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Bone Histomorphometry in Children and Adolescents with ß-Thalassemia Disease: Iron-Associated Focal Osteomalacia

Departments of Pediatrics (P.M., A.C.), Pathology (V.S., P.K.), Radiology (R.S.), and Medicine (S.D., R.R.), Research Center (L.C., A.S.), Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand

Address all correspondence and requests for reprints to: Dr. Pat Mahachoklertwattana, Department of Pediatrics, Ramathibodi Hospital, Faculty of Medicine, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. E-mail: rapmw{at}mahidol.ac.th.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Thalassemia/hemoglobinopathy is a hereditary disease that causes chronic anemia and increased erythropoiesis. Consequently, an expansion of bone marrow spaces may contribute to osteopenia/osteoporosis. However, the pathogenesis of bone changes is not yet known. We, therefore, carried out the study on bone histomorphometry and biochemical and hormonal profiles in children and adolescents with suboptimally treated ß-thalassemia disease with the hope of gaining some new insight into the cellular and structural alterations of thalassemic bone. Seventeen patients underwent iliac crest bone biopsy for histomorphometric analyses. Bone mineral density (BMD) measurements were performed by dual energy x-ray absorptiometry. Most patients had growth retardation and delayed bone age. BMD was low especially at the lumbar spine. Serum IGF-I levels were almost always low. Bone histomorphometry revealed increased osteoid thickness, osteoid maturation time, and mineralization lag time, which indicate impaired bone matrix maturation and defective mineralization. In addition, iron deposits appeared along mineralization fronts and osteoid surfaces. Moreover, focal thickened osteoid seams were found together with focal iron deposits. Dynamic bone formation study revealed reduced bone formation rate. These findings indicate that delayed bone maturation and focal osteomalacia are the pathogenesis of bone disease in suboptimally blood-transfused thalassemics with iron overload. Iron deposits in bone and low circulating IGF-I levels may partly contribute to the above findings.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THALASSEMIA/hemoglobinopathy is a hereditary disease caused by defective globin synthesis resulting in abnormal as well as decreased quantity of globin chains. Ineffective erythropoiesis, hemolysis, and increased red blood cell turnover ensue (1). Increased erythropoiesis causes the expansion of bone marrow cavities, which may contribute to the reduction of bone mass and the increased incidence of fractures (2, 3, 4, 5, 6, 7).

The pathogenesis of bone changes in thalassemia/hemoglobinopathy is not yet known. One explanation has been offered that increased erythropoiesis demands more bone marrow space through reduction of trabecular bone tissue (8). Previous studies have shown that multiple factors may act in concert to produce bone disease in thalassemia. These include hypogonadism (9), delayed puberty (10), defective GH-IGF-I axis (4, 11, 12, 13), iron deposit in bone (14), vitamin D deficiency (15, 16, 17), and desferrioxamine toxicity in bone (18). However, the interactions among iron, hemopoietic cells, osteoblasts, and osteoclasts in bone tissue have never been explained.

Recent publications reported increased bone resorption markers in the urine of thalassemic patients (12, 19). Although, this finding indirectly indicates elevated bone resorption, a direct evidence of accelerated bone resorption, namely increased osteoclastic activity in bone, has not been demonstrated.

Osteoclasts are derived from hemopoietic granulocyte-macrophage lineages. The cytokines that are involved in hemopoiesis are also involved in the development of osteoclasts (20). Therefore, it is possible that the mechanism that stimulates hemopoiesis in the thalassemic bone may also stimulate osteoclastic formation and/or activity, which, in turn, increases bone resorption and reduces bone mass.

Bone histomorphometry is a useful tool to study the pathogenesis of bone changes. Histomorphometric studies of bone have been performed in only a limited number of children with thalassemia; the results are equivocal (2, 8, 21, 22). Moreover, to our knowledge, a dynamic bone histomorphometric study in children with thalassemia disease has never been reported in the English literature.

We, therefore, carried out the present study on bone histology and biochemical and hormonal profiles in children with thalassemia, a disease prevalent in Thailand, with the hope of gaining some new insight into the cellular and structural alterations of thalassemic bone. The present communication reports the findings of this study.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Twelve patients with ß-thalassemia/hemoglobin E and five with ß-thalassemia major were studied. Diagnosis was confirmed by hemoglobin electrophoresis typing. The z-scores of growth parameters were calculated using National Standard Growth Curve of the Ministry of Public Health, Thailand, 1999.

