The Bone Formation Defect in Idiopathic Juvenile Osteoporosis Is Surface-specific

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1 The Bone Formation Defect in Idiopathic Juvenile Osteoporosis Is Surface-specific F. RAUCH, 1 R. TRAVERS, 1 M. E. NORMAN, 2 A. TAYLOR, 2 A. M. PARFITT, 3 and F. H. GLORIEUX 1 1 Genetics Unit, Shriners Hospital, McGill University, Montréal, Québec, Canada 2 A. I. du Pont Institute, Wilmington, DE, USA 3 Division of Endocrinology and Metabolism, University of Arkansas for Medical Sciences, Little Rock, AR, USA We have previously shown that idiopathic juvenile osteoporosis (IJO) is characterized by a decreased cancellous bone volume and a very low bone formation rate on cancellous surfaces. Whether IJO similarly affects cortical bone is unknown. We therefore compared tetracycline double-labeled transfixing iliac-crest bone biopsies from eight children with typical clinical features of IJO (six girls; age years) and from nine children (four girls; age 9 12 years) without metabolic bone disease. No differences in intracortical remodeling activity were detected. Both structural parameters reflecting intracortical remodeling (cortical porosity, active canal diameter, and quiescent canal diameter) and bone surface-based metabolic parameters (osteoid, osteoblast, mineralizing, osteoclast and eroded surfaces, and bone formation rate) were similar in IJO patients and controls (p > 0.2 each, t-test). Although the internal cortex of the biopsy was thinner in IJO patients than in controls ( m vs m; p 0.02), there was no difference in the width of the external cortex (p 0.36). In growing children, both cortices exhibit an external modeling drift. Therefore, the difference in internal cortical width point to a decreased modeling activity on the endocortical surface of the internal cortex. In fact, bone formation rate on this surface was 48% lower in IJO patients than in controls (82 45 m 3 / m 2 per year vs m 3 / m 2 per year). However, this difference did not achieve statistical significance (p 0.21) due to the high variability of bone formation rate on modeling surfaces. The disturbance of bone remodeling in IJO is limited to cancellous bone, but there may be a modeling defect affecting the internal cortex. Thus, the process causing IJO appears to mainly affect bone surfaces that are in contact with the bone marrow cavity. (Bone 31:85 89; 2002) 2002 by Elsevier Science Inc. All rights reserved. Key Words: Cortex; Histomorphometry; Idiopathic juvenile osteoporosis (IJO); Ilium; Modeling; Remodeling. Introduction Idiopathic juvenile osteoporosis (IJO) is a transient, nonhereditary form of childhood osteoporosis without extraskeletal involvement. 11 IJO typically develops in prepubertal, previously Address for correspondence and reprints: Dr. Frank Rauch, Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montréal, Québec H3G 1A6 Canada. frauch@shriners.mcgill.ca healthy children of either gender. 2 Long bone fractures, mostly at metaphyseal sites, may occur and vertebral compression fractures are frequent. Spontaneous recovery is the rule after 3 5 years of evolution, although spine deformities and severe functional impairment may persist. 13 In a previous study we presented a detailed histomorphometric analysis of iliac cancellous bone in IJO. 12 Cancellous bone volume was low, due to abnormally thin trabeculae and a decreased number of trabeculae. Bone remodeling was clearly disturbed in IJO, with a very low bone formation rate. These analyses were limited to remodeling in cancellous bone, which is the traditional focus of interest in adult bone histomorphometry. However, in children, bones not only undergo remodeling, but also change in size and shape in a process for which Frost coined the term modeling. 3 Remodeling and modeling differ in a variety of histologic characteristics. In remodeling, bone formation is spatially and temporally linked to a prior episode of bone resorption. 7 In bone modeling, formation and resorption continue for a long period of time at the same location and over an extensive region of bone surface. 3,6 We have recently shown that the ileal cross section increases through outward modeling drifts of internal and external cortices. 10 The corresponding histoanatomical features are shown in Figure 1. The modeling drift of the inner cortex is slower than that of the external cortex, which leads to a slow increase in the distance between the two cortices. Cortical width is determined by the balance between modeling on the periosteal and endocortical surfaces of a cortex. 10 Simultaneous to these modeling processes affecting periosteal and endocortical surfaces, cortical bone is remodeled on intracortical surfaces. 3,6 It is unknown at present whether the remodeling defects in IJO are limited to cancellous bone or whether intracortical remodeling is disturbed as well. It is also unclear as to whether the disease process affects modeling. These are important gaps in our understanding of the disease, because cortical modeling and remodeling are essential for maintaining cortical stability. 3,6 Using the same biopsies as in our previous study, we therefore analyzed histomorphometric parameters of iliac cortical bone modeling and remodeling. Subjects and Methods Subjects The present analyses of cortical bone were performed in the same biopsies that had been used earlier to study cancellous bone. 12 The earlier study comprised material from 9 patients with IJO 2002 by Elsevier Science Inc /02/$22.00 All rights reserved. PII S (02)

