Abstract
This study has established the normal reference intervals for bone histomorphometric measurements derived from healthy premenopausal women, which is rarely available. We presented the static and dynamic bone histomorphometric data from trans-iliac bone biopsies in 62 healthy premenopausal women (19 blacks and 43 whites, ages 20–53 years). There were no significant differences in age and BMI between black and white women. Since there was no significant difference in bone remodeling between the two ethnic groups, we pooled data of all 62 premenopausal women to establish normal reference intervals for bone histomorphometry. The results provide normal reference intervals for both static and dynamic histomorphometric variables in cancellous and cortical bone of the ilium. None of the bone remodeling-related variables correlated with age or BMI. This study provides reference intervals for bone histomorphometric measurements in both cancellous and cortical bone of the ilium, which would be helpful in the evaluation of bone health in women.
Keywords: Histomorphometry, Reference intervals, Premenopausal women, Cancellous bone, Cortical bone
INTRODUCTION
Histomorphometric examination of transiliac bone biopsies after in vivo double fluorochrome labeling can provide critical information about the status of iliac bone remodeling and modeling, and its microarchitecture simultaneously [1, 2]. In humans, anterior-superior part of the ilium is selected as the site for bone biopsy, because of its easy accessibility with minimal or no adverse effects and similarity of microarchitecture to the vertebral body which is predisposed to osteoporosis [1, 3, 4]. Iliac bone histomorphometry is the principle method of identifying mineralization defects and tissues level bone turnover [5–8]. In addition, bone histomorphometry can be used to assess the therapeutic and adverse effects of new and existing therapeutic agents [9–12].
However, comparison with “normal” histomorphometric data is both critical and essential to understand the bone status affected by diseases and/or treatment [13, 14]. Accordingly, reliable reference intervals for bone histomorphometric data from healthy populations is very important for proper interpretation of the results from individual patients. We and others have reported histomorphometric normative data pertaining to the mature and aging skeleton [2, 15–17], but the data for normal reference intervals are scarce. Melsen et al [13] reported normative values rather than reference intervals for iliac bone histomorphometry measured from 41 subjects that included 12 men and 29 women with different menstrual status. Recker et al [14, 18, 19] reported bone histomorphometric reference intervals for healthy premenopausal women, healthy postmenopausal women, and elderly men. These studies only focused on cancellous bone.
Although bone histomorphometric reference intervals from healthy postmenopausal women or elderly men are useful to determine the status of bone health in subjects with similar demographic characteristics, they may not represent normality because older people are more likely to suffer from undiagnosed diseases that potentially affect bone and mineral metabolism. In addition, estrogen status of pre and post-menopausal women differs vastly and significantly, and estrogen deficiency in postmenopausal women results in bone loss due to an increase in remodeling rate, in which the increase in bone resorption exceeds the increase in formation [19]. Therefore, the normal bone histomorphometric reference intervals for women should ideally be obtained from healthy premenopausal women. In the present study, we developed a set of normal reference intervals for bone histomorphometric data, which were derived from iliac bone biopsies from 62 healthy premenopausal volunteers recruited in a study that was solely for the purpose of investigating postmenopausal osteoporosis.
MATERIALS AND METHODS
Subjects
Sixty-two premenopausal women, 19 blacks and 43 whites, were recruited between 1981 and 1993 as part of a larger study of the effect of age and menopause on bone structure and remodeling, the details of which were reported previously [20]. All women were menstruating regularly and determined to be skeletally healthy, and had normal physical examination by the senior author (DSR), who performed all trans-iliac bone biopsies. They had normal laboratory values for liver and renal function, normal thyroid and parathyroid function. They also had normal spinal radiographs and normal values for forearm bone mineral density. In each subject, standing shoeless height (ht) was measured with the Harpenden stadiometer and clothed weight (wt) with a standard clinical beam scale. Body mass index (BMI) was calculated as wt/ht2 (kg/m2). The study was approved by Institutional Review Board of the Henry Ford Hospital and a written informed consent was provided by all the participants.
