Comparative effects of teriparatide and strontium ranelate in the periosteum of iliac crest biopsies in postmenopausal women with osteoporosis

Bone 48 (2011) 972–978

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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 e v i e r. c o m / l o c a t e / b o n e

Comparative effects of teriparatide and strontium ranelate in the periosteum of iliac crest biopsies in postmenopausal women with osteoporosis☆,☆☆ Yanfei L. Ma a, Fernando Marin b, Jan Stepan c, Sophia Ish-Shalom d, Rüdiger Möricke e, Federico Hawkins f, Georgios Kapetanos g, María P. de la Peña h, Jörn Kekow i, Guillermo Martínez f, Jorge Malouf j, Qing Q. Zeng a, Xiaohai Wan a, Robert R. Recker k,⁎ a

Lilly Research Laboratories, Indianapolis, USA Lilly Research Centre, Windlesham, UK c Institute of Rheumatology, Charles University Faculty of Medicine, Prague, Czech Republic d Bone and Mineral Metabolism Unit, Rambam Health Care Campus, Technion Faculty of Medicine, Haifa, Israel e Endocrinology Clinical Research Laboratory, Magdeburg, Germany f Department of Endocrinology, Hospital 12 de Octubre, Madrid, Spain g Department of Orthopedic Surgery, Aristotelion University, Thessaloniki, Greece h Clínica Médica Monraz, Guadalajara, Mexico i Department of Rheumatology, Speciality Hospital, Vogelsang-Gommern, Germany j Department of Internal Medicine, Hospital Santa Creu i Sant Pau, Barcelona, Spain k Osteoporosis Research Centre, Creighton University, Omaha, Nebraska, USA b

a r t i c l e

i n f o

Article history: Received 19 August 2010 Revised 10 January 2011 Accepted 12 January 2011 Available online 22 January 2011 Edited by: R. Baron Keywords: Bone biopsy Osteoporosis Periosteum Strontium Teriparatide

a b s t r a c t The periosteum contains osteogenic cells that regulate the outer shape of bone and contribute to determine its cortical thickness, size and position. We assessed the effects of subcutaneous injections of teriparatide (TPTD, 20 μg/day) or oral strontium ranelate (SrR, 2 g/day) in postmenopausal women with osteoporosis on new bone formation activity at the periosteal and endosteal bone surfaces using dynamic histomorphometric measurements. Evaluable tetracycline-labeled transiliac crest bone biopsies were analyzed from 27 patients in the TPTD group, and 22 in the SrR group after six months of treatment. Measurements were conducted on the thicker and thinner cortices separately, and comparisons between the thicker, thinner and combined cortices were carried out. At the combined periosteal cortex, the mineralization surface as a percent of bone surface (MS/BS%) was greater for TPTD (mean± SE: 8.08± 1.22%) than SrR (3.22 ± 1.05%) (p b 0.005). The difference in mineral apposition rate (MAR) between TPTD (0.35 ± 0.06 μm/day) and SrR (0.14 ± 0.06 μm/day) was also significant (p b 0.05), while that of bone formation rate per bone surface (BFR/BS) between TPTD (0.014 ± 0.004 mm3/mm2/ year) and SrR (0.004± 0.003 mm3/mm2/year) was not (p = 0.057). Statistically significant differences between the two treatments were also observed for MS/BS%, BFR/BS, MAR and the double-labeled perimeter in the periosteum of the thicker, but not thinner, iliac crest cortices. The comparison between the thicker and thinner cortices of both periosteal and endosteal surfaces showed statistically significant differences for MAR and the double-labeled perimeter for TPTD treated women. There were no statistically significant differences in any bone formation dynamic measurements between the two cortices in the SrR group. In conclusion, most of the bone formation and mineralization variables were significantly higher for TPTD- than SrR-treated women at both the periosteal and endosteal combined cortices. The response to TPTD for dynamic bone formation measurements in the periosteal surface was greater for the thicker than thinner cortex, but this difference was not significant in SrR treated patients. This may reflect a greater ability of TPTD to enhance responsiveness of bone to the mechanical loading environment. These effects on bone formation may underlie the improvement in bone quality in patients with osteoporosis treated with TPTD. © 2011 Elsevier Inc. All rights reserved.