2 86 F. Rauch et al. Bone Vol. 31, No. 1 Cortical bone histomorphometry in IJO and/or legal guardian. The study protocol was approved by the ethics committees of the participating institutions (Shriners Hospital and du Pont Institute). Histomorphometry Figure 1. Schematic representation of a transiliac bone biopsy specimen of a growing child. There is a modeling drift in an external direction (upward in the diagram) on both cortices. Vigorous bone formation activity (osteoid seams, long double labels) is usually evident on the periosteal surface of the external cortex and on the endocortical surface of the internal cortex. Conversely, resorption predominates on the endocortical surface of the external cortex and on the periosteal surface of the internal cortex. Corresponding to this modeling drift pattern, primary bone (containing Sharpey fibers and consisting of lamellae that are parallel to the periosteal surface) is found almost exclusively on the external cortex. On the internal cortex, lamellae parallel to the periosteal surface are seen occasionally, but there are no Sharpey fibers. This bone might result from periosteal remodeling events. The dotted lines indicate the endocortical surfaces of the two cortices, which exemplifies how these surfaces were identified in the present study. and 12 control subjects. For analysis of cortical bone, biopsies from one of the IJO patients and from three control subjects had to be excluded because the cortices were not adequately preserved. Thus, the present study comprised eight children with IJO, aged years (Table 1), who presented the typical features of IJO, as described previously. 12 The control population consisted of nine age-matched children (age years; Table 1). These were part of a larger group of children and adolescents without metabolic bone disease, who were biopsied with the aim to establish reference data, as described previously. 4 Informed consent was obtained in each instance from the subject Table 1. Study groups and dimensions of the biopsy specimens Ctrl IJO p n (M/F) 9 (5/4) 8 (2/6) Age (years) Biopsy dimensions C.Wi (mm) Mean Ct.Wi (mm) Ct.Wi/C.Wi (%) Data expressed as mean SD; p values calculated by U-test. KEY: ctrl, control; IJO, infantile juvenile osteoporosis; mean Ct.Wi, averaged width of external and internal cortex. The biopsy procedure and processing of these samples have been described previously. 4,12 Cortical analyses were performed separately in external and internal cortices. The external cortex was identified by the presence of abundant muscle cells on the periosteal surface. The following primary measures were obtained: width of the entire biopsy core; cortical width; cortical tissue area; cortical bone area; bone perimeter; osteoid area; osteoid perimeter; eroded perimeter; osteoblast perimeter; osteoclast perimeter; osteoclast number; double-label perimeter; interlabel distance (measured at multiple locations spaced by 50 m); single-label perimeter; haversian canal diameter; and area of primary bone. Primary bone was defined as cortical bone that was not part of an osteonal system, exhibited a lamellation parallel to the periosteal surface, and contained Sharpey fibers (Figure 1). 5 The quantification of primary bone was limited to the external cortex, because the inner cortex of most biopsies contained no or very small amounts of primary bone. All measures of perimeters, as well as osteoid area, osteoclast number, and interlabel distance, were obtained separately for the three surface types of each cortex. Haversian canal diameter was measured as the shortest diameter of the canal cross section, as described elsewhere. 1 This was measured separately for quiescent secondary osteons and active secondary osteons (Figure 1). Osteons were regarded as active when osteoid or erosions were present on the canal wall. Core width was measured directly at 500 m intervals as the distance between the periosteal surfaces of the two cortices. Cortical width was determined similarly as the distance between the periosteal surface and the endocortical surface of each cortex. The distinction between cortical and cancellous bone can be difficult and is subject to observer disagreement. 