Chemistries and hormones

Serum chemistries and hormones were determined with routine and special laboratory methods. They were calcium (Ca), phosphate (P), ferritin, bone-specific alkaline phosphatase (BAP), osteocalcin (OC), 25-hydroxyvitamin D (25-OHD), intact PTH, IGF-I, free T4 (FT₄) and TSH. Morning urine samples were analyzed for Ca, creatinine (Cr), and deoxypyridinoline (Dpd).

Serum IGF-I, intact PTH, and ferritin levels were measured by immunoradiometric assay using reagents purchased from Cis Biointernational ORIS Group (France); intraassay coefficients of variation (CVs) were 4.0%, 4.3%, and 4.2%, and interassay CVs were 1.3%, 3.8%, and 14.9%, respectively. Serum 25-OHD and FT₄ concentrations were measured by RIA using reagents purchased from Dia Sorin (Stillwater, MN) and Diagnostic Products (Los Angeles, CA), respectively; intraassay CVs were 11.0% and 4.7%, and interassay CVs were 6.0% and 10.1%, respectively. Serum TSH concentrations were determined by immunoradiometric assay using reagents purchased from Diagnostic Products; intra- and interassay CVs were 5.3% and 11.0%, respectively. Serum BAP, OC, and urinary Dpd concentrations were measured by enzyme immunoassay using reagents purchased from Metra Biosystems (Mountain View, CA); intraassay CVs were 3.2%, 6.6%, and 3.2%, and mean interassay CVs were 9.1%, 11.8%, and 4.6%, respectively. The values of bone markers were compared with our normative data for Thai children of the same chronological ages.

Bone mineral density (BMD) and bone age (BA)

BA was assessed by x-ray of the left hand and was compared with x-ray photographs in the atlas of Greulich and Pyle (23). All x-rays for BA were read twice by the same observer (P.M.). BMD and total body fat were determined by dual energy x-ray absorptiometry (DPX-L, version 4.6D, Lunar Corp., Madison, WI). The volumetric BMD (vBMD) of the lumbar spine was estimated by a method previously described (24). The BMDs of the lumbar spine (L₂–L₄), femoral neck, distal radius, and total body and the vBMD of the lumbar spine were expressed as z-scores based on our normative BMD data for Thai children of the same chronological ages (n = 600) (25).

Bone histomorphometry

Each patient underwent in vivo double tetracycline labeling, with an interlabel time of 14 d. The schedule for labeling was as follows: oxytetracycline (500 mg) was given orally twice a day for 2 d, followed by a 12-d free interval, then oxytetracycline (500 mg twice a day) was again given for 2 d. Transiliac crest bone biopsy was performed a day after completion of tetracycline labeling with a bone biopsy needle 5 mm in internal diameter. The specimen was placed immediately in 95% ethanol, embedded in hard plastic blocks, and cut into 5- and 15-µm-thick sections with a heavy-duty microtome.

Bone structures were measured in 5-µm-thick sections stained with Goldner-Trichrome. The extent of iron deposit was measured in sections stained by the method of Gomori. The rate of bone formation was determined as the distance between the two tetracycline labels in unstained 15-µm-thick sections visualized under a fluorescence light microscope.

All static parameters of bone formation and resorption in each patient were determined in one section with a measurable area ranging from 20–36 mm² (mean, 24 mm²). Two patients had two sections measured with measurable areas of 42 and 48 mm². Only cancellous bone was measured. Mineralized bone was defined as a green structure containing osteocytes (Goldner stain). Osteoid was defined as red-staining seams at least 1.5 µm wide at the bone-bone marrow interface. Thickened osteoid was defined as osteoid seams more than 10 µm wide (>2 SD of normal). Eroded surface was identified as scalloped or ragged appearance at the bone-bone marrow interface with or without the presence of osteoclasts. Osteoblasts were identified as cells directly apposed to osteoid. Osteoclasts were multinucleated cells in the neighborhood of an eroded surface. Iron was seen as blue-staining lines at the interface between the marrow and the mineralized or osteoid perimeters.

All parameters were analyzed using a semiautomatic image analyzer (Osteomeasure software, Osteometrics, Atlanta, GA). About 25–30 fields were analyzed in 1 bone specimen. All nomenclature, abbreviations, and standard formulas follow the recommendations of the American Society for Bone and Mineral Research (26, 27).

