Cortical bone development in black and white South African children: Iliac crest histomorphometry

Bone 44 (2009) 603–611

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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e

Cortical bone development in black and white South African children: Iliac crest histomorphometry☆ C.M. Schnitzler a,⁎, J.M. Mesquita a,b, J.M. Pettifor a,c a b c

MRC Mineral Metabolism Research Unit, University of the Witwatersrand, Johannesburg , South Africa Division of Orthopaedic Surgery, University of the Witwatersrand, Johannesburg, South Africa Department of Paediatrics, Chris Hani Baragwanath Hospital, University of the Witwatersrand, Johannesburg, South Africa

a r t i c l e

i n f o

Article history: Received 19 June 2008 Revised 10 November 2008 Accepted 2 December 2008 Available online 24 December 2008 Edited by: J. Kanis Keywords: Cortex Children Ethnicity Histomorphometry Modeling

a b s t r a c t Fragility fracture rates in South Africa are lower in blacks (B) than in whites (W) both in adults and in children. In adults this difference may in part be explained by histomorphometric findings in iliac crest cortical bone of B of thicker, less porous cortices, greater endocortical (Ec) wall thickness, fewer canals and greater osteoid thickness accompanied by greater mineral apposition rate and bone formation rate compared to W. Since no comparative data for B and W children are available we examined iliac crest cortical bone of 57 B and 56 W aged 0–23 yrs by routine histomorphometry. Results: The effects of growth as expressed in differences between external and internal cortex were similar in B and W children. Cortical thickness increased with age similarly in B and W until about age 15 whereafter it continued to increase only in B. Ec wall thickness rose with age in B but did not change in W. After age 11 canal number was lower in B. Cortical porosity was highest between ages 6 and 15 with a tendency to lower values in the external cortex in B. Thus structural differences reported in adults were evident in children. Bone turnover as reflected in osteoid surface and eroded surface declined with age similarly in B and W but osteoid thickness did not change with age. Greater osteoid thickness in B children could reflect greater vigor of osteoblasts and greater osteoblast team performance as it did in B adults and may have contributed to the structural advantage in B children. Conclusion: B children showed greater values for osteoid thickness, endocortical wall thickness and cortical thickness, and a tendency to lower porosity compared to W children. These features may contribute to lower fragility fracture rates in B children. Differing environmental influences and possibly genetic effects may play a role. © 2008 Elsevier Inc. All rights reserved.

Introduction Fragility fracture rates in black (B) women are lower than in white (W) women in both Africa [1–3] and the US [4]. Recently published longitudinal fracture data in South African (SA) children also showed lower fracture rates in B compared to W [5]. In both countries lower hip fracture rates in B women can be explained by higher bone mass [4,6] and thicker femoral neck cortices [4]. Furthermore, in adults lower fracture rates in B may be explained by differences in a number of histomorphometric features of iliac crest trabecular and cortical bone. Trabecular bone in B SA men and women showed greater values for bone volume, trabecular thickness, osteoid thickness, osteoid surface and eroded surface [7], and in B US women greater bone

☆ This study was funded by the Medical Research Council of South Africa and the University of the Witwatersrand, Johannesburg, South Africa. ⁎ Corresponding author. 608 Helderberg Village, Private Bag X19, Somerset West 7129, South Africa. Fax: +27 21 855 2389. E-mail address: [email protected] (C.M. Schnitzler). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.12.009

volume and trabecular thickness [8] than in their W counterparts. Cortical histomorphometric variables in B SA adults revealed thicker, less porous cortices, greater endocortical wall thickness, fewer intracortical osteons, greater values for osteoid thickness, osteoid surface, endocortical mineral apposition rate and endocortical bone formation rate, and lower values for eroded surface [9]; cortical data in US B women differed from those in W only by greater osteoclast surface [8,10]. In children whole body bone mineral content measured by DXA is also greater in B in both countries, after adjusting for differences in body size [11]. However, published iliac crest histomorphometric data in normal children are confined to those in white Canadian children [12–15], and no comparative data for B and W children are available. This histomorphometric study therefore aims to examine iliac crest cortical bone from normal SA B and W children for effects of growth and age on structural and bone turnover variables, and for possible racial differences that might further our understanding of the difference in fracture rates between B and W children. Cortical bone was chosen because at many anatomical sites bone strength depends mainly on cortical bone [16,17].

