Histomorphometric and biochemical characterization of bone following acute severe burns in children

Bone Vol. 17, No. 5 November 1995:455~,60

ELSEVIER

Histomorphometric and Biochemical Characterization of Bone Following Acute Severe Burns in Children G. L. K L E I N , 1'4 D. N. H E R N D O N , 2'4 W. G. G O O D M A N , 5 C. B. L A N G M A N , 6 W. A. PHILLIPS, 3'4 I. R. D I C K S O N , 7 R. E A S T E L L , 8 K. E. N A Y L O R , s N. A. M A L O N E Y , 9 M. D E S A I , 2'4 D. B E N J A M I N , 4 and A. C. A L F R E Y 1° Departments of 1Pediatrics, 2Surgery, and 3Orthopedic Surgery, University of Texas Medical Branch and 4Shriners Burns Institute, Galveston, TX, USA 5 Department of Radiology, UCLA Center for the Health Sciences, Los Angeles, CA, USA 6 Department of Pediatrics, Northwestern University Medical School, Chicago, 1L, USA 7 Department of Biology and Biochemistry, Brunel University, Middlesex, UK s Department of Human Metabolism and Clinical Biochemistry, University of Sheffield, Sheffield, UK 9 Medical and Research Services, VA Medical Center, Seattle, WA, USA m Medical and Research Services, VA Medical Center and the University of Colorado School of Medicine, Denver, CO, USA

duced without a concomitant decrease in resorption; osteoid area is reduced and stainable aluminum is present at the bone surface. 16 While this uncoupling hypothetically increases the risk of bone loss, the long-term effects of bum injury on bone density are unknown in these patients. In contrast, we have shown that children with burns >40% TBSA have reduced bone density measurements within eight weeks of the burn injury and these data are indistinguishable from cross-sectional study from a population of similarly burned children at a mean of five years after bum injury, suggesting that the low bone density persists. 15 However, no histomorphometric measurements have been performed on bone biopsies in these burned children compared with findings in adults. Moreover, with the increasing use of serum and urinary markers of bone formation and resorption, it would be important to determine not only the nature of bum-related bone injury in children but also the accuracy of biochemical markers of bone turnover in reflecting these changes. The present study reports the histomorphometric characterization of the acute response of the bone to bum injury in children and the relationship of biochemical markers of bone formation and resorption to the bone histomorphometry. Moreover, individual data on bone density in these patients, while previously published as part of a larger study on bone density, 15have been provided here for purposes of comparison with bone histomorphometry.

Severe burns in adults is associated with an uncoupling of normal remodeling, low bone formation without reduced resorption. The risk of osteopenia that may occur under such circumstances is heightened by our detection in a crosssectional study of low bone mass in severely burned children. We report here the acute histomorphometric and biochemical response of bone to severe burn injury, as well as bone mass in severely burned children. We enrolled 24 patients ages 5.8 to 17.5 years following burns of 63 - 16% (SD) body surface area. Serum and urine were collected weekly until iliac crest bone biopsy was obtained 26 -+ 10 days postburn. Seventeen of 18 patients, including 5 patients receiving growth hormone treatment to accelerate wound healing, failed to take up doxycycline in trabecular bone, and had no detectable osteoblasts at the osteoid seam, while eroded surface was normal and osteoblasts were documented by staining. Thus, bone formation was virtually absent. There was an eightfold elevation in urinary free cortisol excretion and high serum levels of acute phase reactants and interleukin-115 and -6. Biochemical markers of bone formation, osteocalcin, and type I procollagen propeptide were low, as were resorptive markers urinary pyridinoline and deoxypyridinoline. However, there was no correlation with resorptive surface. Mean age-related z-score for bone mass was - 1 . 0 6 - 1.05, 40 days postburn. Immobilization and endogenous corticosteroid production may be the main factors responsible for acutely reduced bone formation while inflammatory cytokines may mediate resorption. (Bone 17:455--460; 1995)

Patients and Methods

Key Words: Bone formation; Bone resorption; Bum; Corticosteroids; Immobilization.