Written informed consent for the present study was obtained from the legal guardians and, when appropriate, from the children before entering the study. The study protocol was approved by the ethics committee of the Faculty of Medicine, Ramathibodi Hospital, Mahidol University.

Statistics

Height and weight for chronological age were expressed as median z-scores and ranges. Biochemical and hormonal values were presented as the mean ± SD and ranges. Bone age, BMD for chronological age, and BMD adjusted for BA were expressed as mean z-scores ± SD and ranges. Spearman’s nonparametric correlation was used to determine the correlation.

Histomorphometric data were presented as the mean ± SD and range. Unpaired t test was used to determine differences in parameters between our patients and the normal children reported by Glorieux et al. (26). Iron deposits with and without focal thickened osteoid were compared by t test for proportions. P < 0.05 denotes statistical significance.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The mean age (±SD) of the patients was 13.4 (±2.4) yr. There were 11 males and 6 females. Twelve patients had ß-thalassemia/hemoglobin E disease, and the remainder had ß-thalassemia major. Of the 17 patients, seven had spontaneous puberty, and 10 of 17 patients were in the prepubertal stage. Seven of the latter had delayed puberty (no signs of puberty after 14 yr in males or after 13 yr in females).

Eight patients required regular packed red cell transfusion every 1–2 months to maintain hemoglobin levels above 6.5–7.0 g/dl. These transfusion-dependent patients received desferrioxamine therapy 1–20 times a month. Their median pretransfusion hemoglobin level was 7.0 g/dl (range, 6.0–7.9 g/dl). The other nine patients needed blood transfusions much less frequently, i.e. every 3–12 months; they were considered transfusion independent. Their hemoglobin levels ranged from 7.5–8.5 g/dl. Due to financial reasons, the former were treated with suboptimal transfusion and iron chelation. Therefore, the average hemoglobin values were almost equal in both groups.

Growth and body fat

Almost all patients had stunted growth. The median z-scores of their height and weight were, respectively, -3.18 (range, -7.02 to -0.31) and -2.85 (range, -4.87 to 0.61). The severity of growth failure increased with advancing age. Body fat was estimated by dual energy x-ray absorptiometry. All patients had percentage of total body fat within ±1 SD for sex- and age-matched normative data, which indirectly indicated that none was malnourished.

Biochemistry and hormones

Biochemical and hormonal profiles are shown in Table 1. As expected, serum ferritin levels were markedly elevated, ranging from 1,485–16,215 µg/liter in the transfusion-dependent patients compared with only 137–1365 µg/liter in the transfusion-independent patients. The serum Ca, P, BAP, OC, 25-OHD, intact PTH, FT₄, and TSH levels as well as urinary Ca and Dpd were all within normal limits in all patients. There was no correlation between serum 25-OHD and intact PTH levels. Serum IGF-I levels were markedly low; of the 17 patients, 15 (88%) had serum IGF-I values below the fifth percentile of normal Thai children and adolescents (28).

Bone maturation, as determined by x-ray for BA, was markedly delayed, as shown in Table 2. The mean z-scores of BMD of lumbar spines, femoral neck, radius, and total body were -3.82, -2.34, -1.29, and -2.47, respectively. The z-scores of BMD were also adjusted for BA, because delayed BA is one of the factors causing reduced apparent areal BMD. Even adjusted for BA, the mean z-scores of BMD were still low especially of the lumbar spine, femoral neck, and total body (-2.56, -1.67, and -1.53, respectively; Table 2). Similarly, the vBMD of the lumbar spine was also low. There were no differences in BMD between transfusion-dependent and transfusion-independent patients or between boys and girls.

Bone histomorphometric data are shown in Table 3. Structural parameters, namely, cancellous bone volume relative to total bone volume, mineralized bone volume, and trabecular thickness, were slightly less than corresponding values in normal children, albeit insignificantly. Trabecular number, trabecular separation, and bone surface (BS) did not differ from normal values. Bone marrow fibrosis was not seen in the bone sections of all patients.

View larger version (70K): [in this window] [in a new window]   FIG. 1. The photomicrograph (von Kossa stain; magnification, x500) of undecalcified bone section shows mineralized bone (black) demarcated from the thick unmineralized osteoid matrix (pink) by a mineralization front (arrows). The unmineralized bone is also rimmed with active osteoblasts (arrowheads).