Bone Histomorphometry
Before biopsy, all subjects received in vivo double tetracycline labeling with an inter-label interval time of 14 days (3 days ON - 11 days OFF - 3 days ON). A cylindrical trans-iliac bone biopsy core with intact and unfragmented cortices was obtained using a 7.5 mm trephine. All bone biopsies in our series contained two intact cortices. The samples were processed, embedded, sectioned, stained and examined as previously reported [2, 20]. Briefly, in two serial 5 μm sections obtained from one region of the biopsy specimen, the static variables were measured in a section stained with modified toluidine blue method using bright-light microscopy and dynamic variables were measured in an unstained section using fluorescent (UV light) microscopy. All Bone histomorphometric variables were designated in accordance with the nomenclature recommended by the American Society for Bone and Mineral Research (ASBMR) [1].
The parameters related to bone structures included total bone volume per tissue volume (BV/TV, %), trabecular thickness (Tb.Th, μm) and number (Tb.N, #/mm2), and cortical thickness (Ct.Th, μm). Tb.Th and Tb.N were calculated indirectly from the bone surface to volume ratio and BV/TV. Static and remodeling indices were measured separately on the cancellous, intracortical and endocortical surfaces. The static indices included osteoid and eroded surfaces as a fraction of bone surface (OS/BS, % and ES/BS, %); wall thickness (W.Th, μm); the average distance between the cement line and the quiescent bone surface); and osteoid thickness (O.Th, μm), measured directly on the bone surface with osteoid. The surface lengths covered by osteoblasts and osteoclasts (Ob.S and Oc.S) were measured separately and expressed as a fraction of bone surface (Ob.S/BS, % and Oc.S/BS, %), as well as a fraction of osteoid surface for Ob.S (Ob.S/OS, %).
The dynamic remodeling indices were also measured separately on the cancellous, intracortical and endocortical surfaces. The double and single tetracycline-labeled surfaces represented the extent of bone surface where mineralization was in progress (mineralizing surface (MS) = (length of 1st label + length of 2nd label)/2 during the period of tetracycline administration, from which the MS as a fraction of total bone and of osteoid surfaces (MS/BS, % and MS/OS, %) was calculated. Mineral apposition rate (MAR, μm/day) was obtained from the average distance between the two tetracycline labels divided by the interval of administration (14 days in our study). Adjusted apposition rate (Aj.Ar, μm/day), osteoid maturation time (Omt,), mineralization lag time (Mlt,) and formation period (FP,) were calculated as MAR*MS/OS, O.Th/MAR, O.Th/Aj.AR and W.Th/Aj.AR, respectively. Bone formation rate at the surface level (BFR/BS, μm3/μm2/year) was calculated as MAR*(MS/BS). Activation frequency (Ac.f, #/year), the annual probability of activation of a new remodeling site at any given locus on the bone surfaces, was derived from 1/Total Period (1/Tt.P). Total period includes resorption, reversal and formation periods. For the surface containing only a single label, a minimum value of 0.3 μm/day was assigned to MAR [21, 22]. This number was corrected for section obliquity by multiplying by π/4, which yields a minimum value of 0.236 μm/day for MAR [2]. If no label was present, MAR, Aj.Ar, Omt, Mlt and FP were treated as a missing value, whereas MS/BS, BFR/BS, BFR/BV and Ac.f were assigned a zero [2].
Distances, areas, and perimeters were measured separately in both cancellous and cortical regions and on cancellous, intracortical and endocortical surfaces. The data were collected and analyzed in cancellous and cortical bone. For cortical bone, the data were present for intracortical and endocortical bone envelope separately as well as combined. The total tissue areas for histomorphometry measurements ranged from 29 to 73 mm2. The cancellous bone areas were 2.5–15 mm2 and cortical bone areas were 7.3 – 28 mm2. The surface lengths were 35–154 mm for cancellous envelope, 11–74 mm for intracortical envelope and 7.6–18.6 mm for endocortical envelope. The histological material in bone sections was sufficient for the studies on bone histomorphometry [2, 20].
Statistics
The age, height, weight and body mass index (BMI) were compared between black and white subjects using student t tests. Mann–Whitney test was used when a variable was not normally distributed. Correlations of age and BMI with each histomorphometric variable were analyzed by nonparametric Spearman rank correlation tests.
The reference intervals were calculated using a robust method following Box-Cox transformation of data. The upper and lower end points covering 95% of the reference value of the analytes were determined with their respective 90% confidence intervals (CIs). The D’Agostino-Pearson (DAP) test was used to assess normality of distributions of histomorphometric values.