Patients

We studied 24 patients following bum injury />40% TBSA, mean 63 +-- 16%, range 40%-94%, 17 boys (71%) and 7 girls (29%) ages 5.8 to 17.5 years, mean 11.5 -+ 3.6 (SD). No patients had prior underlying bone, renal, or endocrine disorders as determined by interview and medical history. All but one were normally nourished as defined by weight for height criteria and all had their bone mass reported previously. 15 Nutrition support in the form of milk infused through a nasogastric tube was begun within 24 h of admission, supplying approximately 2.7 g/m2 of calcium and 1.3 g/m2 of phosphate per day. 14'15 All patients

Introduction Burn injury to >50% of total body surface area (TBSA) in adults results in uncoupled bone remodeling. Bone formation is reAddress for correspondence and reprints: Gordon L. Klein, M.D., Pediatric Gastroenterology Division, Children's Hospital, University of Texas Medical Branch, Galveston, TX 77555-0352.

© 1995 by ElsevierScienceInc.

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underwent burn wound excision and skin grafting as early as possible following admission and were confined to bed for 5-6 days following each operation to allow the skin grafts to take and the donor sites to heal.l'~ Serum and a 24-h urine collection were obtained beginning at admission and repeated weekly for the first two to three weeks postburn. Specimens were analyzed for the following: Biochemical markers of bone turnover; Formation: type I collagen propeptide (PICP); Resorption: 24-h urine collection for urinary excretion of type I collagen crosslinks, pyridinoline and deoxypyridinoline, and calcium and creatinine determinations; Biochemical markers of inflammation: serum alpha-l-antitrypsin; alpha-l-acid glycoprotein, albumin, a negative acute phase reactant, and serum alpha-2 HS glycoprotein, a plasma protein concentrated extravascularly in bone matrix9; and interleukin 1 beta (IL-I[3) and IL-6; Markers of stress: free cortisol in urine; Markers of toxicity: serum and urine aluminum. Additionally, intraoperative iliac crest bone biopsies were performed in l 8 of the patients between 15 and 44 days postburn to ascertain the bone histomorphometry and aluminum content. Coincidentally, five of the biopsied patients received recombinant human growth hormone (rHGH) subcutaneously (Genentech, South San Francisco, CA), 0.2 mg/kg/day throughout the course of their hospitalization, including the period of bone biopsy, to facilitate wound healing. 12

Bone Vol. 17, No. 5 November 1995:455460 diameter was obtained. Two to three weeks prior to biopsy, all patients received doxycycline intravenously at therapeutic doses ranging from 150-300 mg per day for two days followed by another identical course of intravenous doxycycline 8-14 days later. Specimens were placed in 10% formalin for 24 h and then transferred to 70% ethanol. Processing, embedding, and analysis of the specimens for the static and dynamic parameters of bone remodeling were carried out as previously published. 25 Normal values for children (N = 29) were obtained from iliac crest bone biopsies in children 2.5 to 18 years of age who had received oral tetracycline and then underwent elective orthopedic surgery. Approval by the Human Subject Protection Committee at UCLA and informed consent were obtained. While the histomorphometry in normal and burned children were analyzed by the same histomorphometrist (W.G.G.), the normal children were not specifically selected as controls for this study. A smaller number of normal children had their bone histomorphometry results previously reported. 26 Lumbar spine bone mineral density (BMD) was performed using the QDR 1000 W absorptiometer (Hologic, Waltham, MA). 15 This study was approved by the Institutional Review Board at the University of Texas Medical Branch at Galveston and by the Shriners Hospitals for Crippled Children in Tampa, FL. Results

