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Experimental Studies |
Department of Endocrinology (N.B., H.d.B., E.A.v.d.V., P.L.), and Department of Nuclear Medicine (J.C.R.), Academic Hospital Vrije Universiteit, 1007 MB Amsterdam, The Netherlands; and Department of Oral Cell Biology (P.H.), Academic Center of Dentistry, 1081 BT Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: P. Lips, Department of Endocrinology, Free University Hospital, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are few data concerning the histomorphometry of GHD patients. In a previous study, we compared histomorphometric variables of untreated adults, with childhood-onset GHD, with those of a control population (8). It seemed that bone formation variables were relatively low, whereas bone resorption variables were moderately high. The trabecular bone volume in the iliac crest was not lower than in controls but even rather high in some patients. However, in these patients, relatively low values for lumbar spine and femoral neck BMD were found (9).
In this paper, we report the effect of GH treatment in the same group of patients, on the histomorphometric variables of bone mass and bone turnover. Our hypothesis was that GH would increase both bone formation and resorption. We tested whether the stimulated bone turnover would result in a higher bone volume caused by an anabolic effect of GH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study population consisted of 36 GHD men (age range, 19–35 yr). Thirteen patients had isolated GHD and 23 patients had multiple pituitary hormone deficiencies. The latter patients were on adequate and stable replacement therapy for pituitary deficiencies other than GH. All patients had childhood-onset GHD and had received GH treatment during childhood under the supervision of the Dutch Growth Foundation for a period of 8 ± 3.8 yr. The ethiology of the GHD has been described previously (9). Criteria for GHD were: serum insulin-like growth factor I (IGF-I) less than 15 nmol/L, and maximal GH response to GHRH or insulin-induced hypoglycemia less than 7 µg/L). In hypogonadal patients, androgen replacement therapy had been withheld until the age of 18. The mean off-treatment period for GH was 7.4 ± 4.2 yr (range, 1–21 yr) before the baseline measurements, including the first bone biopsy. At baseline, the patients were randomly divided into 4 groups: groups 1, 2, and 3 received 1, 2, or 3 IU/m2/day (2.9, 5.8, 8.7 mg/m2/day) of recombinant human GH (rhGH), respectively, for 1 yr; and group 4 received placebo for 6 months, followed by rhGH 2 IU/m2/day (5.8 mg/m2/day) for the subsequent 6 months. The rhGH was kindly provided by Novo Nordisk, Gentofte, Denmark (Norditropin). The different treatment dosages were monitored by measuring serum markers (10) and bioimpedance analysis (11). Most patients required a dose reduction because of side effects; therefore, patients were regrouped for statistical analysis. The patients underwent a bone biopsy twice (at baseline and after 1 yr of treatment). The biopsy was taken at the standard location, 2 cm behind the superior anterior iliac spine and 2 cm under the edge of the iliac crest. The first biopsy was obtained at the right side and the second at the left side. Before each biopsy, patients received tetracycline double labeling with a dose of 250 mg, four times a day, on days 1, 2, and 13 and 14 (2–10-2). Between 2 and 7 days after the last tetracycline administration, the patients underwent a transiliac bone biopsy, under local anesthesia, at the standard location (12).
Biochemistry
The serum and/or urine calcium, creatinin, and alkaline phosphatase were measured with routine laboratory methods. Serum osteocalcin concentrations were measured by RIA using a kit of Incstar Corp. (Stillwater interassay CV 13%). With this assay, the intact molecule and the 1–43 fragment were measured. Urinary hydroxyproline was measured as described elsewhere (13). Serum IGF-I was measured using an immunoradiometric assay from Medgenics, Fleurus, Belgium.
Assessment of bone mineral density (BMD)
Bone mineral content (BMC) of the lumbar spine and the femoral neck (nondominant hip) was measured by dual x-ray absorptiometry (DXA, Norland XR-26), as described previously (9). The long-term precision of the method was 2.4% for the lumbar spine and 2.3% for the femoral neck. BMD is calculated by the software program and presented as the aerial density, expressed in g/cm2, or as the difference in SD from the normal mean of age- and sex-matched healthy subjects (Z score).