RESULTS
Clinical data
Demographic data for the cohort are shown in Table 1. The mean age of premenopausal women was 37.1±7.37 years, ranging between 20 and 53 years and all were still regularly menstruating. The mean BMI was 26.5±7.42. The age, height, weight and BMI were similar in black and white women without significant differences (Table 1).
Table 1.
Demographic data for the cohort
| Number | Age (years) | Height (cm) | Weight (kg) | BMI (kg/m2) | |
|---|---|---|---|---|---|
| All | 62 | 37.1 (7.37) | 164.0 (6.30) | 71.5 (21.3) | 26.5 (7.42) |
| Black | 19 | 36.5 (6.33) | 164.2 (5.88) | 71.5 (15.5) | 26.5 (5.22) |
| White | 43 | 37.3 (7.83) | 163.0 (6.54) | 71.5 (23.6) | 26.5 (8.28) |
| p | NS | NS | NS | NS |
Data expressed as mean (SD); p values: Black vs White
Correlations between bone histomorphometric variables and age and BMI
All the correlations with age and BMI were modest, and none would be statistically significant if a strict multiple comparisons correction was applied. There was a modest positive correlation between age and BMI in the entire cohort (r = 0.259, p = 0.04; Table 2). However, none of the p-values for BMI correlations were below 0.05 for any of the histomorphometric variables in either cancellous or cortical bone. There were modest inverse correlations between age and BV/TV in both cancellous and cortical bone (r = 0.274 and r = 0.275, respectively, both p = 0.031). The nominally strongest correlation was between age and Tb.N in cancellous bone (r = −0.331, p = 0.009).
Table 2.
Correlations between bone histomorphometric variables and age and BMI
| Cancellous bone | Cortical bone | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Age | BV/TV | OV/BV | Tb.Th | Tb.N | W.Th | OS/BS | Age | BV/TV | OV/BV | Ct.Th | W.Th | OS/BS | ES/BS | ||
| (year) | (%) | (%) | (μm) | (#/mm) | (μm) | (%) | (year) | (%) | (%) | (μm) | (μm) | (%) | (%) | ||
| BMI | 0.259* | 0.141 | −0.102 | 0.231 | 0.007 | 0.014 | 0.052 | BMI | 0.259* | −0.217 | 0.163 | −0.004 | 0.0635 | 0.133 | 0.046 |
| Age | −0.274* | 0.019 | −0.081 | −0.331** | −0.074 | 0.132 | Age | −0.275* | 0.176 | −0.115 | −0.074 | 0.085 | −0.043 | ||
| ES/BS | Ob.S/BS | MS/BS | Oc.S/BS | O.Th | MAR | Aj.AR | Ob.S/BS | MS/BS | Oc.S/BS | O.Th | MAR | Aj.AR | Omt | ||
| (%) | (%) | (%) | (%) | (μm) | (μm/day) | (μm/day) | (%) | (%) | (%) | (μm) | (μm/day) | (μm/day) | (days) | ||
| BMI | −0.021 | 0.103 | −0.022 | 0.213 | −0.077 | −0.114 | −0.109 | BMI | 0.099 | −0.105 | 0.143 | 0.066 | 0.001 | −0.093 | 0.031 |
| Age | 0.142 | 0.059 | 0.086 | 0.160 | −0.160 | −0.064 | −0.062 | Age | −0.075 | −0.057 | −0.066 | −0.064 | 0.120 | −0.120 | −0.112 |
| −0.075 | −0.057 | −0.066 | −0.064 | 0.120 | −0.12 | −0.112 | |||||||||
| Omt | MLT | FP | BFRs | BFRv | Ac.f | MLT | FP | BFRs | BFRv | Ac.f | |||||
| (days) | (days) | (days) | (μm3/μm2/y) | %/year | #/year | (days) | (days) | (μm3/μm2/y) | %/year | #/year | |||||
| BMI | 0.045 | 0.114 | 0.104 | −0.066 | −0.132 | −0.069 | BMI | 0.130 | 0.113 | −0.105 | −0.050 | −0.112 | |||
| Age | −0.111 | 0.057 | 0.066 | 0.036 | 0.056 | 0.045 | Age | 0.123 | 0.078 | −0.049 | 0.072 | −0.020 | |||
Reference intervals of bone histomorphometric variables
Of the 62 biopsy specimens, 60 had double and 2 had only single tetracycline labeling in cancellous bone. In cortical bone, 59 subjects had double labeling, 2 had only single labeling and 1 had no labeling. In the entire cohort, 58 subjects had double labeling in both cancellous and cortical bone. Fluorochrome labeling of some form was found in either cortical or trabecular bone of each specimen. The reference intervals for various histomorphometric measurements in cancellous and cortical bone are shown in Table 3 and Table 4, respectively, including the values for median, mean, 95% reference interval, and the lower and upper bounds for the 95% confidence intervals. Furthermore, the reference intervals for histomorphometric measurements in cortical bone are present for intracortical and endocortical envelope separately (Table 5 & 6).