Regarding bone resorption indexes, both eroded surface relative to bone surface (ES/BS) and number of osteoclasts were significantly decreased (P < 0.001).

Bone sections stained for iron showed extensive iron deposits on mineralization fronts, osteoid surfaces, and bone marrow spaces in the transfusion-dependent patients (Fig. 2) in contrast to the minimal iron deposits seen in the transfusion-independent patients. The extent of iron deposit was estimated by measuring bone surfaces stained positively for iron relative to BS (FeS/BS). It varied from 7–90% of the total BS.

View larger version (157K): [in this window] [in a new window]   FIG. 2. The photomicrograph (iron stain; magnification, x500) of undecalcified bone section illustrates the thick linear deposition of iron (blue line) along the mineralization front (thick arrows). The unmineralized zone is also stained blue, but with lighter intensity (areas between thin arrows). Active osteoblasts (arrowheads) are also present along the osteoid surface.

The bone histomorphometric parameters of transfusion-dependent and transfusion-independent patients were not different (data not shown).

Correlation

There was no correlation among BMD, hemoglobin, 25-OHD, and IGF-I levels. O.Th had a negative correlation with height and BMD of femoral neck. In contrast, O.Th had a positive correlation with FeS/BS (Table 4). There was a correlation between FeS/BS and ferritin levels (r = 0.49; P < 0.05). However, there was no correlation among ferritin levels, O.Th, and BFR/BS. The osteoid maturation time had a negative correlation with BMD of the femoral neck, lumbar spine and radius, and height (Table 4). ES/BS, a bone resorptive index, was correlated with MAR (r = 0.53; P < 0.05) and BFR/BS (r = 0.58; P < 0.05).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

It is well known that the more severe the disease, the more bone changes/bone loss are apparent. The severity of the disease varies with the severity of anemia. In the present study the more severe (transfusion-dependent) patients received much more frequent blood transfusions, but only to raise the hemoglobin level above a critical value of approximately 7 g/dl; this was necessitated by economic reasons and explained the insignificant difference in hemoglobin values between the more severe transfusion-dependent and the less severe transfusion-independent patients.

The BMD depends upon bone mineral content and bone size (29). In addition, delayed growth and puberty may decrease peak bone mass acquisition, as seen in adults with a history of delayed puberty (30). Moreover, axial BMD increases more rapidly than appendicular BMD during puberty (31). Therefore, stunted growth, delayed puberty and BA, and small bone size simply and collectively contribute to the markedly low BMD seen in the thalassemic patients. The marked reduction in BMD was still apparent when the BMD was adjusted for BA. The significance of low BMD was substantiated by low vBMD values of the lumbar spine. The lumbar spine, which is exclusively trabecular bone and consists of wide bone marrow spaces, was the most affected part. Thus, the structure of bone itself may directly affect BMD. The least affected part was the radius, which consists of more cortical and less trabecular bone.

Bone histomorphometric indexes revealed increased O.Th, predominantly at the iron-deposited areas, and lengthened osteoid maturation time, which indicated decreased osteoblast recruitment and/or delayed mineralization. In 1990, Rioja and co-workers (8) published bone histomorphometry for 17 thalassemic children, the largest number ever reported in the English literature. Of the 17 patients, 7 had increased O.Th. When the O.Th values of these children were reevaluated, taking into consideration the normative data for age reported by Glorieux et al. (26), 12 of 17 patients had increased O.Th. These findings together with ours confirm thickened osteoid seams in thalassemic bones. Moreover, the present study also demonstrated decreased mineralizing surface and lengthened mineralization lag time, which indicated defective mineralization. Thus, all of the above-mentioned findings are compatible with osteomalacia. In addition, the BFR was significantly lower than normal. The normal MAR and the decreased BFR indicate normal osteoblast function, but decreased osteoblast recruitment or differentiation. This might also be suggested by the decreased osteoid surface and increased O.Th. Decreased eroded surfaces and number of osteoclasts suggest a reduction of bone resorption. These findings are in contrast with previous studies (12, 19) that suggested increased bone resorption by demonstrating a rise in bone resorption markers in the urine. However, increased bone resorption markers were more pronounced in thalassemic adults with hypogonadism (19); this was not apparent in thalassemic children in the present study. In addition, increased bone resorption and bone formation markers in those studies were demonstrated in optimally blood-transfused patients (12, 19). Moreover, treatment with hypertransfusion can increase bone turnover in thalassemic patients (2). In contrast to the previous studies, the patients in the present study were treated with suboptimal blood transfusion and iron chelation, which could explain our findings of reduced both bone formation and resorption as well as substantial iron deposition in bone tissue. The bone resorptive index (ES/BS) was correlated with dynamic bone formation indexes (MAR and BFR/BS), which indicated a parallel reduction of both bone resorption and formation.