☆ Trial Registration: clinicaltrials.gov identifier NCT00239629. ☆☆ Funding Sources: Funding was provided by Lilly Research Center, Europe. ⁎ Corresponding author at: Osteoporosis Research Center, Creighton University School of Medicine, 601 N. 30th St., Suite 6730, Omaha, Nebraska NE 68131, USA. Fax: +1 402 280 5034. E-mail addresses: [email protected] (Y.L. Ma), [email protected] (F. Marin), [email protected] (J. Stepan), [email protected] (S. Ish-Shalom), [email protected] (R. Möricke), [email protected] (F. Hawkins), [email protected] (G. Kapetanos), [email protected] (M.P. de la Peña), [email protected] (J. Kekow), [email protected] (G. Martínez), [email protected] (J. Malouf), [email protected] (Q.Q. Zeng), [email protected] (X. Wan), [email protected] (R.R. Recker). 8756-3282/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2011.01.012

Y.L. Ma et al. / Bone 48 (2011) 972–978

Introduction

Subjects and methods

Therapeutic drugs for the treatment of osteoporosis increase bone strength and, as a result, decrease the incidence of fragility fractures. Antiresorptive agents such as calcitonin, estrogen, selective estrogen receptor modulators, bisphosphonates or denosumab reduce bone turnover, thereby, decreasing the remodeling space and number of basic multicellular units (BMUs), resulting in a preservation of bone microarchitecture. In contrast, the so-called bone anabolic agents increase bone turnover with a greater stimulation of formation than resorption, resulting in a net positive bone balance [1,2]. Histomorphometry of human bone biopsies provides critical data for understanding the mechanisms of action of anti-osteoporosis therapies at the bone tissue level. However, until now, most histomorphometric analyses reported in humans have focused on the effects of these drugs at the cancellous and endocortical surfaces, and there is limited information on the effects of osteoporosis therapies on the periosteal surface of bone in humans. The periosteum covers the surface of most bones and lies between the cortical bone and overlying soft tissue or muscle. It contains osteoblasts and mesenchymal precursors that work in coordination with osteocytes and the inner cortical endosteum to regulate cortical thickness and bone geometry [3,4]. The estrogen deficiency that occurs during menopause is associated with increased periosteal perimeter and concomitant loss of bone from endocortical and cancellous surfaces [5,6]. As the modification of bone dimensions and geometric properties associated with periosteal expansion increase bone strength, especially at sites such as the lumbar spine and femoral neck, there is a reduction in fracture risk that can be independent of bone mineral density (BMD) [6,7]. Thus, any enhancement of this expansion could have significant clinical benefits; indeed the addition of only one-third of the amount of bone lost from the endocortical bone surface needs to be added to the periosteal surface to achieve equivalent biomechanical properties [8]. In paired biopsy studies in humans, bone anabolic agents such as teriparatide (TPTD), given as once daily injections, increased cortical bone width through preferential modeling of both endosteal and periosteal surfaces [9–12]. In vitro and in vivo animal studies have suggested that strontium ranelate (SrR) may dissociate coupled bone remodeling by stimulating bone formation and decreasing bone resorption [13–15], and periosteal expansion has been described in rats [16]. However, SrR bone anabolic effects in humans have remained controversial due to insufficient data from paired biopsy studies [17], and conflicting reports of its effects on biochemical markers of bone remodeling [18,19]. To date, there have been no published studies on the effects of SrR on the human periosteum. The two cortices of human iliac bone biopsies from postmenopausal women often differ in thickness. Based on the observation that bone shape and growth adapt to mechanical demand [20,21], it has been hypothesized that the thicker cortex may be associated with greater loading than the thinner cortex [22]. Further, pre-clinical studies have shown that bone anabolic agents such as parathyroid hormone (PTH) or prostaglandin E2 (PGE2) and mechanical loading work together to have synergistic effects on bone [23–26]. The present report is a post-hoc analysis following a previously reported study that compared the efficacy of daily TPTD or SrR on bone remodeling and histomorphometry at the trabecular and endocortical surfaces in postmenopausal women with osteoporosis [18]. The primary aim was to extend this previous in-depth study to an investigation of the effects of TPTD and SrR on dynamic histomorphometric measurements on the periosteal and endosteal surfaces in these biopsies. The secondary aim was to compare the histomorphometric bone formation and mineralization variables at the thicker and thinner cortices of the human ilium after treatment with these two therapies.