Bone Histomorphometry MeNo~ PICP analysis was carried out by modification of an existing equilibrium radioimmunoassay (Incstar, Stillwater, MN); coefficients of variation for the assays reported here were 4% intraassay and 7% interassay, lo Urine pyridinoline (Pyd) and deoxypyridinoline (DPyd) crosslinks were determined by high performance liquid chromatographic (HPLC) separation using the method of Colwell et a1.,4'6 with intra-assay variation of 7% and interassay variation of 12%; creatinine was determined by HPLC separation using a kinetic Jaffe reaction. 4'6 Calcium was analyzed colorimetrically on an Ektachrome 700 analyzer (Eastman Kodak, Rochester, NY). Serum c~-l-antitrypsin, o~-l-acid glycoprotein, and c~-2-HS glycoprotein and albumin were determined by radial immunodiffusion or by one-dimensional electrophoresis. 8 IL-l[3 was determined by an immunoenzymometric assay "sandwich" technique (Amersham Biokine, Northbrook, IL) with intra- and interassay coefficients of variation of 6% and 11%, respectively, over the range studied. 17 IL-6 was measured by an ultrasensitive enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), 22 with similar intra- and interassay variations. Tumor necrosis factor-a (TNF a) was measured by double antibody enzyme immunoassay (R&D Systems, Minneapolis, MN) as previously described. 16 Urinary free cortisol was determined by HPLC. 5 Serum and urine aluminum determinations were carried out using flameless atomic absorption spectroscopy as previously published. 19 Biochemical markers of bone formation and resorption, along with the interleukins and TNF a, were carried out in the laboratory o f C . B. Langman. Urinary collagen crosslinks were determined in the laboratory of R. Eastell and K. Naylor; markers of inflammation were analyzed in the laboratory of I.R. Dickson, and aluminum levels were analyzed in the laboratory of A.C. Alfrey. lntraoperative iliac crest bone biopsy was obtained using a Korb biopsy needle (Zimmer, Warsaw, IN) following separate informed consent and a core of tissue approximately 6 mm in

Bone biopsies were carried out 26 + 10 days postburn with time interval from last urine or serum marker of bone turnover and biopsy being 7 -+ 5 days, range 3 to 19 days. All patients were noted to have a hypercellular, reactive marrow. Static histomorphometric variables (Table 1) such as bone and osteoid area, osteoid and eroded surfaces, and trabecular width and spacing were not different from normal; however, there was a trend toward lower osteoid areas in the burned patients. Staining of the bone surfaces for tartrate-resistant acid phosphatase (TRAP) revealed osteoclasts at the resorptive surfaces (Figure 1). However, only one of the 18 patients biopsied had trabecular bone that took up the doxycycline, leading to a subnormal rate of bone formation (Table 1). Five of the patients (28%) had at least a single doxycycline label in cortical bone. No cuboidal osteoblastic cells were identified adjacent to surface osteoid seams. None of the patients had histologic evidence of osteopenia as indicated by bone volume, trabecular width, and spacing, all of which fell within the normal range. Also, no patient had osteomalacia, as no excess osteoid was observed.

Biochemical Markers of Bone Remodeling Both the marker for bone formation, PICP, and the markers for bone resorption, urinary pyridinoline and deoxypyridinoline, were low (Table 2). There were no differences based on pubertal status and no correlations with eroded perimeter. Also of note is that urinary deoxypyridinoline and pyridinoline were low, while previously reported data for type I collagen telopeptide (ICTP), ~5 another marker of resorption, was high (Table 2). in the same patients. In contrast, formation markers PICP and osteocalcin, the latter previously reported,15 were both reduced in the same patients. Urinary calcium excretion, previously shown to be elevated in these patients, 15 1.22 -+ 0.81 mmol/kg/day (4.89 -+ 3.24 mg/kg/day), was unrelated to histomorphometric and biochemical indices of bone resorption.

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G.L. Klein et al. Bone in severe burns

457

Table 1. Histomorphometric features Patients (N) Bone area (% tissue area) Osteoid area (% bone area) Osteoid perimeter (% bone perimeter) Eroded perimeter (% bone perimeter) Trabecular width (mm) Trabecular spacing (mm) Bone formation rate (~m2/mm2/p.m) Mineral apposition rate (txm/day) Single label perimeter (% of total)

18 18 18 18 18 18 18 18 18

Mean (-+SD) 21.23 1.68 15.2 2.94 137 508

± 4.3 ~ ± 1.35 ± 10.9 ± 1.89 ± 27 - 154 0a 0d 0.3 ± 0.940

Range

Normals (N)