8 Therefore, all cortical measurements were performed by the same investigator. Endocortical and cancellous bone surfaces were distinguished based on the sizes of the intracortical spaces, their distance from the bone surface, and the diameters of the trabeculae (Figure 1). To eliminate the influence of section obliquity, cortical width was also analyzed relative to biopsy core width. The other measurements were performed as described earlier for cancellous bone. 4,12 Apart from core width and cortical width, which were measured at a magnification of 32, all analyses were carried out at a magnification of 200. A digitizing table with OS- TEOMEASURE software (Osteometrics, Inc., Atlanta, GA) was used for all measurements. Three-dimensional parameters were derived from the primary measures using standard formulae. 4 Nomenclature and abbreviations follow the recommendations of the American Society for Bone and Mineral Research. 9 Statistical Analysis Results are given as mean and SD for normally distributed data and median and interquartile range for nonnormally distributed data. Differences between IJO patients and controls were tested for significance using the Mann Whitney U-test. All tests were two-tailed, and throughout the study p 0.05 was considered statistically significant. These calculations were performed using SPSS software, version 6.0 for Windows (SPSS, Inc., Chicago, IL).

3 F. Rauch et al. Cortical bone histomorphometry in IJO 87 Table 2. Structural parameters of external and internal cortices Ct.Wi (mm) Ct.Wi/C.Wi (%) Ct.Po (%) a.ca.dm ( m) Q.Ca.Dm ( m) 37 [28 56] 39 [32 45] [22 27] 27 [24 33] 0.44 Primary BV/total BV (%) Data expressed as mean SD or median [interquartile range. p values calculated by U-test. See Results for abbreviations of bone parameters, and Table 1 for others. Boldface values highlight significant differences bewteen Ctrl and IJO. Results Biopsy Dimensions Core width (C.Wi) was similar in IJO patients and in healthy controls (Table 1). Mean cortical width (Ct.Wi) tended to be smaller in the IJO group, but the difference did not achieve statistical significance. The subsequent analyses were performed for internal and external cortices separately, as the two cortices are clearly different in children. This is because bone modeling has opposite effects on the two cortices. 10 Structural Parameters of External and Internal Cortices In the external cortex, width and porosity (Ct.Po) were similar in IJO patients and healthy children (Table 2). There were no differences in the diameter of active (a.ca.dm) or quiescent haversian canals (Q.Ca.Dm). The fraction of the external cortex consisting of primary bone was somewhat smaller in IJO patients, but the difference vs. controls did not reach significance. As to the internal cortex, its width was decreased by 33% in IJO patients relative to control subjects. The other structural parameters of the internal cortex were similar in the two groups. Formation and Resorption Parameters Periosteal bone formation and resorption parameters are shown in Table 3. The differences between results in IJO patients and controls reached significance only for osteoid surface extent (OS/BS) on the external cortex. Results for endocortical surfaces are listed in Table 4. No significant differences between IJO patients and healthy children were found. However, mean bone formation rate was 48% lower on the endocortical surface of the internal cortex of IJO patients. Parameters reflecting intracortical remodeling are given in Table 5. To facilitate the comparison of intracortical and cancellous remodeling parameters, Table 5 also contains previously published results for cancellous bone. 12 These values are slightly different from the earlier report, because three control biopsies and one IJO biopsy were excluded from the present analysis (see Subjects and Methods). Osteoid thickness (O.Th) was increased in the external cortex of IJO patients, whereas it was decreased in the internal cortex. In addition, mineral apposition rate (MAR) was significantly decreased in the external cortex of IJO patients. No difference was found for the other parameters. Discussion Bone Modeling During growth the width of the biopsy core is determined by modeling movements of the two periosteal surfaces relative to each other (Figure 1). 10 Cortical width is determined by the relationship between modeling movements on the periosteal and endocortical surfaces of a cortex. 