Study design

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The design and study population of this phase 4, multicenter, open-label, randomized, active-comparator study of TPTD 20 μg/day administered subcutaneously and oral SrR 2 g/day in the treatment of postmenopausal women with osteoporosis has been described previously [18]. Briefly, the study consisted of approximately a 1.5month screening phase and a 6-month treatment phase at which point bone biopsies were obtained. Patients received 1000 mg/day elemental calcium and 800 IU/day of vitamin D. The study received ethical review board approval at all sites, and all patients gave informed written consent. The study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and were consistent with good clinical practices and applicable laws and regulations. Study population Postmenopausal, ambulatory women, between 45 and 90 years of age, with osteoporosis that was defined as a T-score of −2.5 or lower for the posterior–anterior lumbar spine (L1 through L4) BMD and/or femoral neck BMD and/or total hip BMD when measured with DXA, were treated with TPTD or SrR. Patients had last menses or bilateral oophorectomy at least 5 years before entry, no severe or chronically disabling conditions other than osteoporosis, and normal or clinically insignificantly abnormal laboratory values, including serum calcium, PTH (1–84), and total alkaline phosphatase. Other exclusion criteria have been described previously [18]. Bone histomorphometry Transiliac crest bone biopsies were obtained using a Bordier needle or similar large bore (6–8 mm) trephine system between 4 and 6 days after in vivo double tetracycline labeling in a 2:12:2 day sequence. Because SrR can form complexes with oral tetracyclines at the gastrointestinal level and reduce their absorption, the administration of SrR and TPTD (as the comparator) was stopped during the tetracycline labeling days. Biopsy specimens were prepared for histological sections by the Osteoporosis Research Center's standard operational procedures [18]. For the current study, one section with Goldner's stain and one unstained section used in our previous analyses [18] were re-used for bright field or fluorescence microscopic analyses. All 49 biopsies analyzed for bone mineralization and dynamic parameters of bone formation in the previous analysis were included in the current study, even though some specimens only exhibited the fluorochrome label on cancellous surfaces and not on endocortical or periosteal surfaces. If the first unstained section did not show any tetracycline fluorescence or exhibited single label only, then the second section was used for fluorochrome label measurements. Histomorphometric analyses were performed semi-automatically by a direct tracing method using a Digitizing Image Analysis System. This consisted of an epifluorescent microscope and digitizing pad (Summagraphic, Fairfield, CT, USA) coupled to a computer with histomorphometry software (KSS Scientific Consultants, Magna, UT, USA) [11]. Qualitative identification of the thicker and thinner cortices was possible in the majority of sections but where this was not possible the decision was made on quantitative measurements of cortical thickness. All measurements were done under ×100 magnification on the entire periosteal and endocortical bone envelopes on the two cortices separately. The assessments were performed by a single reader who was blinded to patients' treatment allocation (QQZ; Lilly Research Laboratories, Indianapolis, IN).