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Materials and methods Experimental subjects In this cross-sectional study cortices of transiliac bone samples from 113 normal B (31 males, 26 females) and W (38 males, 18 females) individuals aged 6 weeks to 23 years were examined by routine histomorphometry. Except for a small number of W children in the 6–11 year age group the ages were fairly evenly represented (Table 1). Male and female data were pooled because of uneven gender-distribution among the age groups. It was not a goal of this study to examine gender-specific differences. The bone samples were taken from 142 available samples (73 B, 69 W) of which 29 were excluded (16 B, 13 W, p = 0.649) because of damaged cortices or longitudinally cut Haversian systems. The subjects of the 21 to 23 year age group were partly identical with those of our previous study of cortical bone in B and W adults [9]. Sixteen (all B) of the 113 analysed bone samples were obtained from patients who underwent limb surgery for conditions other than a recent fracture or metabolic bone disease but who were otherwise healthy and ambulant. Written informed consent was obtained from subjects or their legal guardian. The remaining 97 bone samples (41 B, 56 W) were obtained from cadavers of previously healthy individuals who had died suddenly and who at autopsy had shown no organic disease that could have affected bone. Bone samples were obtained within two days after death, rarely after three days. The study was approved by the Committee for Research in Human Subjects of the University of the Witwatersrand, Johannesburg, South Africa. All subjects came from the same population of the urban and periurban areas of Johannesburg, SA as the subjects of our previous bone histomorphometric studies in adults [7,9]. Socio-economic and lifestyle factors differ between B and W. Whereas W children came from families who had a European or North American lifestyle, many B children came from larger families with poorer socio-economic background and often walked long distances from an early age. The nutritional status of urban B children is generally adequate but calcium intakes are lower than those of their W peers (approximately 340 mg/d and 730 mg/d, respectively) [18]. No data on age at initiation of puberty were available for the subjects. The age at initiation of puberty (Tanner stages 1 to 2) in the general population of Johannesburg did not differ significantly between boys and girls or B and W, and ranged between 9.8 and 10.5 years [19]. Pre-biopsy tetracycline double bone labeling was carried out in 16 B subjects using demethylchlortetracycline 300 mg twice a day in subjects aged 12 years and over, and tetracycline hydrochloride 20 mg/kg/d in divided doses in younger children according to the schedule 2 days on, 7–12 days off, 2–5 days on and pre-biopsy interval of 2–10 days. The age of labeled (13.3 y) did not differ from unlabeled (11.4 y, p = 0.43) subjects. No tetracycline labeling was available in W subjects. Bone specimens Cylindrical transiliac bone samples consisting of both cortices and intervening cancellous bone were taken from the standard biopsy site

and processed undecalcified as previously described [7]. For each specimen three sections were stained with Goldner's trichrome stain and one or more were left unstained. Sections had to have two complete cortices suitable for histomorphometric analysis. Both cortices of two sections (4 cortices) were analysed in each case. The total cortical bone area available for examination depended on cortical thickness and varied between 3.4 and 44.5 mm2. Histomorphometric analysis All primary and calculated variables were obtained as previously described [9]. Nomenclature, abbreviations and symbols of terms are those approved by the American Society for Bone and Mineral Research [20]. All specimens were examined by the same investigator (CMS). Intraobserver variability has been reported previously [9]. External (ext) and internal (int) cortices were analysed separately. Since soft tissue on both periosteal surfaces had been removed, criteria other than presence of muscle [15] had to be used in the designation of ext and int cortex. The cortex with the greater thickness of subperiosteal primary intramembranous bone (referred to as primary bone) was taken to be the ext cortex, as was presence of primary bone within interstitial bone. Remnants of incorporated trabeculae were taken to denote int cortex. Primary bone was characterized by fine collagen lamellae of low birefringence, running parallel to the periosteal surface and being traversed at angles of 45–60° by highly birefringent Sharpey's fibres. The examined and calculated variables of cortical structure and bone turnover are listed in Tables 2a and b. Haversian (H) osteons presented as asymmetric or primary H osteons, and concentric or secondary H osteons. Asymmetric H osteons had an eccentric or marginal canal whereas concentric osteons had a central canal. Measurements of H osteonal dimensions were confined to complete concentric inactive osteons (neither osteoid nor erosion on the canal surface) as previously described [9,21]; dimensions of asymmetric H osteons were not measured. The canal diameter of concentric H osteons was measured and is listed as “Inactive canal diameter”. Canal diameter of all active and inactive canals combined is listed as “Canal diameter (active and inactive)” and was calculated as previously described [9]; it includes all canals, concentric and asymmetric, and reflects the average size of all intracortical voids. Canal number is the number of all canals (active and inactive) of concentric and asymmetric H osteons and reflects the number of intracortical voids per mm2. Density of concentric H osteons is given as number per mm2. The frequency distributions of concentric and asymmetric H osteons were assessed semi-quantitatively and represented as a score on a scale of 0 to 5: 0 = 0%; 1 = 5–10%; 2 = 20–30%; 3 = 40–60%; 4 = 70–80%; 5 = 90–100%. Special care was taken in the measurement of endocortical (Ec) wall thickness (Ec.W.Th, μm) in children under age 18 in whom compaction, a feature of modeling made definition of the intracortical boundary of endocortical Ec osteons at times difficult. Compaction is the process of continuous bone formation layer after layer, seemingly uninterrupted by rest periods or resorption, on the int Ec envelope [12,14]. As a result, cement lines are either difficult to identify or

Table 1 Age distribution of 113 normal black and white individuals Age group

Age (years) Blacks

0–5 y 6–11 y 12–17 y 18–23 y Total

p-values B vs W

Whites

N

Mean ± SD

Median [range]

N

Mean ± SD

Median [range]

16 11 14 16 57

1.9 ± 1.6 9.4 ± 1.6 14.6 ± 1.8 21.4 ± 1.2 12 ± 7.7

1.2 10 14.5 22 12

20 3 13 20 56

2.3 ± 1.6 8 ± 1.7 15.5 ± 1.3 20.8 ± 1 12.3 ± 8.2

2.3 7 16 21 15

[0.1–4] [6–11] [12–17] [19–23] [0.1–23]

[0.3–5] [7–10] [13–17] [19–23] [0.3–23]

0.432 0.228 0.154 0.093 0.822

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605

Table 2a Iliac crest histomorphometric data for external and internal cortex in 27 black and 23 white children aged 0.1 to 11 years Variables

Blacks N

Whites External

N

Internal

N

p-values External

N

Internal

B vs Wa

Ext vs Int B

Structure Age (years) Core length (mm) Cortical thickness (μm) Ec wall thickness (μm) Ps.1°B.Th/Ct.Th (%) Cortical porosity (%) Canal number (N/mm2) Canal diameter(active and inactive) (μm) Inactive canal diameter (μm) H osteonal diameter (μm) H wall thickness (μm) H concentric osteonal density (N/mm2)