Table 3.
Histomorphometric reference intervals in cancellous envelope
| Variable | Median | Mean | 95% Reference interval | Lower Limit | Upper Limit |
|---|---|---|---|---|---|
| 90% CI | 90% CI | ||||
| BV/TV (%) | 24.9 | 25.0 | 13.4–42.5 | 12.0; 15.0 | 39.0; 45.9 |
| OV/BV (%) | 1.46 | 1.53 | 0.259–5.48 | 0.172; 0.393 | 4.43; 6.65 |
| Tb.Th (μm) | 139 | 142 | 98.8–217 | 93.4; 106 | 197; 238 |
| Tb.N (#/mm) | 1.70 | 1.72 | 1.18–2.56 | 1.11; 1.26 | 2.38; 2.73 |
| W.Th (μm) | 36.2 | 36.4 | 29.5–45.4 | 28.5; 30.6 | 43.3; 47.4 |
| OS/BS (%) | 11.1 | 12.2 | 2.39–39.7 | 1.70; 3.38 | 32.0; 47.6 |
| ES/BS (%) | 5.91 | 6.07 | 1.24–14.5 | 0.796; 1.82 | 12.7; 16.1 |
| Ob.S/BS (%) | 3.54 | 3.55 | 0.107–14.1 | 0.009; 0.361 | 11.3; 17.1 |
| MS/BS (%) | 5.24 | 4.97 | 0.541–16.4 | 0.253; 1.05 | 13.9; 19.1 |
| Oc.S/BS (%) | 0.460 | 0.524 | 0.008–3.45 | 0.001; 0.026 | 2.49; 4.50 |
| O.Th (μm) | 8.84 | 8.83 | 5.69–13.8 | 5.29; 6.17 | 12.7; 14.9 |
| MAR (μm/day) | 0.586 | 0.576 | 0.295–0.873 | 0.228; 0.364 | 0.807; 0.929 |
| Aj.AR (μm/day) | 0.257 | 0.243 | 0.024–0.708 | 0.010; 0.047 | 0.616; 0.792 |
| Omt (days) | 15.3 | 15.6 | 8.67–33.3 | 7.87; 9.69 | 27.6;40.0 |
| MLT (days) | 35.7 | 37.4 | 13.2–277 | 11.6; 15.4 | 158; 549 |
| FP (days) | 142 | 151 | 53.7–1381 | 47.8; 62.2 | 733; 3088 |
| BFRs (μm3/μm2/year) | 11.0 | 10.6 | 0.755–34.8 | 0.153; 1.99 | 29.7; 40.6 |
| BFRv (%/year) | 12.2 | 11.7 | 0.696–39.8 | 0.176; 1.81 | 33.8; 46.4 |
| Ac.f (#/year) | 0.319 | 0.292 | 0.020–0.933 | 0.004; 0.056 | 0.799; 1.08 |
Table 4.
