortical surface (Fig. 1, row B), whereas no differences in either the outer zone area or the inner zone area were found between the two groups. Treatment with rhGH enhanced the entire bone area by 22%, and this increase was ascribed to an expansion of the outer zone area, whereas no difference in the inner zone area was observed between the rhGH-treated group and the OVX vehicle-injected animals (Fig. 1, row C). Combined treatment with PTH(1-34) plus rhGH enhanced the entire bone area by 53% in proportion to the OVX vehicle-injected group, and this increase was caused both by an expansion of the outer zone area and by deposition of new bone at the endocortical surface (Fig. 1, row D). The two groups given PTH(1-34) showed deposition of new bone at the endocortical surface, hereby forming a new endocortical zone (Fig. 1). However, this deposition was less in the group treated with PTH(1-34) alone compared with the combined PTH(1-34) plus rhGH group, although rhGH treatment by itself had no influence on endocortical bone formation.
Changes in the femoral middiaphysial cross-sectional bone areas caused by age and OVX are given in Table 2. Compared with the 10-month-old baseline control rats, the Sham 18-month-old and 20-month-old animals had increased the entire bone area (14% and 21%, respectively) by increasing the outer zone area, whereas no differences in inner zone area and marrow cavity area were found between these three groups. The OVX rats increased their entire bone area to the same extent as that of the Sham animals. The OVX caused an expansion of the marrow cavity, both in the 18-month-old and 20-month-old OVX rats.
At the periosteal surface, both the OVX groups given either rhGH alone or rhGH in combination with PTH(1-34) showed a pronounced increase in MS/TS, MAR, and MBFR when compared with the OVX vehicle-injected animals. Treatment with PTH(1-34) alone resulted in an enhancement of MS/TS whereas the increase in MAR and MBFR were found to be modest. No differences in MAR and MBFR were seen between the rhGH group and the rhGH plus PTH(1-34) group.
At the endocortical surface, a pronounced increase in MS/TS, MAR, and MBFR was seen in both PTH(1-34)-treated groups. However, the combined PTH(1-34) plus rhGH treatment increased MAR and MBFR more than did the PTH(1-34) treatment alone (53% and 55%, respectively), despite the fact that rhGH treatment alone had no influence at all on MS/TV, MAR, and MBFR (Fig. 2).
In the 10-month-old baseline controls, bone formation took place at the periosteal surface (Table 4). In the 18-month-old Sham and OVX rats, bone formation still took place at the periosteal surface, although this was reduced to only one-third of the bone formation found in the 10-month-old baseline controls. In the 20-month-old animals, very little bone formation was found.
For animals injected with hormones, the results of the mechanical testing are given in Table 5. Ultimate load was increased by 12% in the group given PTH(1-34) alone, by 23% in the group given rhGH alone, and by 48% in the combined PTH(1-34) plus rhGH group compared with the OVX vehicle-injected rats. Furthermore, ultimate load was increased in the combined PTH(1-34) plus rhGH group compared with the groups given PTH(1-34) alone and rhGH alone (31% and 20%, respectively). No difference in ultimate load was found between the group given PTH(1-34) alone and the group given rhGH alone. Ultimate stiffness was increased by 23% in the group given PTH(1-34) alone, by 29% in the group given rhGH alone, and by 43% in the combined PTH(1-34) plus rhGH group compared with the OVX vehicle-injected rats. Furthermore, ultimate stiffness was increased in the combined PTH(1-34) plus rhGH group compared with the groups given PTH(1-34) alone and rhGH alone (16% and 11%, respectively). No difference in ultimate stiffness was found between the group given PTH(1-34) alone and the group given rhGH alone.
When the mechanical quality of the osseous tissue was calculated by correcting for dimensions of the bone at the load insertion point, ultimate stress and Young's modulus were increased by 17% and 31%, respectively, in the group given PTH(1-34) alone compared with the OVX vehicle-injected group, and by 20% and 38%, respectively, compared with the group given rhGH alone. Correction for dimensions was not performed in the combined PTH(1-34) plus rhGH group because the cross-sectional shape of the marrow cavity could not be assumed to be elliptical (Fig. 2D).