(15-28) b (0.06-5.20) (1.14-35.86) (0-7.42) (99-245) (346-770) (0-128) ~ (0-1.1) ~ (0-3.46)

29 29 29 29 29 29 29 29 29

Mean (±SD) 20.59 2.36 18.2 2.13 138 486 316 1.2 5.4

± ± ± ± ± ± ±

Range

5.7 1.38 8.5 0.89 32 104 270 0.2 0.4

(9-34) (0.23-5.83) (4.3-37.5) (0.5-4.3) (90-220) (301-737) (93-613) (0.8-1.5) (0.4-12.6)

~Mean - SD. bRange. ~Upper range is the only patient with detectable double-label doxycycline uptake. dp < 0.001 vs. normal values.

Biochemical Determination of lndices of Stress, Inflammation, and Aluminum Toxicity

IL-6 in serum were elevated, the latter markedly (Table 3), while TNF a was normal in 8 of 11 (73%) patients. Serum levels of a-l-antitrypsin and a - l - a c i d glycoprotein, both acute phase reactants, were high, while serum concentrations of albumin and a-2-HS glycoprotein, a negative acute phase reactant,18 were low (Table 3). Serum and urinary aluminum were only minimally elevated (Table 3) and no aluminum was detected in the bone biopsies by histochemical staining.

Urinary free cortisol was elevated an average of eightfold above the upper limits of normal for the first three weeks postbum without sign of falling. Mean urinary free cortisol varied inversely with osteoid area (r = - 0 . 5 7 , p < 0.05) but did not correlate with biochemical markers of bone formation. IL-113 and

/ J

Figure 1. Photomicrograph of iliac crest bone biopsy of a burned child shortly postburn TRAP stained (red) for osteoclasts.

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Bone Vol. 17, No. 5 November 1995:455-460

Table 2. Biochemical markers of bone remodelinga Formation (serum) PICP Osteocalcinu (ng/mL) (ng/mL)

N Patients Range

21

210 ± 10C ( 109-611 )

Normals

Resorption (urine) Pyridinoline Deoxypyridinoline (nmol/mmol creatinine) Prepuberty 12 99.8 ± 26.3 (46.6--129.2) Postpuberty 7 100.8 -+ 29.4 a (63.3-153.4) d Prepubeny 324 ± 52.5 J

3.1 ± 2.2 (0.5-7.6)

(200-700)

(10-25) < 12 years (2-8) > 13 years

28.5 -+ 7.5 (13.3-36.9)

N 21

(Serum) ICTP b (ng/mL) 82.2 ± 43.1 (23.9-186.5)

28.8 ± 8.4 (17.8-43.7) 92.6 ± 30.2

<30

Postpuberty 174 ± 36.5 d aMean of 1-4 values per patient. bFrom Ref. 2 reported for comparison. CMean ± SD. dExtrapolated from normal data for HPLC determination of pyridinoline/deoxypyridinoline

Bone Mass Bone density of the lumbar spine was reduced (Table 4) with only 2 of 16 patients having a positive age-related z score. Finally, no difference could be seen in bone histomorphometry or bone-specific biochemical markers between the five patients who received therapeutic rHGH and those who did not. Discussions

Our present study in children confirms the histomorphometric findings in adults of reduced bone formation without clear evidence for a comparable decrease in resorption.16 The reduction of bone formation is more severe in the children, however, as reflected by the lack of doxycycline uptake in trabecular bone in all but one patient and the lack of detectable osteoblasts at the osteoid seams. In addition, this study documents the large quantities of circulating inflammatory cytokines and endogenous corticosteroids in the children and the relationship of biochemical markers to both static and dynamic histomorphometric measurements. These data provide a more detailed assessment than in the adults of the response of bone to burn injury. The documentation of low bone mass in these same children supports the lack of bone formation as being clinically significant. Data obtained in

49.8 ± 19.9

= 3.5/1.