27 27 27 21 27 27 27 27 15 15 15 27

4.9 ± 4.1 6.44 ± 2.09 579 ± 294 38.5 ± 10.2 55.9 ± 30.8 8.32 ± 4.85 12.7 ± 9.3 103 ± 41 30.4 ± 12.1 119 ± 32 44.4 ± 13.2 0.6 ± 0.72

27 26 27 27 27 27 11 11 11 26

Static bone turnover H osteoid thickness (μm) H osteoid surface (%) H eroded surface (%) Ec osteoid thickness (μm) Ec osteoid surface (%) Ec eroded surface (%) Ps osteoid surface (%) Ps eroded surface (%)

27 27 27 27 27 27 27 27

10.8 ± 3.3 38.4 ± 20.6 28.9 ± 21.4 11.2 ± 4.2 19 ± 13 34.2 ± 20.4 60.3 ± 34.5 2.9 ± 5.8

27 27 27 27 27 27 27 27

W

Ext

Int

0.647 0.233 0.181 0.899 0.444 0.973 0.513 0.114 0.087 0.279

0.471 0.644 0.619 0.055 0.814 0.123 0.674 0.728

599 ± 301 41.1 ± 10.3 27.1 ± 32.9 6.56 ± 4.6 11.4 ± 8.6 92 ± 44 25.9 ± 8.1 104 ± 14 39 ± 5.2 0.56 ± 1

23 23 23 19 23 23 23 23 13 13 13 22

3.1 ± 2.5 5.57 ± 1.37 659 ± 345 37.2 ± 8.6 52.6 ± 29.4 9.06 ± 6.19 12.8 ± 6.4 99 ± 58 32 ± 11.4 115 ± 16 41.6 ± 6.9 0.89 ± 1.08

23 18 23 23 23 23 13 13 13 22

543 ± 239 36.1 ± 8.6 22.4 ± 28.1 5.71 ± 3.58 11.6 ± 4.2 80 ± 36 26.3 ± 8 110 ± 25 41.6 ± 9.6 0.93 ± 1.21

0.722 0.479 0.0002 0.135 0.356 0.177 0.396 0.2 0.24 0.782

0.066 0.879 b 0.0001 0.003 0.375 0.05 0.452 0.71 0.932 0.879

0.059 0.656 0.068 0.855 0.131 0.447 0.309 0.636 0.464 0.217 0.301 0.057

9.9 ± 3 38 ± 19.4 28.4 ± 18.9 12.1 ± 3 45.6 ± 25.7 16.6 ± 16 29.1 ± 35.1 26.2 ± 31.9

23 23 23 23 23 23 23 23

9.9 ± 2.6 39 ± 20.9 25.9 ± 16.3 8.4 ± 3.1 20.2 ± 15.9 37.2 ± 21.4 58.5 ± 34.6 3.47 ± 9

23 23 23 23 23 23 23 23

8.8 ± 1.9 34.2 ± 15.6 24.6 ± 15.1 10.3 ± 3 48.5 ± 27.8 11.6 ± 12.1 35 ± 35.5 26.9 ± 31.6

0.113 0.936 0.92 0.244 b 0.0001 0.0007 0.0034 0.001

0.1 0.405 0.698 0.03 b 0.0001 b 0.0001 0.012 0.001

0.948 0.998 0.94 0.07 0.371 0.805 0.5 0.736

Data are given as mean ± SD. a Comparison of B vs W with correction for age.

absent altogether, and collagen fibres rarely change orientation abruptly — features that, if present help to delineate the Ec osteon from underlying cortical bone. However, converging collagen fibres at either end of the osteon aided in the delineation of its intracortical boundary. Ec osteons with an osteoid surface were included in measurements, provided the thickness of the osteoid seam did not exceed 20% of the Ec.WTh. Approximately 81 Ec W.Th measurements were made on about 9 Ec osteons per bone sample. Thickness of double collagen lamellae (one bright, one dark under polarized light) was measured [22] at three sites: in 1.) subperiosteal

primary bone, 2.) compacting bone and 3.) secondary Ec osteons in a subgroup of patients whose age distribution reflected that of the total sample. Statistical analysis Statistical analysis was carried out with the aid of the Statistical Analysis System (SAS) program (SAS Institute, Cary, NC). Age differences between B and W children within age groups were tested by 1-way ANOVA (Table 1). Ext and int cortices were compared by the

Table 2b Iliac crest histomorphometric data for external and internal cortex in 30 black and 33 white individuals aged 12 to 23 years Variables

Blacks N

Whites External

N

Internal

N

p-values External

N

Internal

Structure Age (years) Core length (mm) Cortical thickness (μm) Ec wall thickness (μm) Ps.1°B.Th/Ct.Th (%) Cortical porosity (%) Canal number (N/mm2) Canal diameter(active and inactive) (μm) Inactive canal diameter (μm) H osteonal diameter (μm) H wall thickness (μm) H concentric osteonal density (N/mm2)

30 30 30 30 30 30 30 30 29 29 29 29

18.3 ± 3.8 9.07 ± 2.09 1115 ± 369 53.4 ± 12 11.9 ± 12.6 5.41 ± 3.24 10 ± 4.1 85 ± 32 31 ± 9.7 131 ± 20 50 ± 7.6 2.28 ± 1.41

30 29 30 30 30 30 28 28 28 29

Static bone turnover H osteoid thickness (μm) H osteoid surface (%) H eroded surface (%) Ec osteoid thickness (μm) Ec osteoid surface (%) Ec eroded surface (%) Ps osteoid surface (%) Ps eroded surface (%)

29 30 30 30 30 30 30 30

10.8 ± 4 15.3 ± 15.6 14.2 ± 10.1 11.1 ± 4.6 11.3 ± 8.1 11.7 ± 11.2 38.9 ± 30.3 10.4 ± 16.6

30 30 30 30 30 30 30 30

Data are given as mean ± SD. a Comparison of B vs W with correction for age.