Regarding iron toxicity, a study of the effect of iron overload on bone remodeling in animals showed decreased osteoblast recruitment and collagen synthesis, resulting in decreased BFR (32). In addition, a previous study in rats revealed that iron deposition along the mineralization fronts was associated with increased osteoid seams (33). In the present study focal thickened osteoid seams were found together with focal iron deposits along osteoid surfaces, which is identical to the latter study. In addition, FeS/BS was correlated with O.Th. Therefore, iron deposition in bone may impair osteoid maturation and inhibit mineralization locally, resulting in focal osteomalacia, which can be seen in some cases of iron overload (34), aluminum toxicity (35), or etidronate use (36). The mechanism by which iron interferes osteoid maturation and mineralization may be explained by the incorporation of iron into crystals of calcium hydroxyapatite (37), which consequently affects the growth of calcium hydroxyapatite crystals and increases osteoid in bone tissue.

It is speculated that increased erythropoiesis in bone marrow, i.e. increased generation of cells in the erythropoietic lineage, may adversely affect the proliferation and maturation of cells in the osteogenic lineage. In addition, low serum IGF-I levels may decrease osteoblastic cell proliferation and bone matrix formation. Moreover, low IGF-I levels may decrease the activation of osteoclasts (38). Therefore, low IGF-I levels may contribute to the decreases in both bone formation and resorption in the patients studied. The previous study in GH-deficient patients with low circulating IGF-I levels demonstrated that there was a positive correlation between the vBMD of the lumbar spine and the IGF-I concentration (39). However, serum IGF-I levels were not correlated with BMD in the present study. This may be due to the wide normal range of serum IGF-I levels during childhood and adolescence.

In conclusion, osteopenia in suboptimally blood-transfused thalassemics with iron overload is primarily caused by focal osteomalacia as well as decreased bone formation without evidence of increased bone resorption. Iron deposits in bone and low circulating IGF-I levels may partly contribute to the above findings. However, the interactions among iron, osteogenic cells, and hemopoietic cells in the bone and bone marrow on the pathogenesis of bone loss in thalassemia disease are not yet understood. Further studies are required to elucidate the cellular mechanism of the pathogenesis of thalassemic bone diseases.

	Acknowledgments

 
We thank Dr. Umaporn Suthutvoravut for providing normative data of BMD of Thai children and Dr. Phienvit Tantibhedhyangkul for the suggestion and comment.

	Footnotes

 
This work was supported by Thailand Research Fund.

Abbreviations: BA, Bone age; BAP, bone-specific alkaline phosphatase; BFR, bone formation rate relative to bone surface; BMD, bone mineral density; BS, bone surface; Ca, calcium; Cr, creatinine; CV, coefficient of variation; Dpd, deoxypyridinoline; ES/BS, eroded surface relative to bone surface; FeS, iron-stained bone surface; FT₄, free T₄; MAR, mineral apposition rate; OC, osteocalcin; 25-OHD, 25-hydroxyvitamin D; O.Th, osteoid thickness; P, phosphate; vBMD, volumetric bone mineral density.

Received October 3, 2002.