10 As modeling is a discontinuous process with an on-off mechanism, 3 variability of formation and resorption parameters is higher on modeling than on remodeling surfaces. For example, the interindividual coefficient of variation (SD divided by mean value, in percent) for bone formation rate on the external periosteal surface was 120% in the IJO group and 87% in healthy controls (calculated from Table 3). In contrast, the variability of bone formation rate on intracortical surfaces of the external cortex was only 38% in IJO patients and 50% in controls (Table 5). Due to this high variability, large sample numbers are necessary to detect group differences on modeling surfaces. This makes it difficult to study modeling abnormalities in rare diseases such as IJO. Core width was normal in IJO, showing that the relationship between the two periosteal surfaces was preserved. Accordingly, Table 3. Periosteal surfaces: Formation and resorption parameters O.Th ( m) OS/BS (%) Ob.S/BS (%) MS/BS (%) BFR/BS ( m 3 / m 2 per year) MAR ( m/day) N.Oc/B.Pm (/mm) Oc.S/BS (%) ES/BS (%) Data expressed as mean SD; p values calculated by U-test. See Results and Table 1 for abbreviations.

4 88 F. Rauch et al. Bone Vol. 31, No. 1 Cortical bone histomorphometry in IJO Table 4. Endocortical surfaces: Formation and resorption parameters O.Th ( m) OS/BS (%) Ob.S/BS (%) MS/BS (%) BFR/BS ( m 3 / m 2 per year) MAR ( m/day) N.Oc/B.Pm (/mm) Oc.S/BS (%) ES/BS (%) Data expressed as mean SD. p values calculated by U-test. See Results and Table 1 for abbreviations. there were no consistent abnormalities in periosteal bone formation or resorption. External cortical width of IJO patients was close to results in controls, suggesting that periosteal apposition and endocortical resorption were in equilibrium on this cortex. This corresponds to the clinical observation that long bone diaphyses, which develop by the same mechanism as the external iliac cortex, are typically not affected in IJO. In contrast, the width of the internal cortex was significantly decreased in IJO patients. The width of this cortex is determined by endocortical formation and periosteal resorption. 10 As mean bone formation rate on the endocortical surface was decreased by about 48% in the IJO group (although nonsignificantly), the decreased width of the internal cortex was likely due to decreased endocortical apposition. Bone Remodeling In our previous study we found that trabeculae were thinner in IJO patients than in controls and that wall thickness was decreased. 12 These findings suggest that more bone was removed than added during a remodeling cycle. If bone were similarly lost during intracortical remodeling, the consequence should be an increase in cortical porosity and a larger diameter of quiescent osteonal canals. However, cortical porosity and canal diameter were similar to the results in healthy children. Thus, there was no indication that bone was lost by intracortical remodeling. In addition, parameters of intracortical bone formation and resorption were generally close to the control values. In the same biopsies, bone formation rate on cancellous bone was decreased to 36% of the results of controls (Table 5). Thus, the bone remodeling defect in IJO appears to be limited to cancellous surfaces. Although this was an unexpected finding, the discrepancy between intracortical and cancellous bone remodeling is not unique to IJO. In postmenopausal osteoporosis intracortical remodeling is preserved even though bone formation rate is decreased on cancellous surfaces. 8 Nevertheless, the analogy between juvenile and postmenopausal osteoporosis does not extend to endocortical surfaces. On the endocortical surface, bone formation rate is elevated in postmenopausal osteoporosis, 8 but tends to be low in IJO. It is difficult to provide a mechanistic hypothesis for the cause of these differences between bone surfaces, because the factors that determine site-specific bone cell activity have not been elucidated. Synthesis The combined results of the present study and our earlier analyses of the same biopsies 12 demonstrate that the pathophysiologic processes associated with IJO affect various bone surfaces differently. Cancellous bone remodeling is clearly abnormal, whereas intracortical remodeling appears to be unaffected. Endocortical modeling tends to be slow on the internal cortex, leading to decreased internal cortical width. No major defect in periosteal modeling was found and, accordingly, biopsy core width was normal. Taken together, these findings suggest that IJO is caused by a factor that predominantly affects bone formation on surfaces exposed to the bone marrow environment. Table 5. Intracortical and cancellous surfaces: Formation and resorption parameters Cancellous bone Ctrl IJO p O.Th ( m) OS/BS (%) OV/BV (%) Ob.S/BS (%) MS/BS (%) MAR ( m/day) BFR/BS ( m 3 / m per year) BFR/BV (%/year) N.Oc/B.Pm (/mm) Oc.S/BS (%) ES/BS (%) Data expressed as mean SD. p values calculated by U-test. See Results and Table 1 for abbreviations. Boldface values highlight significant differences between Ctrl and IJO.