B vs Wa

Ext vs Int B

1114 ± 469 49.1 ± 11.8 5.3 ± 9.9 4.77 ± 3 11.1 ± 3.8 76 ± 31 27.3 ± 4.8 125 ± 22 48.8 ± 9.5 3.12 ± 1.88

33 33 33 32 33 33 33 33 31 31 31 33

18.7 ± 2.8 8.9 ± 2.08 881 ± 406 41 ± 8.03 14.2 ± 13 6 ± 5.14 12.3 ± 5 81 ± 50 29.9 ± 7.8 127 ± 22 48.4 ± 9.4 2.83 ± 1.7

33 32 33 33 33 33 33 33 33 33

914 ± 406 40.7 ± 10.8 0.37 ± 1.32 5.5 ± 2.7 13.5 ± 4.1 72 ± 21 25.9 ± 5.9 120 ± 19 46.9 ± 8.9 3.3 ± 2.48

0.995 0.1 0.002 0.264 0.094 0.113 0.081 0.152 0.46 0.022

11 ± 4.1 14.7 ± 11.7 11 ± 10.4 11.6 ± 4.6 20 ± 15.1 8.9 ± 8.8 22.3 ± 19.6 12 ± 18.3

33 33 33 33 33 33 33 33

8.9 ± 3.4 17.2 ± 16.9 15.9 ± 15.6 8.9 ± 3.6 16.7 ± 16.4 13.1 ± 8.5 38.9 ± 29.1 8.4 ± 15.6

33 33 33 33 33 33 33 33

8.8 ± 2.7 21.4 ± 14.3 15.6 ± 12.2 9.3 ± 2.9 26.7 ± 24.7 8 ± 6.4 18.7 ± 22.1 18.3 ± 25.2

0.792 0.767 0.008 0.567 0.0006 0.16 0.014 0.681

W

Ext

Int

0.67 0.893 b0.0001 0.581 0.104 0.289 0.005 0.066 0.315 0.286

0.583 0.764 0.019 b 0.0001 0.206 0.31 0.058 0.992 0.626 0.337 0.346 0.235

0.054 0.006 0.006 0.259 0.026 0.737 0.32 0.221 0.299 0.909

0.861 0.085 0.893 0.573 0.01 0.011 0.003 0.044

0.065 0.282 0.341 0.053 0.064 0.455 0.903 0.599

0.014 0.014 0.02 0.027 0.096 0.723 0.487 0.241

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Table 3 Collagen lamellar thickness in μm in cortical bone of growing individuals Site of measurement

1° bonea Compaction boneb 2° Ec osteonsc a b c

Blacks N 11 8 10

Whites Ext

N

4.99 ± 0.28 6.04 ± 0.39 6.34 ± 0.51

6 10 11

Int 5.45 ± 0.62 5.88 ± 0.52 6.09 ± 0.55

N

p-values Ext

11 10 11

N

5.24 ± 0.27 6.08 ± 0.21 6.09 ± 0.37

5 10 11

Int 5.43 ± 0.71 5.86 ± 0.39 6.07 ± 0.58

Ex vs In

B vs W

B

W

Ext

Int

0.206 0.1 0.168

0.527 0.152 0.931

0.035 0.753 0.212

0.991 0.908 0.95

1° bone vs compaction bone B p = 0.033, W p = 0.036. Compaction bone vs 2° Ec osteons B p = 0.01, W p = NS. Ec osteons vs 1° bone B p = 0.0009, W p = b 0.0001.

paired t-test (Tables 2a, b, 3). One-way ANOVA with correction for age was used for the comparison of B and W children (Tables 2a, b, 3). Correlations (Tables 4 and 5) were tested by correlation analysis (Pearson correlation coefficient). Differences in proportions were tested by the chi-square test.

puberty: 0.1–11 years (Table 2a) also referred to as “younger” children, and 12–23 years (Table 2b) also referred to as “older” children. External vs internal cortex

Results

The effects of growth are expressed in differences between ext and int cortex and Ec and Ps surfaces (Tables 2a, b and 3).

Data are presented in Tables 1–5. Although gender distribution between B and W children did not differ (M/F B 31/38, W 26/18, p = 0.142, chisq), gender distribution among age groups was uneven. Data for males and females were therefore analysed together. Data of structural and static bone turnover variables of ext and int cortex are presented and analysed in two age groups to account for effects of

Structure Cortical thickness, Ec wall thickness, canal number, H wall thickness, H osteonal diameter and lamellar thickness did not differ between the cortices. Since primary bone thickness was among identification criteria for the ext cortex, its thickness as a percentage of cortical thickness was greater in the ext cortex in both age groups of B

Table 4 Linear regression equations and correlation coefficients of the relationship between age and cortical histomorphometric variables in 57 black and 56 white individuals aged 0.1 to 23 years Variables

External cortex

Internal cortex

Intercept

Slope

r

p

Intercept

Slope

r

p

Blacks Core length Cortical thickness Ec wall thickness Ps.1°B.Th/Ct.Tha Cortical porosity Canal number Canal diameter (active and inactive) Inactive canal diameter H osteonal diameter H wall thickness H osteoid thickness H osteoid surface H eroded surface Ec osteoid thickness Ec osteoid surface Ec eroded surface Ps osteoid surface Ps eroded surface