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The variables measured included endocortical and periosteal perimeters, single-label length and double-label length and width. These measurements were later used to calculate mineralizing surface as a percentage of bone surface (MS/BS%), mineral appositional rate (MAR), and bone formation rate surface-based (BFR/BS). For the specimens without single- or double-label, a zero was assigned and their respective MAR and BFR/BS were accordingly zero. No imputed values were assigned for the specimens with undetectable fluorochrome label at the cortical surfaces. To generate the combination data, raw values of bone surface lengths, single-label lengths, and double-label lengths from the thicker and thinner cortices were summed for each section. The consequent values were later used to calculate MS/BS% and BFR/BS. Inter-label width was the average value of the two cortices from each section. The measurements and calculations used for this study were in accordance with those recommended by the subcommittee on bone histomorphometry of the American Society for Bone and Mineral Research [27]. Statistical analyses Statistical analyses were based on a modified intent-to-treat (ITT) principle: the randomized and treated patients with valid biopsies were analyzed according to the assigned treatments. The sample size was calculated and chosen to show any superiority of TPTD relative to SrR for MS/BS% after 6 months of treatment as previously described [18]. For the comparison between the two treatment arms, bone histomorphometric results were analyzed using a one-way ANOVA model with a fixed effect for assigned treatment. As a safeguard against potential non-normality of the data, the predefined primary statistical analysis used an exact permutation test using Monte Carlo simulation to derive the p-value for the two-sample t-test. The test

was two-sided using the method of equal differences from the mean rather than doubling the one-sided p-value. The mid-p continuity correction was applied to all reported p-values. The comparison between the histomorphometric parameters of the thicker, thinner and combined cortices in both treatment groups was performed with a paired t-test. All statistical testing was two-sided, with an alpha value of 0.05. Results The baseline characteristics, therapy compliance and clinical outcomes of all randomized patients were described previously [18]. There were no significant differences between the TPTD- and SrRtreated groups at baseline in patient characteristics, including serum levels of 25-hydroxyvitamin D and endogenous PTH, BMD, or bone markers. Of the 57 biopsies obtained, 6 biopsy specimens were excluded from histomorphometric analysis because they were fragmented or had insufficient tissue (all in the SrR group) (Fig. 1). All 49 evaluable biopsy samples for mineralization and dynamic parameters of bone formation used in the previous study (27 in the TPTD group and 22 in the SrR group) were also valid for the dynamic histomorphometric measurements performed in the present study. One TPTD biopsy contained only one intact cortex, and this was assigned to the thicker cortex group. The numbers of TPTD- and SrR-treated subjects who had samples with detectable single- and double-labels are given in Table 1. The percent of specimens that exhibited double-labeling was higher in the TPTD (70%) than the SrR group (27%; p b 0.005, Fisher's exact test) at the periosteal surface. Specimens that showed single-labeling were also more frequent in the TPTD (93%) than in the SrR (64%) group (p b 0.05) at the periosteal surface. The details of the quantitative histomorphometric comparisons between the TPTD and SrR groups for bone formation and

107 subjects screened 27 screen failures 80 subjects randomized 1 randomized withdrew before receiving study drug 79 randomized and treated

6 discontinued Adverse event n=2 Patient decision n=2 Other n=2

39 started teriparatide

33 completed teriparatide

40 started strontium ranelate

32 completed strontium ranelate

4 rejected biopsy

8 discontinued Adverse event n=4 Patient decision n=3 Other n=1

4 rejected biopsy 29 biopsies taken

28 biopsies taken 6 non-evaluable biopsies*

2 non-evaluable biopsies** 27 evaluable biopsies for dynamic parameters

22 evaluable biopsies for dynamic parameters

*fragmented, insufficient tissue. **triple tetracycline label, no label. Fig. 1. Disposition of patients screened and treated with teriparatide or strontium ranelate for the evaluation of dynamic histomorphometric variables.