5 F. Rauch et al. Cortical bone histomorphometry in IJO 89 Acknowledgments: The authors thank Guy Charette for technical assistance with sample processing and Mark Lepik for preparation of Figure 1. This study was supported by the Shriners of North America. References 1. Brockstedt, H., Kassem, M., Eriksen, E. F., Mosekilde, L., and Melsen, F. Ageand sex-related changes in iliac cortical bone mass and remodeling. Bone 14: ; Dent, C. E. and Friedman, M. Idiopathic juvenile osteoporosis. Q J Med 34: ; Frost, H. M. Skeletal structural adaptations to mechanical usage (SATMU): 1. Redefining Wolff s law: The bone modeling problem. Anat Rec 226: ; Glorieux, F. H., Travers, R., Taylor, A., Bowen, J. R., Rauch, F., Norman, M., and Parfitt, A. M. Normative data for iliac bone histomorphometry in growing children. Bone 26: ; Jones, S. J., Glorieux, F. H., Travers, R., and Boyde, A. The microscopic structure of bone in normal children and patients with osteogenesis imperfecta: A survey using backscattered electron imaging. Calcif Tissue Int 64:8 17; Parfitt, A. M. Bone-forming cells in clinical conditions. In: Hall B. K., ed. The Osteoblast and Osteocyte. Bone: A Treatise 1. Caldwell, NJ: Telford; ; Parfitt, A. M. The cellular basis of bone remodeling: The quantum concept reexamined in light of recent advances in the cell biology of bone. Calcif Tissue Int 36(Suppl. 1):S37 S45; Parfitt, A. M. Surface specific bone remodeling in health and disease. In: Kleerekoper M., & Krane S. M., eds. Clinical Disorders of Bone and Mineral Metabolism. New York: Liebert; 7 14; Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H., Meunier, P. J., Ott, S. M., and Recker, R. R. Bone histomorphometry: standardization of nomenclature, symbols, and units: Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2: ; Parfitt, A. M., Travers, R., Rauch, F., and Glorieux, F. H. Structural and cellular changes during bone growth in healthy children. Bone 27: ; Rauch, F. and Glorieux, F. H. Osteoporosis in childhood. In: Henderson J. E., & Goltzman D., eds. The Osteoporosis Primer. Cambridge, UK: Cambridge University; ; Rauch, F., Travers, R., Norman, M. E., Taylor, A., Parfitt, A. M., and Glorieux, F. H. Deficient bone formation in idiopathic juvenile osteoporosis: A histomorphometric study of cancellous iliac bone. J Bone Miner Res 15: ; Smith, R. Idiopathic juvenile osteoporosis: Experience of twenty-one patients. Br J Rheumatol 34:68 77; Date Received: November 2, 2001 Date Revised: February 14, 2002 Date Accepted: February 18, 2002