this study are insufficient to determine whether burn patients lose bone or fail to accrue bone mineral normally due to low bone formation. Inasmuch as there is no documented trabecular bone loss any actual loss that might occur may come from cortical bone. Alternatively, bone mineral accretion may behave in a manner similar to growth velocity, which is reduced after a s e vere burn. 23 In support of the latter possibility, preliminary data are obtained from follow-up bone biopsies in four patients 10 to 20 months following the burn injury and 9 to 19 months following the initial bone biopsy. There was still no tetracycline uptake by the bone in two of the patients, suggesting long-term persistence of reduced bone formation. The first consideration is the technical adequacy of doxycycline administration for bone fluorochrome labeling. Successful bone labeling with doxycycline was carried out in adults who underwent bone biopsy, the results of which have been reported previously, l In addition, one of us (N. A.M.) reviewed the doxycycline labeling in that report for fluorescence and length of label compared to tetracycline. Doxycycline was used as a first label in two of the patients and tetracycline as a second label. While the fluorescence was somewhat reduced compared to tetracycline, as has been reported elsewhere, 1~ it was readily detected at 105 × magnification and the length of label differed from tetracycline by only 3%.

Table 3. Biochemical parameters of stress, inflammation, and aluminum toxicity Seruma N Patients Normal range

14

IL-I[3 L-6 (pg/mL) 3.4 ± 1.9 <1

126 ± 58 <1

N 16

a- 1antitrypsin (g/L)

a- 1-acid glycoprotein (g/L)

a-2-HS glycoprotein (g/L)

Albumin (g/L)

3.69 ± 1.0l 1.9-3.5

2.00 ± 0.34 0.55-1.40

0.28 ± 0.08 0.40-0.85

22.8 ± 3.7 35.0-55.0

Urine"

N Patients Normal range

12

aMean of 1-3 determinations/patient.

Free cortisol* (mg/24 h) 395 ± 284 8J,7

N 7

Aluminum* (ixmol/24 h) 5.0 ± 2.1 < 1.0

N (~mol/L)

Aluminum 0.8 ± 0.2 <0.37

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G . L . Klein et al. Bone in severe burns

Table 4. Lumbar spine bone mineral density in relation to burn i~ury and bone biopsy

Patienta

BMD z-score for age

Time after burn (days)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

- 2.67 + 0.19 + 1.07 - 0.29 - 1.06 - 1.38 - 1.16 - 2.90 - 0.99 0.79 - 1.92 -0.62 -0.18 - 1.42 -0.49 - 2.38

21 30 38 59 48 43 57 45 49 52 18 45 27 59 28 22

Mean ±SD

- 1.06 ±1.05

40 ±14

Interval between BMD and biopsy (days) - 16b 15 13 43 25 24 28 8 14 15 8 13 6 33 8 10 15 ±13

~BMD obtained in only 16 of the 18 patients biopsied. BMD = bone mineral density. bBone biopsy occurred first in 15 of the 16 patients. The number preceded by a minus sign indicates that the BMD measurement occurred first. In this study doxycycline was given in therapeutic quantities. This should result in therapeutic blood levels o f doxycycline, which should be sufficient for bone uptake. Blood levels o f doxycycline were not measured in this study. However, there was cortical bone uptake o f at least a single label in five o f the patients, suggesting that the dose is capable of being taken up by bone. Furthermore, osteoblasts were not seen and osteoblast number correlates well with double-labeled tetracycline surfaces in previous studies from this laboratory (r = 0.9). 27 Moreover, supportive data for reduced bone formation was obtained from low serum levels o f both biochemical markers o f bone formation, PICP, and osteocalcin. T w o major factors contributing to low bone formation, which cannot at this time be separated, are immobilization and increased e n d o g e n o u s corticosteroid production, t3 All children are confined to bed following burn injury during the process of early wound excision and grafting. Arnaud et al. 2 have reported reduced bone formation in young adult volunteers after only one week o f bedrest. Immobilization may contribute to the bone pathology by reducing osteoblast recruitment and by reducing osteoblast precursor cell gene expression o f insulin-like growth factor (IGF) II, a putative factor coupling bone formation and resorption. 28 W e have confirmed the striking increase in endogenous corticosteroid production in our patients, although this is a previously w e l l - d o c u m e n t e d occurrence. 16.2o The inverse relationship b e t w e e n urinary free cortisol and osteoid area suggests a possible suppressive role in bone formation o f endogenous corticosteroids. With reference to aluminum as a potential cause o f the histological lesion, the bone changes in children, in contrast to adults, occur in the absence o f any significant aluminum loading. It is uncertain h o w much resorption there is in light o f severely reduced bone formation and the normal serum levels o f parathyroid h o r m o n e previously reported in these patients. 15 Moreover, markers o f bone formation and resorption are propor-