Y.L. Ma et al. / Bone 48 (2011) 972–978 Table 1 Incidence of detectable fluorochrome label on the cortical bone specimens. Thicker cortex

Thinner cortex

Combined cortices

S-label

S-label

S-label

Periosteal surface TPTD 22/27 SrR 9/22 Endocortical surface TPTD 26/27 SrR 18/22

D-label

D-label

D-label

15/27 5/22

21/26 11/22

6/26 2/22

25/27 14/22

19/27 6/22

24/27 11/22

24/26 18/22

18/26 14/22

27/27 20/22

25/27 17/22

S-label: Single-label, D-label: Double-label.

mineralization in the thicker, thinner and combined cortices at the periosteal and endosteal levels are presented in Table 2. Overall, the bone formation and bone mineralization measurements were approximately 2.5-fold higher for the combined cortices in the TPTD group than the SrR group at the periosteal level, and 1.5to 2-fold at the endosteal level. These differences were statistically significant at the periosteal and endosteal levels for MS/BS%, singlelabeled perimeter, and MAR (Table 2). Similar differences were observed between the two treatment groups for MS/BS%, BFR/BS and the double-labeled perimeter for the thicker cortex (Table 2). The higher value of MS/BS% in the TPTD group compared with the SrR group for the periosteal surface at the thinner cortex attained near significance (p = 0.051). For both therapies, the absolute values of the measured dynamic variables were higher at the endosteal surface than at the periosteal layer. Comparisons between the thicker, thinner and combined cortices for TPTD- and SrR-treated patients are given in Table 3. Significantly greater values for double-labeled perimeter, BFR/BS, and MAR were observed in the thicker cortex compared with the combined cortices at both the periosteal and endosteal levels in the TPTD group. There were no statistically significant differences between the three comparisons (thicker versus thinner, combined versus thicker and combined versus thinner) for the single-labeled perimeter or MS/BS% in either the TPTD- or SrR-treated groups at either the periosteal or endosteal levels. No statistically significant differences of the histomorphometric dynamic values between any of the cortices for any variable in the SrR-treated group were observed (Table 3). Discussion From a biomechanical perspective, agents that stimulate periosteal expansion may make a greater contribution to the improvement of bone strength and resistance to fracture than those that primarily target endosteal and cancellous bone cell activity [28,29]. However, there is limited information on the effect of osteoporosis therapies on the periosteum in humans. In the present study we analyzed the comparative effects of TPTD and SrR on the dynamic indices of bone formation and mineralization at the periosteal and endocortical surfaces of transiliac crest biopsies from postmenopausal women with osteoporosis. Six months treatment with TPTD resulted in significantly greater values of several dynamic measures of bone formation and mineralization at the periosteal and endosteal surfaces of the combined cortices of transiliac crest biopsies. Of note, the values for bone formation indices and MAR were greater at the endocortical surface than at the periosteal layer in both treatment groups, suggesting more active turnover at the endocortical surface of the iliac crest, as previously described [4]. In our previous analysis [18], we did not observe any significant differences in the bone dynamic and structural parameters of the cancellous bone between the two treatment groups, with the exception of an increase in cortical porosity in the TPTD group,

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Table 2 Histomorphometric parameters in postmenopausal women treated with teriparatide or strontium ranelate for 6 months at the periosteal and endosteal levels. Histomorphometric parameters