Changes in mechanical strength caused by age and OVX are given in Table 6. Compared with the 10-month-old baseline controls, ultimate load and ultimate stiffness were increased both in Sham and in OVX animals, whereas ultimate load and ultimate stiffness did not differ between the Sham and the OVX groups. When the mechanical quality was calculated by correcting for bone dimensions, no differences between any of the groups were seen. However, when stepwise multiple regression analysis was performed between the OVX and the Sham groups, a slight decrease in ultimate stress was observed in the OVX animals (p = 0.04). However, the E modulus of the OVX animals was not decreased (p = 0.06).
This experiment clearly shows that combined treatment with PTH(1-34) plus rhGH results in substantial bone deposition both at the periosteal and at the endocortical surfaces. The periosteal bone deposition found in the PTH(1-34) plus rhGH group coincides with the bone deposition measured in the rhGH group. However, the endocortical bone deposition is more pronounced in the PTH(1-34) plus rhGH group than in the PTH(1-34) alone group, although rhGH treatment by itself does not influence endocortical bone deposition. At present, we do not have any explanation as to the reason for this increased endocortical response found in the combined treatment group. However, it is worth considering whether the relationship between PTH(1-34) administration and increased insulin-like growth factor I (IGF-I) production, caused by rhGH administration, could be of interest. Using osteoblast-like cells, Spencer et al. have shown that the mitogen effect of IGF-I is potentiated when IGF-I is combined with PTH(1-34).(21) They also administered IGF-I and PTH(1–34) alone and in combination to rats in vivo and found that the combination of IGF-I and PTH(1-34) potentiated bone formation at cancellous bone surfaces. In vitro studies have shown that GH is able to stimulate local production of IGF-I in cultured bone tissue.(22–24) The extent to which the local IGF-I production has been stimulated in the present experiment is not known. Serum levels of IFG-I are GH dependent, and the rhGH dose used in this experiment has been shown to double the serum IGF-I value in rats.(10)
Treatment with PTH and/or GH has previously been investigated.(25,26) Both studies reported the characteristic anabolic effect of PTH on bone. However, no effect of GH administration was found, either when GH was given alone or in combination with PTH. The authors used ovine GH (specificity not stated) in a dose of 1 mg/kg per day. One reason for the lack of response to the GH treatment could be the use of a GH dose that was too low. In dose-response studies, bone formation and mechanical strength have been found to remain unaffected at GH doses lower than 2 mg/kg per day,(9,27) whereas several investigations have reported the anabolic effect of GH treatment at GH doses in the range of 2–10 mg/kg per day.(9,11,14,27,28)
In this experiment, the enhanced bone deposition caused by the treatments results in enhanced mechanical strength of the bone. rhGH alone increases periosteal bone deposition, whereas PTH(1-34) alone increases endocortical bone deposition, both leading to enhanced mechanical strength of the bone. As the combined treatment with rhGH and PTH(1-34) increases both periosteal and endocortical bone deposition, the mechanical strength of the bone is increased further. This shows that the combined administration of the two hormones affects the bone strength in an additive way. This additive effect on bone strength by combined treatment with PTH and GH recently has been reported.(29)
Resistance of tubular bones to bending and torsion is influenced by the bone geometry, the most effectual shape of the bone being that in which the osseous tissue is distributed far from the neutral axis (the center of the medullary cavity).(30,31) Bending and torsional loadings induce the highest stresses in the human appendicular skeleton, and because osseous tissue in males is located farther from the neutral axis than that of females, male resistance to these loading modes becomes stronger.(32–34) What makes the combined treatment with PTH(1-34) plus rhGH interesting is the fact that both the anabolic bone effects of PTH(1-34) and the periosteal bone deposition of rhGH are induced by this treatment. The periosteal bone deposition is distributed far from the neutral axis, and our results show that resistance to bending is exceptionally enhanced in the combined treatment group.