459

tionately reduced (Table 2). Whatever residual resorption exists is likely to result from the high circulating levels o f resorptive cytokines, IL-113 and especially IL-6, the latter o f which may further stimulate production o f e n d o g e n o u s corticosteroids. 2t We have documented both a hypercellular reactive marrow in our patients and elevated serum levels of acute phase reactants. Therefore, the production o f the cytokines by inflammatory cells is to be expected. However, staining o f bone surface for tartrate resistant acid phosphatase, while demonstrating the presence o f osteoclasts, does not demonstrate that they are actively resorbing bone. The use o f biochemical markers o f bone remodeling in these patients may have some value. Both serum osteocalcin and PICP levels were low in these patients but could not be correlated with either bone formation or mineral apposition rates because o f the absence o f doxycycline uptake. Urinary pyridinoline and deoxypyridinoline excretion when normalized for creatinine were low and suggest reduced bone resorption, as did the low osteoclast number reported in the adults. 16 The intraindividual variability of the pyridinolines as a marker may limit the usefulness o f single determinations as an adequate index o f bone r e s o r p t i o n ) However, they appear to be more indicative o f bone resorption activity in these patients than serum ICTP levels, which were previously reported to be elevated. 15 This discrepancy may result from cleavage o f the carboxy terminal o f type I collagen in dermis by proteases released from the skin as a result o f the extensive burn injury. The elevated urinary calcium excretion is not likely to be indicative o f bone resorption not only due to the lack o f correlation with eroded surface but also due to the large quantities of protein and calcium c o n s u m e d in the diet, 15 both o f which may contribute to hypercalciuria. Finally, the patients receiving therapeutic rHGH could not be distinguished from any o f the others either by bone histomorphometry or biochemical markers. W e did not observe the characteristic rise in serum osteocalcin 7"24 and PICP concentration. 24 This suggests that rHGH given at this dose over a short-term period cannot prevent the reduced bone formation.

Acknowledgments: The work was supported in part by Shriners Hospitals for Crippled Children Grant No. SHCC 15877 (G.L.K.), an unrestricted grant form the Otto Sprague Memorial Fund, and the General Clinical Research Center (RROOO48), (C.B.L.) and RR 00865 (W.G.G.), National Institutes of Health. This study was presented in part at the XIIth International Conference on Calcium Regulating Hormones, Melbourne, Australia, February 14-19, 1995; the presentation was facilitated by an ICCRH Travel Award (G.L.K.). I.R.D. thanks Research Into Ageing for financial support. The authors are grateful for the valuable technical support of Dawn Sailer and Nancy Miller.