Accepted May 18, 2003.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Mohamed N, Jackson N 1998 Severe thalassemia intermedia: clinical problems in the absence of hypertransfusion. Blood Rev 12:163–170[CrossRef][Medline]
2. Pootrakul P, Hungsprenges S, Fucharoen S, Bayink D, Thompson E, English E, Lee M, Burnell J, Finch C 1981 Relation between erythropoiesis and bone metabolism in thalassemia. N Engl J Med 304:1470–1473[Medline]
3. Schettini F, Mele M, Mattia DD, Falcone G, Addabbo AD 1987 Bone metabolism in thalassemia major. J Pediatr 111:158–159[Medline]
4. Soliman AT, Banna NE, Fattah MA, EIZalabani MM, Ansari BM 1998 Bone mineral density in prepubertal children with ß-thalassemia: correlation with growth and hormonal data. Metabolism 47:541–548[CrossRef][Medline]
5. Jensen CE, Tuck SM, Agnew JE, Koneru S, Morris RW, Yardumian A, Prescott E, Hoffbrand AV, Wonke B 1998 High prevalence of low bone mass in thalassemia major. Br J Hematol 103:911–915[CrossRef][Medline]
6. Wonke B 1998 Bone disease in ß-thalassemia major. Br J Haematol 103:897–901[CrossRef][Medline]
7. Ruggiero L, DeSanctis V 1998 Multicentre study on prevalence of fractures in transfusion dependent thalassemic patients. J Pediatr Endocrinol Metab 11:773–778
8. Rioja L, Girot R, Garabedian M, Cournot-Witmer G 1990 Bone disease in children with homozygous ß-thalassemia. Bone Miner 8:69–86[CrossRef][Medline]
9. Anapliotou MLG, Kastanias IT, Psara P, Evangelou EA, Liparaki N, Dimitriou P 1995 The contribution of hypogonadism to the development of osteoporosis in thalassemia major: new therapeutic approaches. Clin Endocrinol (Oxf) 42:279–287[Medline]
10. Saka N, Sukur M, Bundak R, Anak S, Neyzi O, Gedikoglu G 1995 Growth and puberty in thalassemia major. J Pediatr Endocrinol Metab 8:181–186[Medline]
11. Low LCK, Postel-Vinay MC, Kwan EYM, Cheung PT 1998 Serum growth hormone (GH) binding protein, IGF-I and IGFBP-3 in patients with ß-thalassemia major and the effect of GH treatment. Clin Endocrinol (Oxf) 48:641–646[CrossRef][Medline]
12. Pollak RD, Rachmilewitz E, Blumenfeld A, Idelson M, Goldfarb AW 2000 Bone mineral metabolism in adults with ß-thalassemia major and intermedia. Br J Haematol 111:902–907[CrossRef][Medline]
13. Soliman AT, Banna NE, Ansari BM 1998 GH response to provocation and circulating IGF-I and IGF-binding protein-3 concentrations, the IGF-I generation test and clinical response to GH therapy in children with ß-thalassaemia. Eur J Endocrinol 138:394–400[Abstract]
14. Bordat C, Constans A, Bouet O, Blanc I, Trubert CL, Girot R, Cournot G 1993 Iron distribution in thalassemic bone by energy-loss spectroscopy and electron spectroscopic imaging. Calcif Tissue Int 53:29–37[CrossRef][Medline]
15. Dandoma P, Menon RK, Houlder S, Thomas M, Flynn DM 1987 Serum 1,25 dihydroxyvitamin D and osteocalcin concentrations in thalassemia major. Arch Dis Child 62:474–477[Abstract]
16. Aloia JF, Ostuni JA, Yeh JK, Zaino EC 1982 Combined vitamin D parathyroid defect in thalassemia major. Arch Intern Med 142:831–832[Abstract]
17. Moulas A, Challa A, Choliasos N, Lapatsanis PD 1997 Vitmain D metabolites (25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D) and osteocalcin in ß-thalassemia. Acta Paediatr 86:594–599[Medline]
18. DeVirgilis S, Congia M, Frau F, Argiolu F, Diana G, Cucca F, Varsi A, Sanna G, Fodde M, Pirastu GF, Cao A 1988 Deferoxamine-induced growth retardation in patients with thalassemia major. J Pediatr 113:66–69
19. Voskaridou E, Kyrtsonis MC, Terpos E, Skordili M, Theodoropoulos I, Bergele A, Diamanti E, Kalovidouris A, Loutradi A, Loukopoulos D 2001 Bone resorption is increased in young adults with thalassemia major. Br J Haematol 112:36–41[CrossRef][Medline]
20. Manolagas SC, Jilka RL 1995 Bone marrow, cytokines, and bone remodeling. N Engl J Med 332:305–311[Free Full Text]