Histomorphometric reference intervals in combined cortical envelope
| Variable | Median | Mean | 95% Reference interval | Lower Limit | Upper Limit |
|---|---|---|---|---|---|
| 90% CI | 90% CI | ||||
| BV/TV (%) | 95.9 | 95.5 | 91.4–100 | 89.9; 93.0 | 98.8; 102 |
| OV/BV (%) | 0.438 | 0.434 | 0.077–1.46 | 0.048; 0.119 | 1.20; 1.75 |
| Ct.Th (μm) | 1.34 | 1.36 | 0.775–2.28 | 0.701; 0.865 | 2.08; 2.49 |
| W.Th (μm) | 39.1 | 40.1 | 27.1–69.9 | 25.6; 28.5 | 62.6; 78.7 |
| OS/BS (%) | 14.2 | 13.6 | 2.98–34.3 | 1.94; 4.49 | 29.8; 39.0 |
| ES/BS (%) | 4.80 | 5.02 | 1.23–14.1 | 0.892; 1.69 | 11.8; 16.3 |
| Ob.S/BS (%) | 4.15 | 4.06 | 0.163–12.8 | 0.000; 0.581 | 11.1; 14.6 |
| MS/BS (%) | 7.43 | 7.07 | 0.550–19.9 | 0.131; 1.30 | 17.2; 22.4 |
| Oc.S/BS (%) | 0.386 | 0.361 | 0.000–2.46 | 0.000; 0.008 | 1.96; 3.02 |
| O.Th (μm) | 9.03 | 9.02 | 5.56–13.6 | 5.07; 6.12 | 12.6; 14.6 |
| MAR (μm/day) | 0.625 | 0.628 | 0.281–0.925 | 0.207; 0.355 | 0.871; 0.975 |
| Aj.AR (μm/day) | 0.293 | 0.306 | 0.071–1.11 | 0.049;0.100 | 0.859;1.43 |
| Omt (days) | 14.6 | 14.7 | 8.72–33.4 | 8.07; 9.52 | 27.2; 42.1 |
| MLT (days) | 31.3 | 30.2 | 8.73–106 | 6.74; 11.4 | 78.4; 142 |
| FP (days) | 125 | 133 | 35.2–618 | 26.9; 46.7 | 399; 949 |
| BFRs (μm3/μm2/year) | 17.6 | 15.9 | 0.492–56.4 | 0.009; 1.91 | 47.6; 65.1 |
| BFRv (%/year) | 4.48 | 4.23 | 0.102–16.6 | 0.001; 0.425 | 13.7; 19.7 |
| Ac.f (#/year) | 0.430 | 0.398 | 0.007–1.43 | 0.000; 0.038 | 1.21; 1.66 |
Table 5.
Histomorphometric reference intervals in intracortical envelope
| Variable | Median | Mean | 95% Reference interval | Lower Limit | Upper Limit |
|---|---|---|---|---|---|
| 90% CI | 90% CI | ||||
| W.Th (μm) | 37.5 | 40.0 | 25.3–76.6 | 24.1; 26.9 | 67.0; 86.4 |
| OS/BS (%) | 13.5 | 12.5 | 1.83–33.1 | 0.904; 3.24 | 28.5; 37.5 |
| ES/BS (%) | 3.40 | 3.60 | 0.222–12.2 | 0.067; 0.531 | 9.79; 14.8 |
| Ob.S/BS (%) | 3.50 | 3.14 | 0.003–15.7 | 0.000; 0.169 | 13.4; 18.9 |
| MS/BS (%) | 6.34 | 6.55 | 0.391–22.3 | 0.119; 0.896 | 18.5; 26.1 |
| Oc.S/BS (%) | 0.364 | 0.217 | 0.000–0.332 | 0.000; 0.001 | 2.58; 4.10 |
| O.Th (μm) | 8.95 | 8.92 | 4.31–14.4 | 3.62; 5.12 | 13.3; 15.5 |
| MAR (μm/day) | 0.623 | 0.608 | 0.228–0.993 | 0.158; 0.310 | 0.934; 1.05 |
| Aj.AR (μm/day) | 0.321 | 0.325 | 0.042–1.10 | 0.022; 0.076 | 0.910; 1.32 |
| Omt (days) | 14.5 | 14.8 | 8.38–37.9 | 7.76; 9.18 | 29.8; 49.7 |
| MLT (days) | 28.5 | 28 | 8.17–150 | 6.71; 10.3 | 94.9; 253 |
| FP (days) | 123 | 128 | 34.6–886 | 28.1; 43.0 | 531; 1603 |
| BFRs (μm3/μm2/year) | 16.3 | 14.3 | 0.331–63.1 | 0.037; 1.22 | 51.5; 74.9 |
| BFRv (%/year) | 3.01 | 2.66 | 0.050–12.9 | 0.005; 0.197 | 10.4; 15.6 |
| Ac.f (#/year) | 0.415 | 0.357 | 0.007–1.60 | 0.000; 0.027 | 1.31; 1.91 |
Table 6.