Biopsies
Transiliac bone biopsies were fixed overnight in 4% phosphate buffered formaldehyde and transferred to 70% alcohol. After dehydration, the bone specimens were embedded, without prior decalcification, in methylmethacrylate supplemented with 20% plastoid-N and 0.13 g/mL perkadox (14). Sections of 5 µm were prepared using a Jung K microtome (Reichert-Jung, Heidelberg, Germany). Sections were stained with Goldner’s trichrome for measurement of eroded surfaces. For measurement of osteoid, sections were stained with solochrome-cyanine R. Tartrate-resistant acid phosphatase staining was performed to visualize osteoclasts (15). Floating standard methylmethacrylate sections were incubated for 1 h in a solution consisting of naphthol As-BI phosphate (0.5 mg/mL), N,N-dimethyl formamide (5%), veronal acetate (25%), NaNO2 (0.016%), pararosaniline (4%, with a pH of 5.0, supplemented with K, Na-tartrate (5.642 mg/mL). The sections were counterstained with light green. Measurement of the tetracycline labels was performed on unstained sections of 5 µm.
Variables
Histomorphometry was performed mainly on trabecular bone. Histomorphometric variables were measured semiautomatically with a microscope equipped with a drawing tube (Leitz, Wetzlar, FRG), cursor, and digitizing tablet, connected to a computer (Zeiss, Oberkochen, FRG). For most measurements, the Osteoplan software was used (Zeiss Kontron, Image Analysis Division, Oberkochen, FRG), except for the cortical thickness and the dynamic variables, which were measured with Videoplan software (Zeiss Kontron, Image Analysis Division). The number of osteoclasts was measured manually using an integrating eyepiece (Zeiss II, Zeiss). The nomenclature is used and calculated according to the American Society for Bone and Mineral Research Nomenclature Committee (16). The following variables were measured: core thickness, cortical thickness, bone volume, bone surface, osteoid surface, osteoid thickness, eroded surface, osteoclast number, wall thickness, mineral apposition rate, and mineralizing surface. The following variables were calculated: trabecular thickness, trabecular number, trabecular separation, osteoid volume, bone formation rate, adjusted apposition rate, mineralization lag time, formation period, remodeling period and activation frequency.
Statistical evaluation
Because of the dose reductions, the patients from groups 1, 2,
and 3 were rearranged into three different dosage groups: A, B, and C,
according to the average dosage of the last 9 months of treatment.
Patients who received an actual dose between 0 and 2.9
mg/m2/day became group A (N = 5), patients with their
actual dose between 2.9 and 5.8 mg/m2/day became group B
(N = 9), and patients with their actual dose between 5.8 and 8.7
mg/m2/day became group C (N = 5). The placebo-treated
patients (group D, N = 6), treated with placebo for 6 months
followed by GH treatment of 5.8 mg/m2/day for 6 months,
were kept as a separate group. The variables in Tables 1–3
are
expressed as the average of all patient groups, including the patients
from group 4 (6 months treatment). Data before and after GH treatment
were compared using a paired Student’s t test. The effect
of the different dosages and the prevalence of other pituitary
deficiencies were tested by ANOVA. The influence of actual dosage and
the GH-induced changes in serum IGF-I were tested with regression
analysis. Correlations were calculated between DXA variables and
histomorphometric variables and were tested using multiple regression
analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Serum IGF-I increased more than 3-fold during GH
treatment. The levels were in the physiological or supraphysiological
range (15% of the patients). Biochemical markers for both bone
formation and bone resorption increased significantly (P <
0.001). The BMD for lumbar spine, femoral neck, and femoral trochanter
did not change significantly in these patients. The increase of femoral
neck BMC was borderline significant. All calculated Z scores were
negative and did not change significantly after GH treatment. The
lumbar spine BMD and femoral neck BMD also were calculated separately
for patients treated for 6 months and patients treated for 12 months.
In both groups, the BMD did not change significantly after GH
treatment. The relationship between the 12-month change of lumbar spine
BMD and femoral neck BMD and the change in trabecular bone volume and
cortical thickness was tested. Neither of these correlations was
significant.
Histomorphometric results on bone structure are summarized in Table 2. Trabecular bone volume and the bone structure
variables (bone surface, trabecular thickness, trabecular number and
trabecular separation) were similar before and after GH treatment.
Cortical thickness increased significantly after GH therapy
(P = 0.005). Both wall thickness and core width did not
change after GH treatment.