Human studies have shown that GH deficiency (GHD) and multiple pituitary insufficiency often result in decreased bone mineral density (BMD) and bone mineral content (BMC).(35–39) A corresponding increase in fracture frequency also has been observed.(40) When patients suffering from these disorders are treated with rhGH, a number of trials have shown decreased lumbar spine and femoral neck BMD and BMC during the first 6–12 months of rhGH therapy,(41–45) although the results are not fully congruent.(46–48) However, trials continuing the rhGH treatment for 2 years or more all revealed increased lumbar spine and femoral neck BMD and BMC levels when compared with initial values.(49–55) Dynamic histomorphometry and serum and urinary biochemical markers have shown enhancement of bone formation and resorption during rhGH therapy.(42,44,46–57) The reported initial decline in BMD and BMC therefore could be explained by an expansion of the remodeling space. These studies also have disclosed that men respond with a greater increase in serum IGF-I levels and markers for bone metabolism than women to equal doses per surface area.(54,58,59) Only a few studies have examined new bone deposition at the cortical surfaces during rhGH treatment in GHD patients. Using histomorphometry, Bravenboer et al. found increased cortical thickness of transiliac bone biopsy specimens after 1 year of rhGH treatment,(56) and by analyzing dual-energy X-ray absorptiometry pictures, Johansson et al. showed increased bone area of both the femoral neck and the lumbar spine after 33 months and 45 months of rhGH treatment.(54)
Studies of postmenopausal osteoporotic women and men with idiopathic osteoporosis have shown that biochemical markers for bone resorption and formation increase within the first week of rhGH administration.(60–62) Bone biopsy specimens from severely osteoporotic men treated with hGH for 8–15 months showed an increase in new periosteal bone formation and intracortical bone resorption.(63) Aloia et al. treated postmenopausal osteoporotic women for up to 2 years with either hGH alone or in combination with salmon calcitonin (sCT).(64–67) Little effect was seen on the total content of body calcium (measured by neutron activation analysis), but a decreased BMC of the radial shaft was found in two of the four studies.(64,66) Combined rhGH and bisphosphonate (pamidronate) therapy for 6 months did not influence BMC of the lumbar spine, femoral neck, proximal radius, or distal radius in postmenopausal osteoporotic women, whereas pamidronate alone increased lumbar spine and distal radius BMC when compared with initial values.(68) Serum osteocalcin and urinary free deoxypyridinoline levels decreased substantially in the group given pamidronate alone, whereas osteocalcin levels increased and free deoxypyridinoline levels remained unchanged in the group receiving the combined treatment. Gonnelli et al. treated postmenopausal osteoporotic women with rhGH for 2 years (daily injection for 1 week every 3 months) and found a decrease in lumbar spine and femoral shaft BMD.(69) When the 1-week rhGH administration was followed by a 3-week sCT treatment period, no decrease in lumbar spine BMD was observed, whereas femoral shaft BMD was decreased. At the end of the treatment periods, an increase in serum osteocalcin and urinary pyridinoline was observed, both in the patients given rhGH alone and in the patients given rhGH followed by sCT. In a recent study, postmenopausal osteoporotic women were treated daily with rhGH.(70) After the first year, lumbar spine BMD was unchanged, whereas femoral neck BMD was decreased. However, after the second year, lumbar spine BMD was increased and femoral neck BMD had returned to initial values.
On the basis of the existing studies, it is evident that rhGH treatment for 2 years or more enhances lumbar spine and femoral neck BMD and BMC in patients with GHD. As far as postmenopausal osteoporotic women are concerned, the rhGH treatment period did not exceed 2 years, and little anabolic effect on BMD and BMC was observed. Longer treatment periods and combined treatment with antiresorptive agents could be of interest. Estimations of bone deposition at periosteal surfaces could similarly be of relevance, because these surfaces are located farther from the neutral axis.
In conclusion, the present study shows that combined treatment with PTH(1-34) and rhGH enhances bone deposition at the periosteal surface as seen when rhGH is given alone. However, at the endocortical surface, the combined treatment with PTH(1-34) and rhGH results in an increased bone deposition, which is more pronounced than that of the PTH(1-34) treatment alone, although rhGH treatment by itself has no influence on endocortical bone formation. Treatment with either PTH(1-34) or rhGH increases the mechanical strength of the bone, and an additive increase in the mechanical strength of the bone is seen when combined treatment is given.
This work was supported by The Danish Health Research Council, grants 9500922 (Center for Molecular Gerontology) and 9600822 (Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration); the Aarhus University Research Foundation; and the Novo Nordisk Foundation. We are grateful to M. Fischer, P.K. Nielsen, and J. Utoft for excellent technical assistance.
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