5.47 417 34.2 72 9.38 14.9 106 26.95 101 37 10.7 45.8 31.5 10.7 18.8 43 71 −0.29

0.197 37.2 1 −3.3 −0.22 −0.31 −1.06 0.266 1.791 0.763 0.0054 −1.63 −0.87 0.038 −0.32 −1.75 −1.86 0.598

0.619 0.672 0.548 − 0.803 − 0.392 − 0.334 − 0.219 0.167 0.47 0.494 0.011 − 0.59 − 0.376 0.067 − 0.222 − 0.689 − 0.426 0.354

b 0.0001 b 0.0001 b 0.0001 b 0.0001 0.003 0.011 0.101 0.278 0.001 0.0007 0.933 b 0.0001 0.004 0.623 0.098 b 0.0001 0.001 0.007

376 38.7 38 6.54 12.8 88 23.28 86.7 31.7 9.43 42.8 33 12.3 55 20.4 34 27.2

41.4 0.54 −1.87 −0.077 −0.13 −0.36 0.238 2.1 0.934 0.089 −1.43 −1.16 −0.039 −1.9 −0.66 −0.74 −0.71

0.678 0.359 −0.558 −0.152 −0.16 −0.073 0.265 0.63 0.636 0.19 −0.565 −0.52 −0.076 −0.609 −0.39 −0.205 −0.21

b 0.0001 0.007 b 0.0001 0.258 0.234 0.588 0.103 b 0.0001 b 0.0001 0.158 b 0.0001 b 0.0001 0.572 b 0.0001 0.003 0.125 0.117

Whites Core length Cortical thickness Ec wall thickness Ps.1°B.Th/Ct.Tha Cortical porosity Canal number Canal diameter (active and inactive) Inactive canal diameter H osteonal diameter H wall thickness H osteoid thickness H osteoid surface H eroded surface Ec osteoid thickness Ec osteoid surface Ec eroded surface Ps osteoid surface Ps eroded surface

5.12 627 38.4 61 9.88 12.5 100 34.13 112 39.1 9.95 44.4 28.4 8.64 25 40.2 63 4.01

0.196 13.4 0.16 −2.53 −0.21 0.007 −1.14 −0.254 0.77 0.512 −0.064 −1.52 −0.7 0.017 −0.42 −1.44 −1.32 0.27

0.66 0.304 0.153 − 0.739 − 0.313 0.022 − 0.22 − 0.213 0.275 0.416 − 0.214 − 0.605 − 0.361 0.038 − 0.167 − 0.632 − 0.328 0.123

b 0.0001 0.023 0.283 b 0.0001 0.019 0.874 0.103 0.165 0.071 0.005 0.113 b 0.0001 0.006 0.782 0.218 b 0.0001 0.014 0.365

468 35.2 27 5.75 10.9 86 26.9 102.4 37.8 9.45 38.1 28 10.9 54 12.9 33 34.2

23 0.23 −1.42 −0.015 0.14 −0.68 −0.06 1 0.532 −0.042 −0.902 −0.66 −0.106 −1.62 −0.25 −0.635 −1.08

0.47 0.174 −0.54 −0.021 0.257 −0.112 −0.07 0.36 0.431 −0.088 −0.434 −0.370 −0.290 −0.503 −0.183 −0.184 −0.3

0.0003 0.228 b 0.0001 0.881 0.056 0.411 0.646 0.014 0.003 0.519 0.0008 0.005 0.03 b 0.0001 0.176 0.175 0.027

a

Ps.1°B.Th/Ct.Th, periosteal primary bone thickness as percentage of cortical thickness.

C.M. Schnitzler et al. / Bone 44 (2009) 603–611 Table 5 Correlations between age versus cortical porosity, canal number and canal diameter (active and inactive) for both cortices combined in 57 black and 56 white children aged 0.1 to 23 years analysed in three age groups Variables

Agegroup 0–5 years r

6–15 years P

r

Blacks Cortical porosity Canal diameter Canal number

N = 16 0.167 0.696 − 0.642

0.536 0.003 0.007

N = 20 −0.498 −0.583 0.361

Whites Cortical porosity Canal diameter Canal number

N = 20 0.543 0.747 − 0.43

0.013 0.0002 0.059

N=9 −0.091 0.04 −0.372

16–23 years p

r

p

0.025 0.007 0.118

N = 21 −0.579 −0.449 −0.077

0.006 0.041 0.741

0.816 0.918 0.324

N = 27 −0.429 −0.478 0.382

0.026 0.012 0.05

and W. Cortical porosity and canal diameter (active and inactive) were greater in the ext cortex of younger W children. Inactive canal diameter too was greater in the ext cortex in older children but this was significant only in W. The density of concentric H systems in older B was greater in the int than the ext cortex.

607

After age 18 both cortices became increasingly indistinguishable from one another and began to resemble adult bone in both B and W subjects. Collagen lamellar thickness did not differ between the cortices (Table 3) but was lower in primary than in compacting bone and secondary Ec osteons; it was also lower in compacting bone than in secondary Ec osteons. Static bone turnover Ext and int cortices did not differ with respect to H osteoid thickness and H osteoid surface. In older B H eroded surface was greater in the ext than the int cortex. In young W Ec osteoid thickness was greater in the int than the ext cortex. Ec osteoid surface and Ps eroded surface were greater in the int cortex, and Ec eroded surface and Ps osteoid surface were greater in the ext cortex in both age groups of B and W, but in older B greater Ec eroded surface and lower Ps eroded surface in the ext cortex failed to reach statistical significance. Effects of age Linear regression analyses of the relationships between age and histomorphometric variables for ext and int cortex in B and W are given in Table 4. Since the magnitude of differences between ext and

Fig. 1. Scatterplots and polynomial regression lines on age for core length, cortical thickness, endocortical wall thickness, cortical porosity and canal diameter of transiliac bone samples from black and white children. Blacks: black diamonds and bold regression lines. Whites: empty squares and thin regression lines.