Teriparatide

Strontium ranelate

n

Mean ± SE

n

13.07 ± 2.39 9.72 ± 2.23 12.17 ± 2.08

22 22 22

6.22 ± 2.99 3.71 ± 1.50 4.97 ± 1.66

0.072 0.033 0.009

3.03 ± 0.82 0.73 ± 0.32 1.99 ± 0.51

22 22 22

0.94 ± 0.55 0.55 ± 0.45 0.73 ± 0.49

0.047 0.762 0.086

0.023 ± 0.006 0.008 ± 0.004 0.014 ± 0.004

22 22 22

0.007 ± 0.004 0.003 ± 0.002 0.004 ± 0.003

0.044 0.239 0.057

9.56 ± 1.60 5.59 ± 1.23 8.08 ± 1.22

22 22 22

4.05 ± 1.70 2.40 ± 0.94 3.22 ± 1.05

0.022 0.051 0.004

0.48 ± 0.09 0.19 ± 0.07 0.35 ± 0.06

22 22 22

0.19 ± 0.08 0.09 ± 0.06 0.14 ± 0.06

0.024 0.281 0.011

12.81 ± 2.74 12.04 ± 1.50 12.39 ± 1.72

22 22 22

7.49 ± 1.66 6.73 ± 1.49 7.23 ± 1.19

0.128 0.018 0.020

13.19 ± 3.23 8.76 ± 2.38 11.01 ± 2.64

22 22 22

4.93 ± 2.06 6.21 ± 1.66 5.56 ± 1.47

0.037 0.431 0.084

0.053 ± 0.013 0.034 ± 0.008 0.041 ± 0.010

22 22 22

0.021 ± 0.008 0.022 ± 0.005 0.019 ± 0.005

0.039 0.220 0.046

19.59 ± 3.89 14.78 ± 2.84 17.20 ± 3.09

22 22 22

8.68 ± 2.66 9.57 ± 1.84 9.18 ± 1.83

0.028 0.148 0.030

0.62 ± 0.05 0.45 ± 0.07 0.54 ± 0.05

22 22 22

0.34 ± 0.08 0.43 ± 0.08 0.38 ± 0.06

0.004 0.800 0.047

Periosteal surface Bone formation Single-labeled perimeter (%) Thicker cortex 27 Thinner cortex 26 Combineda 27 Double-labeled perimeter (%) Thicker cortex 27 Thinner cortex 26 a Combined 27 3 2 BFR/BS (mm /mm /year) Thicker cortex 27 Thinner cortex 26 Combineda 27 MS/BS (%) Thicker cortex 27 Thinner cortex 26 a Combined 27 Bone Mineralization MAR (μm/day) Thicker cortex 27 Thinner cortex 26 Combineda 27 Endosteal surface Bone formation Single-labeled perimeter (%) Thicker cortex 27 Thinner cortex 26 Combineda 27 Double-labeled perimeter (%) Thicker cortex 27 Thinner cortex 26 a Combined 27 3 2 BFR/BS (mm /mm /year) Thicker cortex 27 Thinner cortex 26 Combineda 27 MS/BS (%) Thicker cortex 27 Thinner cortex 26 a Combined 27 Bone Mineralization MAR (μm/day) Thicker cortex 27 Thinner cortex 26 Combineda 27

p⁎

Mean ± SE

n, number of biopsies in which parameter was measured, BFR, bone formation rate; BS, bone surface; MAR, mineral apposition rate; MS, mineralized surface. a Thicker plus thinner. One biopsy only contained one intact cortex, and this was assigned to the thicker cortex group. ⁎ Exact permutation test (using Monte Carlo with N = 25 000).

which is a consequence of increased, transient intracortical remodelings [30,31]. In contrast, in the present analysis, we observed higher MAR and bone formation rates, by a factor of approximately 2.5, at the periosteal surface of the thicker cortex of the transiliac biopsy, in TPTD- compared with SrR-treated women. Interestingly, double tetracycline labels were found in 19 of 27 (70%) TPTD-treated subjects compared with 6 of 22 (27%) of SrRtreated patients at the periosteal surface. In a previous study, a 4fold increase in bone formation rate at the periosteal surface was reported after 28 days of treatment with synthetic human PTH(1– 34) (50 μg/day, subcutaneously) in 27 patients with postmenopausal osteoporosis compared with an historical control group of 13 patients with severe osteoporosis [12]. In contrast to our findings, double labels were seen in only 2 of 27 human PTH(1–34)-treated patients, and none in the control group probably reflecting a greater effect of longer treatment duration on bone formation in our study. Our histomorphometric findings, together with the previous human biopsy study reported by Lindsay et al. [12], and microstructural