Histomorphometric data on bone remodeling are summarized in Table 3. The bone formation variables increased significantly
(OS/BS: P = 0.0002; OV/BV: P = 0.0001),
whereas osteoid thickness did not change. Eroded surface did not
change, but the number of osteoclasts per mm2 increased
significantly (P = 0.0001). All dynamic variables
increased significantly, except for the adjusted apposition rate and
mineralization lag time. The formation period increased, and the
remodeling period decreased, but these changes were not significant.
The relative fraction of the remodeling time used for formation
increased from 0.57 ± 0.16 to 0.71 ± 0.12
(P = 0.0007). The time for resorption and reversal
phase decreased from 65 ± 45 to 40 ± 30 days
(P = 0.02) (Fig. 1). The activation
frequency increased significantly (P < 0.0001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although GH markedly increased remodeling activity, there was no
significant decrease of the remodeling period. However, the ratio of
formation period to resorption period increased significantly in
response to GH treatment. The total time available for both resorption
and reversal phase decreased, indicating that the resorption is moving
faster or the reversal phase is shorter (see Fig. 1). We previously
suggested that the resorption period was prolonged in the untreated GHD
adults (8). The present data suggest that treatment leads to a decrease
of the reversal period, indicating that the coupling between resorption
and formation is more efficient. It was suggested by clinical studies
that IGF-I acts as a coupling agent (17)
The cortical thickness increased significantly, but trabecular bone volume did not change. Results obtained in rats treated with GH confirm these data (18, 19). It is not directly apparent why an increased formation period and a relative decrease of the resorption period does not result in a higher trabecular bone volume. The increased turnover after GH treatment leads to an increment of remodeling space, which results in an initial bone loss. This may explain the unchanged trabecular bone volume and BMD. In newly GH-induced bone multicellular units, the formation period exceeds the resorption period, which may result in an increase of wall thickness and, ultimately, trabecular bone volume. Longer periods of GH treatment in GHD adults have shown increased BMD after 2 and 5 yr (20, 21). In normal bone, approximately 2% is remodeling space (22). The doubled activation frequency indicates that more remodeling units in bone tissue are activated at the same moment, inducing an increase of remodeling space of approximately 2%. However, wall thickness did not change, but an interval of 1 yr may have been too short to demonstrate an increase in wall thickness. Only completed walls were measured, and the probability of finding a new trabecular osteon (new wall thickness) after 1 yr is less than 40%. This means that most walls measured after treatment were old walls.
BMD, as assessed by DXA in the lumbar spine and hip, did not increase significantly after 1 yr treatment with GH. This is in accordance with a previous study (7). The BMC of the femoral neck tended to increase. This site mainly represents cortical bone. It parallels the increase of cortical thickness. GH excess in acromegalic patients results in an increased cortical thickness and, to a lesser extent, in an increased trabecular bone volume (23). In our study, the core width of the iliac crest biopsies tended to increase, suggesting an expanding width of the iliac crest. These results point in the same direction as the data obtained in GH excess.
The highest dosage (8.7 mg/m2/day) resulted in severe side effects, based on fluid retention. Dosage adjustments had to be made, resulting in final dosages that were different in each patient. The dose response relationship was tested in two ways. The actual dosage of the last nine months was calculated in each patient. The first statistical test used the ANOVA. Therefore, the patients were divided according to their actual dosage during the last 9 months, resulting in four groups. Group A received between 0 and 2.9 mg/m2/day in the last 9 months; group B received between 2.9 and 5.8 mg/m2/day in that period; group C between 5.8 and 8.7 mg/m2/day in the same period; and group D received the first 6 months placebo and, thereafter, 5.8 mg/m2/day. A dose-response effect could not be demonstrated with this test, which was probably because of the fact that the groups were too small, especially the group with the highest dosage. The second test to study a dose-response relationship was the regression analysis using the actual GH dosage and the GH-stimulated increase of serum IGF-I as continuous variables. No significant correlations were found between these variables and the histomorphometric variables, although the serum IGF-I showed a wide range from physiological to supraphysiological levels. Group D showed more significant differences in bone remodeling variables than the other three groups. The results of this group may be more pronounced, because this group demonstrated early effects of GH treatment.
In conclusion, GH stimulated bone turnover. It almost doubled activation frequency and decreased the remodeling period, especially the resorption period. After 1 yr, no effect on trabecular bone volume was found. However, the cortical thickness increased significantly.
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Acknowledgments |
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Received July 25, 1996.
Revised January 8, 1997.
Revised February 19, 1997.
Accepted February 23, 1997.
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