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int cortex was generally similar in B and W (Tables 2a, b, 3), data in Fig. 1 are presented for ext and int cortex combined for ease of presentation; in some variables changes with age are better described by polynomial than by linear regression. Structure Core length and cortical thickness of both cortices (Fig. 1) increased with age in B and W. Increase in core length levelled off from age 15 onwards in both B and W. In W cortical thickness also ceased to increase in both cortices around age 15 but in B it continued to rise. Ec wall thickness (Fig. 1) increased with age in both cortices in B but not in W. Primary bone thickness as a percentage of cortical thickness declined to close to zero by age 23 y in both cortices in B and W. Although linear regression analysis showed a decrease with age in cortical porosity of the ext cortex in both races (Table 4) polynomial regression lines suggested a phasic pattern (Fig. 1, Table 5). Whereas in W cortical porosity rose up to age 5, thereafter remained high and only declined after age 15, in B it gradually decreased from age 6 and no rising phase was noted. A similar pattern of age dependence of canal diameter (active and inactive) was observed in both B and W: canals were largest between ages 6 and 15 (Fig. 1, Table 5); linear regression analysis had not reflected these changes (Table 4). Age-related changes of canal diameter were those of active canals since inactive canal diameter did not change with age in B or W (Table 4). Canal number showed the reverse pattern of age dependence (Table 5): in both races canal number was lowest between ages 6 and 15 when canals were largest. H wall thickness and H osteonal diameter increased with age in B and W. H osteons, both concentric and asymmetric were few in the first few months of life. At that age most canals were merely conduits for blood vessels within primary bone and lacked the circumferential collagen lamellae that characterize H osteons [23]. Density of concentric H osteons increased with age in both cortices (ext cortex: B r = 0.676, p = b0.0001; W r = 0.603, p = b0.0001; int cortex: B r = 0.635, p = b0.0001; W r = 0.533, p = b0.0001). Asymmetric H osteons filled resorption cavities eccentrically instead of circumferentially so that the canal of the completed H osteon came to rest in an eccentric or marginal position. This configuration was more prevalent in the ext than the int cortex. The greatest wall thickness of asymmetric osteons was more often nearer the periosteal than the endocortical envelope in both cortices in both races (external cortex: B 5.3, W 6× more often; internal cortex: B 3.8, W 14.3 × more often; B vs W p = NS). The frequency distribution of asymmetric H osteons across age was generally the converse of that of concentric H osteons. Under age 1 asymmetric osteons were few but after age 1 their frequency distribution soon rose to about 90% of all osteons present, and after the mid-teens it declined to 10% or less by age 18 to 23. Concentric H osteons, on the other hand were few until around age 12 but thereafter increased to over 90% of all H osteons present at age 22–23. These age-related changes were similar in B and W. Static bone turnover H osteoid thickness did not change with age in either cortex of either race. H osteoid and eroded surfaces decreased with age in both cortices in B and W; H eroded surface was highest between ages 6 and 15. Ec osteoid thickness did not change with age in B but declined in the int cortex in W. Ec osteoid surface declined with age in the int cortex in B and W. Ec eroded surface declined with age in both cortices in both races but this failed to reach significance in W. Ps osteoid surface declined with age in the ext cortex in both races. Ps eroded surface increased in the ext cortex in B and decreased in the int cortex in W. Effects of race Whereas in younger children (Table 2a) ext cortical thickness tended to be lower in B than in W, in older children cortical thickness

of both cortices was greater in B. Older B also showed greater Ec wall thickness and lower canal number in both cortices, and a greater primary bone fraction in the int cortex than W. Between ages 6 and 15 cortical porosity in the ext cortex tended to be lower in B (p = 0.059) (Fig. 1). Lamellar thickness in primary bone of the ext cortex was lower in B (Table 3). Older B showed greater H osteoid thickness, and in the int cortex lower values for H osteoid surface and H eroded surface. In younger children Ec osteoid thickness in both cortices tended to be greater in B compared to W; these differences were statistically more significant in the older age group (Tables 2a and b). H and Ec osteoid thickness in the 16 B children with tetracycline labeling did not differ from that in the 41 unlabeled B subjects (H.OTh 11.77 ± 2.56 vs 10.15 ± 3.36, p = 0.088; Ec.OTh 12.63 ± 2.74 vs 11.04 ± 3.64, p = 0.122, respectively). The values for mineral apposition rates (MAR) and mineralization lag times (Mlt) were as follows: H.MAR 1.069 ± 0.289, Ec.MAR 0.986 ± 0.286, H.Mlt 12.7 ± 6.38, Ec.Mlt 15.3 ± 10.3. Discussion To our knowledge this study presents the first comparative data on iliac cortical histomorphometry in B and W children. We found B children aged 11 years and younger to show a tendency to greater Ec osteoid thickness. After age 11 B had greater Ec and H osteoid thickness, greater Ec wall thickness, thicker cortices and lower canal number than W children; between ages 6 and 15 cortical porosity in the external cortex tended to be lower in B children. We have thus shown that differences similar to those between B and W adults [9] were becoming apparent in children of the same population. The greater Ec and H osteoid thickness in B children compared to W was unlikely due to a mineralization defect since mineral apposition rates and mineralization lag times at these two envelopes were similar to published normal values for W children [12]. The study of B and W adults [9] from the same population as the children had shown positive correlations between osteoid thickness versus both mineral apposition rate and bone formation rate. Whereas mineral apposition rate reflects the vigor of individual osteoblasts [23], bone formation rate reflects the collective performance of teams of osteoblasts (mineral apposition rate multiplied by mineralizing surface — the latter being dependent on osteoblast recruitment) [24]. These correlations suggest that greater osteoid thickness in B adults may reflect not only greater vigor of individual osteoblasts but also greater collective performance of teams of osteoblasts, i.e. possibly greater osteoblast recruitment (a variable that cannot yet be measured [24]). Osteoblast recruitment is considered to be the major determinant of osteonal wall thickness [24]. Since insufficient tetracycline based data are available in the present study we can but infer that greater osteoid thickness in B children may also reflect greater osteoblast vigor and team performance and that these were responsible for greater Ec wall thickness and greater cortical thickness in B children. A possible role of the osteocyte in these effects may warrant exploration in future studies [25]. Growth Bone growth of the ilium as assessed at the bone sampling site is achieved through modeling which is expressed in differences between ext and int cortices. Our findings of differences between the two cortices were similar in B and W children and accord with data in W Canadian children by Rauch and associates [12,15], as illustrated in their schematic drawing [15]. Modeling encompasses bone enlargement and preservation of the proportions of shape and is characterized by simultaneous bone resorption and formation on different surfaces; in remodeling, on the other hand sequential resorption and formation take place on the same surface. The modeling events at the growing ilium are finely balanced so that as the lateral modeling drift [14] carries both cortices in tandem laterally, the cortical and cancellous compartments enlarge