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Table 3 Paired t-test comparisons between histomorphometric parameters of the thicker, thinner and combined cortices in teriparatide and strontium ranelate treated patients at the periosteal and endosteal levels. Histomorphometric parameters

Periosteal surface Bone formation Single-labeled perimeter (%) Thicker Thinner Combineda Double-labeled perimeter (%) Thicker Thinner Combineda BFR/BS (mm3/mm2/year) Thicker Thinner Combineda MS/BS (%) Thicker Thinner Combineda Bone mineralization MAR (μm/day) Thicker Thinner Combineda Endosteal Bone formation Single-labeled perimeter (%) Thicker Thinner Combineda Double-labeled perimeter (%) Thicker Thinner Combineda BFR/BS (mm3/mm2/year) Thicker Thinner Combineda MS/BS (%) Thicker Thinner Combineda Bone mineralization MAR (μm/day) Thicker Thinner Combineda

Teriparatide Mean ± SE

Strontium ranelate p⁎

Mean ± SE

Thicker vs thinner

Combined vs thicker

0.524

0.530

a

a

Combined vs thinner

0.519

13.07 ± 2.39 9.72 ± 2.23 12.17 ± 2.08

p⁎ Thicker vs thinner

Combineda vs thicker

Combineda vs thinner

0.473

0.464

0.483

0.128

0.110

0.151

0.206

0.104

0.540

0.369

0.353

0.385

0.246

0.246

0.246

0.727

0.803

0.659

0.589

0.577

0.602

0.935

0.536

0.529

0.752

0.713

0.788

0.414

0.414

0.414

6.22 ± 2.99 3.71 ± 1.50 4.97 ± 1.66 0.009

0.009

0.010

3.03 ± 0.82 0.73 ± 0.32 1.99 ± 0.51

0.94 ± 0.55 0.55 ± 0.45 0.73 ± 0.49 0.086

0.020

0.540

0.023 ± 0.006 0.008 ± 0.004 0.014 ± 0.004

0.007 ± 0.004 0.003 ± 0.002 0.004 ± 0.003 0.128

0.131

0.127

9.56 ± 1.60 5.59 ± 1.23 8.08 ± 1.22

4.05 ± 1.70 2.40 ± 0.94 3.22 ± 1.05 0.043

0.044

0.043

0.48 ± 0.09 0.19 ± 0.07 0.35 ± 0.06

0.19 ± 0.08 0.09 ± 0.06 0.14 ± 0.06

0.739

0.755

0.723

12.81 ± 2.74 12.04 ± 1.50 12.39 ± 1.72

7.49 ± 1.66 6.73 ± 1.49 7.23 ± 1.19 0.032

0.035

0.031

13.19 ± 3.23 8.76 ± 2.38 11.01 ± 2.64

4.93 ± 2.06 6.21 ± 1.66 5.56 ± 1.47 0.064

0.032

0.153

0.053 ± 0.013 0.034 ± 0.008 0.041 ± 0.010

0.021 ± 0.008 0.022 ± 0.005 0.019 ± 0.005 0.090

0.095

0.087

19.59 ± 3.89 14.78 ± 2.84 17.20 ± 3.09

8.68 ± 2.66 9.57 ± 1.84 9.18 ± 1.83 0.026

0.027

0.026

0.62 ± 0.05 0.45 ± 0.07 0.54 ± 0.05

0.34 ± 0.08 0.43 ± 0.08 0.38 ± 0.06

BFR, bone formation rate; BS, bone surface; MAR, mineral apposition rate; MS, mineralized surface. a Thicker plus thinner. ⁎ paired t-test.