21. DeVernejoul MC, Girot R, Gueris J, Cancela L, Bang S, Bielakoff J, Mautalen C, Goldberg D, Miravet L 1982 Calcium phosphate metabolism and bone disease in patients with homozygous thalassemia. J Clin Endocrinol Metab 54:276–281[Abstract]
22. DeSanctis V, Stea S, Savarino L, Scialpi V, Traina GC, Chiarelli GM, Sprocati M, Govoni R, Pezzoli D, Gamberini R, Rigolin F 1998 Growth hormone secretion and bone histomorphometric study in thalassemic patients with acquired skeletal dysplasia secondary to desferrioxamine. J Pediatr Endocrinol Metab 11:827–833
23. Gruelich WW, Pyle SI 1959 Radiographic atlas of skeletal development of the hand and wrist, 2nd Ed. Stanford: Stanford University Press
24. Lu PW, Cowell CT, Lloyd-Jones SA, Briody JN, Howman-Giles R 1996 Volumetric bone mineral density in normal subjects, aged 5–27 years. J Clin Endocrinol Metab 81:1586–1590[Abstract]
25. Suthutvoravut U, Charoenkiatkul S, Mahachoklertwattana P, Kosulwat V, Sirisriro R, Intaramarn C, Rojroongwasinkul N, Rajatanavin R 2001 Bone mass development and determinants in Thai children and adolescents aged 9–18 years [Abstract]. Ann Nutr Metab 45(Suppl 1):256
26. Glorieux FH, Travers R, Taylor A, Bowen JR, Rauch F, Norman M, Parfitt AM 2000 Normative data for iliac bone histomorphometry in growing children. Bone 26:103–109[Medline]
27. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2:595–610[Medline]
28. Wacharasindhu S, Chaichanwatanakul K, Likitmaskul S, Angsusinghu K, Punnakanta L, Tuchinda C 1998 Serum IGF-I and IGFBP-3 levels for normal Thai children and their usefulness in clinical practice. J Med Assoc Thai 81:623–633
29. Seeman E 1998 Growth in bone mass and size-are racial and gender differences in bone mineral density more apparent than real? J Clin Endocrinol Metab 83:1414–1419[Free Full Text]
30. Finkelstein JS, Neer RM, Biller BMK, Crawford JD, Klibanski A 1992 Osteopenia in men with a history of delayed puberty. N Engl J Med 326:600–604[Abstract]
31. Rubin K, Schirduan V, Gendreu P, Sarfarazi M, Mendola R, Dalsky G 1993 Predictors of axial and peripheral bone mineral density in healthy children and adolescents, with special attention to the role of puberty. J Pediatr 123:863–870[CrossRef][Medline]
32. De Vernejoul MC, Pointillart A, Golenzer CC, Morieux C, Bielakoff J, Modrowski D, Miravet L 1984 Effects of iron overload on bone remodeling in pigs. Am J Pathol 116:377–384[Abstract]
33. Hiratsuka H, Katsuta O, Toyota N, Tsuchitani M, Akiba T, Marumo F, Umemura T 1997 Iron deposition at mineralization fronts and osteoid formation following chronic cadmium exposure in ovariectomized rats. Toxicol Appl Pharmacol 143:348–356[CrossRef][Medline]
34. Phelps KR, Vigorita VJ, Bansal M, Einhorn TS 1988 Histochemical demonstration of iron but not aluminum in a case of dialysis-associated osteomalacia. Am J Med 84:775–780[CrossRef][Medline]
35. Dunstan CR, Hills E, Norman AW, Bishop JE, Mayer E, Wong SY, Johnson JR, George CR, Collett P, Kalowski S 1985 The pathogenesis of renal osteodystrophy: role of vitamin D, aluminium, parathyroid hormone, calcium and phosphorus. Q J Med 55:127–144[Medline]
36. Boyce BF, Smith L, Fogelman I, Johnston E, Ralston S, Boyle IT 1984 Focal osteomalacia due to low-dose diphosphonate therapy in Paget’s disease. Lancet 1:821–824[Medline]
37. Bauminger E, Ofer S, Gedalia I, Horowitz G, Mayer I 1985 Iron uptake by teeth and bone: a Mossbauer effect study. Calcif Tissue Int 37:386–389[Medline]
38. Conover CA 2000 Insulin-like growth factors and the skeleton. In: Canalis E, ed. Skeletal growth factors. Philadelphia: Lippincott Williams & Wilkins; p. 101–116.
39. De Boer H, Blok GJ, Van Lingen A, Teule GJJ, Lips P, Van der Veen EA 1994 Consequences of childhood-onset growth hormone deficiency for adult bone mass. J Bone Miner Res 9:1319–1326[Medline]

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