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only slightly. The lateral modeling drift is achieved through deposition of primary intramembranous bone on the ext periosteal surface and resorption on the int periosteal surface. To prevent the ext cortex getting ever thicker, its Ec surface undergoes compensatory resorption, and to prevent the int cortex becoming progressively thinner, new bone is deposited on its Ec surface. The ilium thus grows predominantly from the ext Ps and the int Ec surfaces. During this lateral drift new trabeculae are being fashioned from unresorbed Ec bone of the ext cortex, a process referred to as cancellization of the cortex, and existing trabeculae are being incorporated into the int cortex at the Ec envelope, a process referred to as compaction [14]. In addition to modeling a lesser extent of remodeling took place at the Ec envelope as suggested by the presence of osteoid on the ext Ec surface and resorption on the int Ec surface. Any contribution to bone growth by remodeling would require a remodeling imbalance, and would be much smaller than that by modeling [14]. Enlargement of the ilium ceased around age 15 as evidenced by a leveling off of core length increase in both B and W children. In W children this coincided with leveling off of increasing cortical thickness, however in B cortical thickness continued to rise until age 23, the upper age limit of the study. This continued rise in cortical thickness in B subjects in the absence of an increase in core length must have taken place at the Ec envelope as a result of greater Ec wall thickness in B, rather than at the Ps envelope. With diminishing growth after the mid-teens modeling activities at the Ps and Ec envelopes declined as was also observed by Rauch and associates [12]. Cortical porosity Whereas the cellular activities on the Ec and Ps envelopes subserve predominantly modeling, those on the intracortical or H envelope carry out remodeling. This means that bone deposited during modeling on the Ec and Ps surfaces will undergo H remodeling, i.e. it will be resorbed and replaced by H osteons. The largest resorption cavities appeared within subperiosteal primary bone which was more abundant in the ext than the int cortex; this may contribute to the somewhat greater porosity of the external cortex. The thin collagen lamellae of primary bone suggest an immature collagen structure that may render primary bone more readily resorbable than compacting Ec bone with its more mature, albeit not fully mature lamellar structure. In addition, large resorption cavities appear in the subendocortical region of the ext cortex in preparation for the fashioning of new trabeculae from bone left standing between such large resorption cavities [26]. As a result of high intracortical remodeling, the remodeling space, that is the total space of temporarily missing bone is large. This explains the high cortical porosity which may be regarded as a remodeling transient. Following the cessation of bone growth intracortical remodeling declined as judged by the decline in osteoid and eroded surfaces. As a result the remodeling space contracted as evidenced by the decline in canal diameter and cortical porosity, and the cortex became more “solid”. This process of consolidation is further contributed to by a slow increase in cortical thickness until the mid-twenties [14]; in the present study this was found only in B. High cortical porosity between ages 6 and 15 was due to an increase in canal size and not in canal number since the two components of porosity, namely canal size and canal number showed reciprocal age-dependent trends. At this time bone therefore contained larger but fewer canals than before and after that period. Unfortunately, large canals are detrimental to bone strength because they constitute potent stress amplifiers [27]. Development of H osteons Cortical development at the H envelope went through three phases in both cortices of B and W children: 1) vascular conduits, 2) asymmetric H osteons, and 3) concentric H osteons.