Histomorphometric reference intervals in endocortical envelope
| Variable | Median | Mean | 95% Reference interval | Lower Limit | Upper Limit |
|---|---|---|---|---|---|
| 90% CI | 90% CI | ||||
| W.Th (μm) | 39.8 | 40.4 | 28.9–60.0 | 27.4; 30.5 | 55.8; 65.1 |
| OS/BS (%) | 13.2 | 14.9 | 2.06–49.2 | 1.28; 3.28 | 41.3; 57.3 |
| ES/BS (%) | 7.75 | 7.70 | 1.18–25.3 | 0.767; 1.77 | 21.5; 29.3 |
| Ob.S/BS (%) | 3.86 | 3.95 | 0.013; 20.8 | 0.000; 0.160 | 16.9; 24.7 |
| MS/BS (%) | 6.33 | 5.68 | 0.011–30.9 | 0.000; 0.239 | 25.3; 37.6 |
| Oc.S/BS (%) | 0.270 | 0.703 | 0.000–2.580 | 0.000 | 0.000, 3.36 |
| O.Th (μm) | 8.76 | 8.35 | 4.21–15.4 | 3.79; 4.79 | 14.0; 16.7 |
| MAR (μm/day) | 0.578 | 0.557 | 0.215–0.928 | 0.147; 0.297 | 0.863; 0.100 |
| Aj.AR (μm/day) | 0.246 | 0.24 | 0.017–0.831 | 0.006; 0.037 | 0.700; 0.970 |
| Omt (days) | 16.5 | 15.7 | 7.58–41.8 | 6.67; 8.85 | 34.3; 51.2 |
| MLT (days) | 35.8 | 36.4 | 11.7–369 | 10.2; 13.7 | 189; 886 |
| FP (days) | 157 | 166 | 49.3–2766 | 42.9; 57.8 | 1071; 10546 |
| BFRs (μm3/μm2/year) | 14.9 | 11.2 | 0.001–75.9 | 0.000; 0.316 | 61.0–91.0 |
| Ac.f (#/year) | 0.366 | 0.291 | 0.000–1.82 | 0.000; 0.007 | 1.48; 2.23 |
DISCUSSION
Availability of appropriate reference intervals for any quantitative laboratory measure is an indispensable method for medical diagnostics, clinical practice and for monitoring interventions. Comparison of a measurement with its reference interval is a commonly accepted approach for clinical interpretation of laboratory data [23]. There are very few studies on the reference intervals for bone histomorphometry. Until recently, the reference interval values of bone histomorphometry with tetracycline-based dynamic variables have been reported in postmenopausal women [18], premenopausal women [19], elderly men over 45 years [14] and growing children [24]. However, bone histomorphometric values obtained from healthy postmenopausal women may not truly represent the normal status of bone morphology or remodeling due to the effects age and menopause [2, 17, 18, 20]. Bone histomorphometric values obtained from otherwise healthy postmenopausal women may represent “normal postmenopausal ranges”, but many variables, such as OV/BV, ES/BS, MS/BS, MAR, BFR/BS and Ac.f, etc., differ substantially from normal values for premenopausal women [18, 19]. Thus, histomorphometric values obtained from postmenopausal women are not ideal to establish reference intervals. We believe that the subjects enrolled in the present study is a reliable cohort representing the normal U.S. premenopausal women in their 3rd, 4th and 5th decades. The healthy skeleton was determined by the criteria as previously reported [25, 26]. We believe that the reference intervals for bone histomorphometry in our study can be used as reference standard to evaluate women with bone disorders and/or taking medications known to affect bone biology.
In the present study, which included black and white premenopausal women, there were no significant differences in age, height, weight and BMI between the two ethnic groups. The bone histomorphometric measurements in these subjects were reported previously [2, 17, 20]; the results showed that there was no significant difference in the vast majority of variables between black and white premenopausal women, particularly bone remodeling-related variables such as BFR/BS and Ac.f. Parisien et al [27] also reported that there were no significant differences in bone microarchitecture and remodeling in iliac bone biopsies between black and white premenopausal women. Accordingly, we deem that bone histomorphometric data of black and white premenopausal women can be combined to establish reference intervals. Correlation analyses for age, BMI and bone histomorphometric variables in this cohort indicate that both cancellous and cortical bone volumes decrease with aging, suggesting that age-related bone loss would occur in women before menopause. Nevertheless, neither age nor BMI affect cancellous and cortical bone remodeling and mineralization in premenopausal women.