References

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6. Colwell, A., Russell, R. G. G., and Eastell, R. Factors affecting the assay of urinary 3-hydroxy pyridinium crosslinks of collagen as markers of bone resorption. Eur J Clin Invest 23:341 9; 1993. 7. Delmas, P. D., Chatelain, P., Malavah L., and Bonne, G. Serum bone GLAprotein in growth hormone deficient children. J Bone Miner Res 1:333-338: 1986. 8. Dickson, I. R., Bagga, M., and Paterson, C. R. Variations in the serum concentration and urine excretion of alpha 2HS glycoprotein, a bone-related protein in normal individuals and in patients with osteogenesis imperfecta. Calcif Tissue lnt 35:16-20; 1983. 9. Dickson, I. R., Poule, A. R., and Veis, A. Localization of plasma alpha-2 HS glycoprotein in mineralizing human bone. Nature 256:430-242; 1975. 10. Ebeling, P. R., Peterson, J. M., and Riggs, B. L. Utility of type I procollagen propetide assays for assessing abnormalities in metabolic bone disease. J Bone Miner Res 7:1243:-1250; 1992. 11. Fukutani, S., Tsukamoto, Y., and Mori, M. Determination of fluorescent oxytetracycline complexes in dental and skeletal hard tissues by a rapid and accurate quantitative method. Prog Clin Biol Res 187:215-224; 1985. 12. Gilpin, D. A., Barrow, R. E., Rutan, R. L., Broemeling, L. D., and Hemdon, D. N. Recombinant human growth hormone accelerates wound healing in children with large cutaneous burns. Ann Surg 220:19-24; 1994. 13. Hahn, T. J. Steroid and drug induced osteopenia. In: Favus, M. J. Ed. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. New York: Raven P; 250-255; 1993. 14. Herndon, D. N., Rutan, R. L., Alison, W. G., Jr., and Cox, C. C., Jr. Management of burn injuries. In: Eichelberger, M. R., Ed. Pediatric trauma: Prevention, acute care, rehabilitation. St. Louis: Mosby-Yearbook; 568 590; 1993. 15. Klein, G. L., Herndon, D. N., and Langman, C. B. et al. Long term reduction in bone mass following severe burn injury in children. J Pediatr 126:252 256; 1995. 16. Klein, G. L., Herndon, D. N., and Rutan, T. C. et al. Bone disease in burn patients. J Bone Miner Res 8:337-345: 1993. 17. Larrick, J. W., Brindley, L., and Doyle, M. V. An improved assay for the detection of interleukin-l. J Immuno Methods 79:39~.5; 1985. 18. Lebreton, J. P., Joisel, F., Raoult, J. P., Lannuzel, B., Rogez, J. P., and Humbert, G. Serum concentration of human alpha-2 HS glycoprotein during the inflammatory process. J Clin Invest 64:1118-1129: 1979.

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19. Le Gendre, G. R. and Alfrey, A. C. Measuring picogram amounts of aluminum in biological tissue by flameless atomic absorption analysis of a chelate. Clin Chem 22:53-6; 1978. 20. Lephart, E. D., Baxter, C. R., and Parker. C. R., Jr. Effect of bum trauma on adrenal and testicular hormone production. J Clin Endocrinol Metab 64:842848; 1987. 21. Mastorakos, G., Chrousos, G. P., and Weber, J. S. Recombinant interleukin-6 activates the hypothalamic-pituitary-adrenal axis in humans. J Clin Endocrinol Metab 77:1690-4; 1993. 22. Roodman, G. D., Kurihara, N., and Ohsaki, Y. et al. lnterleukin-6 A potential autocrine/paracrine factor in Pagers disease of bone. J Clin Invest 89:46-52: 1992. 23. Rutan, R. L. and Herndon, D. N. Growth delay in postburn patients. Arch Surg 125:392-395: 1990. 24. Saggese, G., Baroncelli, G. I., Bertelloni, S., Cinquanta, L., and Di Nero, G. Effect of long-term treatment with growth hormone on bone and mineral metabolism in children with growth hormone deficiency. J Pediatr 122:3745; 1993. 25. Salusky. I. B., Coburn, J. W., and Brill, J. et al. Bone disease in pediatric patients, undergoing dialysis with CAPD or CCPD Kidney Int 33:975 82: 1988. 26. Sanchez, C. F., Goodman, W. G., Brandli, D. et al. Skeletal response to recombinant human growth hormone (rhGH) in children treated with long-term corticosteroids. J Bone Miner Res 10:2-6: 1995. 27. Sedman, A. B.. Alfrey, A. C.. Miller. N. L., and Goodman, W. G. Tissue and cellular basis for impaired bone formation in aluminum-related osteomalacia in the pig. 79:86-92; 1987. 28. Zhang, R. W., Supowit, S. C.. Klein, G. L. et al. Rat tail suspension regulates gene expression for growth factors and osteopontin and decreases the osteoblastic differentiation of bone marrow stromal cells. J Bone Miner Res 10:415 23: 1995.

Date Received: March 10, 1995 Date Revised: June 12. 1995 Date Accepted: July 14. 1995