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Phase 1 In the first year of life the high number of intracortical canals was accounted for mostly by mere conduits for blood vessels within primary bone with little if any evidence of circumferential collagen lamellae that characterize H osteons [23]. However H systems have been reported in the fetus [28]. Phase 2 Intracortical remodeling was present in the youngest subjects but increased in the years following and remained high to the mid-teens. Most Haversian systems formed during this period were asymmetrical. Active Haversian canals typically showed erosion on the marrow-facing half and osteoid on the periosteum-facing half of the surface. This suggests that osteons are drifting transversely towards the endocortical aspect of the cortex. Osteonal drift has also been described by other investigators [28–32]. Robling and Stout [29] showed in serial sections of individual H osteons in bone from children and young baboons that H erosion proceeds on two fronts: at the cutting cone, advancing the osteon in its longitudinal direction, and along the entire sidewall of the osteon causing the whole osteon to drift sideways towards the endocortical surface, with the direction of drift varying with time by about 90° around the osteon's longitudinal axis. The resultant “giant” resorption cavities, reflected in the present study in the large canal diameter during this period are repaired at their periosteum-facing aspect by a concavo-convex osteon in a manner similar to the compaction process of modeling at the int Ec surface. Indeed, changes in collagen lamellar orientation within some large asymmetric H osteons suggest that successive teams of osteoblasts formed the osteon as in the compaction process of modeling. The concave surface of the sickle-shaped osteon was the bone-forming surface and the thickest part of the completed wall of most osteons faced the periosteum. On cessation of resorption, bone formation closed in on the cavity but left a marginal or eccentric H canal for blood vessels. The reason for the formation of asymmetric H osteons remains unclear. One explanation may lie in the need to fill “giant” resorption cavities. Their surface may be too large and geometrically too irregular to be repaired circumferentially by circular teams of osteoblasts as would be the case in concentric H osteons [23,33]. Nature appears to have resorted to the compacting process of modeling in H remodeling when it comes to the repair of “giant” resorption cavities. It is also thought that the large amounts of calcium needed for longitudinal bone growth are to be supplied by the creation of intracortical resorption cavities which reflect a calcium debt that will only be repaid once growth ceases, i.e. during consolidation [34]. Drift of H osteons directed predominantly towards the marrow cavity has been described previously in flat and long bones of growing individuals [28–32] but its purpose and initiating stimulus remain unknown. Erosion on two fronts may speed up remodeling. It remains to be determined whether the principles of mechanically regulated forces can be invoked [35] to explain the drift and direction of drift of H osteons created during cortical bone remodeling in children. One would have to postulate a strain energy density gradient across the growing cortex that results in lower strain energy density surrounding the marrow-facing half of the resorption cavity where resorption progresses than in the periosteum-facing half of the cavity where new bone is forming [35]. It remains unclear however how the direction of drift, namely toward the marrow cavity can be the same in both cortices despite their structural and modeling differences. With cessation of growth and deposition of primary bone after age 15 “giant” resorption cavities were no longer created as was reflected in the decline in H eroded surface, canal diameter and cortical porosity. Therefore the need to repair cavities by asymmetric H osteons fell away and concentric H osteons could now form. The int cortex always appeared more mature than the ext cortex because of a somewhat higher proportion of concentric osteons. It may be argued

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that in the int cortex concentric osteons could form earlier than in the ext cortex because it contained less primary bone and hence fewer subsequent “giant” resorption cavities that needed to be repaired by asymmetric H osteons. Asymmetric H osteons were the predominant osteonal shape during the years of highest fracture incidence. This suggests that their configuration may be structurally inferior to mature concentric H osteons and that this may contribute to bone fragility in children. Phase 3 Concentric mature H osteons with a central canal were few before the mid-teens but became the predominant form from the late teens onwards after asymmetric osteons had been remodeled. Lower canal number in older B children means fewer H cement lines which are considered potential micro-crack initiators [36]; their lower number in B may be beneficial for bone strength relative to W children. Bone fragility Since bone strength diminishes as a power of increasing cortical porosity [37] high cortical porosity in children must be assumed to be a strong contributory factor to increased fracture risk. Indeed, most fractures in children occur in the 6 to 15 years time span of highest cortical porosity [38]. However, peak fracture incidence in B and W children falls into the latter half of this period, namely around 11 years in girls and 14 years in boys [5,38–40] because of the timing of several maturational events. Peak fracture incidence and peak height velocity with its attendant increase in body mass fall within one year [38]. Although peak bone area velocity also occurs at about this time, peak bone mineral content velocity (PBMCV) lags behind these developments by about 6 months [41]. This lag constitutes a critical period of predisposition to fracture as a body of increasingly adult proportions has to be supported by a child's bones. The lag in PBMCV is explained by large canal size and high cortical porosity, i.e. the large remodeling space. The closure, or consolidation of this remodeling space took place after age 15 in both B and W children in the present study as was reflected in declining H eroded surface, canal diameter and cortical porosity (Fig. 1), and coincided with the change from asymmetric to concentric H systems. Bone weakness in children is thus likely to be contributed to by high porosity, large canals, and possibly by asymmetry of H osteons. The lower cortical porosity in the ext cortex of B children aged 6 to 15 years compared to W was statistically small (p = 0.059) but may be biomechanically significant on account of the inverse power relationship between porosity and bone strength. Thus the trend to lower cortical porosity at that age in B children, together with greater cortical thickness and lower canal number after age 11 may underlie the lower fracture rates in B compared to W children. Limitations of this study are the small number of W subjects in the 6 to 11 year age group. Uneven gender distribution among age groups precluded gender specific analyses. We had no information on body weight, height or pubertal development, and the use of cadaver specimens meant lack of medical histories. Tetracycline based data in W children would have aided in the interpretation of greater osteoid thickness in B children. In summary this study has shown that the effects of growth, expressed in differences between external and internal cortex were similar in B and W children. The dominant age effects in both racial groups were increasing cortical thickness and high bone turnover. The latter resulted in high cortical porosity which was greatest between ages 6 and 15 with a tendency to be lower in B. Together with greater osteoid thickness, and after age 11 greater Ec wall thickness, greater cortical thickness and lower canal number these features may contribute to lower fracture rates in B children. Differing environmental influences and possibly genetic effects may play a role.

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