Traditionally, cancellous bone captured investigators’ attention because disorders in cancellous bone remodeling are commonly responsible for the vast majority of metabolic, genetic, and acquired bone diseases in adults [28]. Thus, the reference intervals of bone histomorphometry are mostly reported for cancellous bone [14, 18, 24]. There is strong evidence that cortical bone plays an important role in maintaining bone strength [29, 30]. Disorders of cortical bone remodeling may compromise bone quality and quantity, leading to loss of bone strength [31–33]. In addition, certain changes in bone remodeling are probably dependent on the macroanatomic type of bone [34, 35]; remodeling in cancellous bone may not represent the status in cortical bone. Thus, excluding cortical bone from analysis may limit the understanding of bone characteristics. To the best of our knowledge, this is the first study to establish normal histomophometric reference intervals for women’s cortical bone.
Bone histomorphometry is a valuable and well-established clinical and research tool for the diagnosis of various metabolic bone diseases, though not osteoporosis, and evaluation of the effects of therapeutic intervention. It provides quantitative information on bone turnover (or remodeling) and microarchitecture [1]. Some bone remodeling theories propose that both extreme high and low bone turnover would increase the risk of fractures [36, 37]. Our previous studies make us concern about the effects of low bone turnover [38]. It is well known that many pathological conditions and medications can suppress bone turnover to extremely low levels, resulting in accumulation of aged bone with compromised material properties [39, 40]. Two specific conditions are associated with extremely low bone turnover: the adynamic bone disease (ABD) in patients with chronic kidney disease and severely suppressed bone turnover (SSBT) in patients after treatment with antiresorptive agents such as bisphosphonates and denosumab [8, 12, 38, 41–43]. However, it is uncertain what degree of bone turnover should be regarded as extremely low. Normal reference intervals of bone histomorphometry is one method to provide boundaries between normal and abnormal bone turnover. Either activation frequency (Ac.f) or bone formation rate (BFR/BS) has been used to estimate bone turnover rate [1, 44, 45]. We propose that bone turnover below the lower limit of the reference interval can be regarded as “extremely low” in the absence of disease and intervention or as “severely suppressed” when caused by diseases such as in ABD or hypoparathyroidism [8, 43, 46] or by medical intervention [12, 38]. Such an approach would encompass both the common occurrence of extremely low bone turnover in otherwise healthy individuals and that caused by diseases or intervention. This will leave the door open for future research to decide whether all three conditions, extremely low bone turnover in untreated individuals, ABD in patients with chronic kidney disease, and SSBT in patients after antiresorptive therapy, are similar or different. In addition, it is important to include cortical bone in the histomorphometric analysis because cortical bone comprises over 80% of the entire skeleton and is the major structural determinant of fracture risk at most skeletal sites [47].
The reference intervals in clinical medicine are a set of values within a certain percentile range (e.g. 2.5–97.5 percentile), which are obtained from an ostensibly healthy population. This reference interval is then used in all medical and therapeutic decision-making. How to obtain appropriate reference intervals depends on the sample size and types of statistics used for analyses. Nonparametric methods are used when >120 samples are available [48]. When the sample size is <120 but >40, a robust method is recommended [48, 49].
The major limitation of this study is a small sample size. Based on the literature and our own experience, we believe it is virtually impossible to obtain >120 samples to establish reference intervals for bone histomorphometry because it is difficult to get such a large number of premenopausal women who are both healthy and willing to donate iliac bone biopsies [14, 18, 24]. Therefore, a robust method was used for statistical analysis in this study. Another limitation is the lack of hip and spine BMD data in this study. Since the iliac bone biopsies were collected between 1989 and 1992. At that time Dual-energy x-ray absorptiometry (DXA) was not available to measure spine and hip bone density at our institution.
In conclusion, we have established reference intervals for bone histomorphometric data based on healthy black and white premenopausal women. The normal reference intervals reported here are appropriate for evaluating the skeletal health in individual woman with or without disease or intervention.
Acknowledgments
GRANT SUPPORT
Research reported in this publication was supported by the National Institutes of Health (AG10381 and AG/AR13918) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (AR062103).
Footnotes
COMPLIANCE WITH ETHICAL STANDARDS
The study was conducted in accordance with the ethical standards of the institutional research committee of Henry Ford Hospital and the 1964 Helsinki Declaration and its later amendments.
CONFLICTS OF INTEREST
Shijing Qiu, George Divine, Elizabeth Warner and Sudhaker Rao declare that they have no conflict of interest.
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