and biomechanical investigations in rabbit and monkey models [30–33], lend support to the concept that daily treatment with TPTD stimulates periosteal bone formation. This stimulation leads to an increase in cortical diameter and, consequently, the cross-sectional moment of inertia that is an important determinant of resistance to fracture [34,35]. The cellular mechanisms responsible for PTH bone anabolism in human periosteal tissue cannot be ascertained from our results and require further investigation. Pro-differentiating and/or pro-survival effects of PTH on post-mitotic pre-osteoblasts have been suggested in mice models [36]. PTH may act directly on periosteal pre-osteoblasts to increase differentiation as suggested by the increased expression of the osteoblast-specific transcription factor Runx2 (Cbfa1), as well as modulating the synthesis of autocrine/paracrine factors, such as insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF)-2 or sclerostin, that regulate the production of osteoblasts. This is in

contrast to the situation in cancellous bone where suppression of osteoblast apoptosis may predominate [36,37]. There are limited data regarding ways in which the periosteal surface is regulated by antiresorptive drugs. Studies of postmenopausal women after risedronate treatment demonstrated that iliac crest cortical width was not significantly altered following reductions in bone remodeling [38]. In a study of transiliac biopsies from pre- and post-menopausal women treated with alendronate, double-fluorochrome-labeling suggested that bone formation in the endocortical envelope was reduced but periosteal expansion continued [39]. Patients treated with high-dose estrogen replacement therapy showed a trend toward increased iliac crest cortical width in a paired biopsy study, but this effect was more likely to have been the result of reduced endocortical resorption than that of increased periosteal apposition [40]. Raloxifene had little or no effect on periosteal apposition in humans [41]. These findings are not unexpected since

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the majority of activity at the periosteal level is formation modeling, and bisphosphonates and estrogen exert their skeletal effects through the suppression of remodeling. Evidence from in vitro and animal studies suggests that SrR may dissociate the coupled bone remodeling process by stimulating bone formation and decreasing bone resorption [13–15], although its anabolic effects at the dose recommended for humans are unclear [18,19,42,43]. To the best of our knowledge, there are no published data on the effects of SrR in the periosteum in humans. Arlot et al. [17] reported a higher (+18%) cortical thickness using microcomputed tomography (μCT) in 20 biopsies from postmenopausal women treated with SrR, but this was based on a non-paired, cross-sectional biopsy analysis, with a placebo-treated group as a control, in which deterioration of the microstructural characteristics occurs over time. In a study with intact rats that had 2 years of SrR treatment starting at 6–7 weeks of age, Amman et al. [16] reported a dose-dependent significant increase (1.8% to 5%) in the periosteal perimeter, whereas endocortical bone perimeter remained unchanged. However, other studies in adult ovariectomized rats treated with SrR showed no cortical effects [44], or suppression of periosteal bone formation rate [45]. In addition to the contradictory data for SrR, rodent cortical data have been shown to have questionable relevance to human cortical physiology because, as rats grow longitudinally for nearly their entire lives, their bones are dominated by modeling. Also, there is a near absence of osteonal remodeling in rat cortical bone [46]. Mechanical loading is known to stimulate periosteal mineral apposition. Moreover, periosteal osteoblasts exhibit greater mechanosensitivity to strain than endosteal osteoblasts [3,47]. In growing subjects the two cortices of an iliac bone specimen differ with regard to bone cell activity on their surfaces [48,49]. Therefore, in an effort to understand better the effects of TPTD and SrR on these two envelopes, we studied the treatment effects on the thicker and thinner cortices of transiliac bone biopsies. Although we could not confirm whether the thicker cortex was inner or outer in our study [22], our results showed that most of the dynamic indices of bone formation and MAR at the periosteal and endosteal levels are statistically higher in the thicker cortex of the TPTD group than in the thinner cortex. Similar findings were previously reported by Balena et al. [22] who found that the bone formation parameters such as wall thickness, osteoid surface, osteoblast surface, mineralizing surface and bone formation rate were all greater on the endocortical, intracortical and periosteal surfaces of the thicker (inner) cortex compared with those parameters on the thinner (outer) cortex in human iliac biopsies of patients with osteoporosis. These authors hypothesized that local factors, such as muscle tension, influenced bone remodeling at the cortical surfaces of human iliac crest [22]. Our findings could support the hypothesis that TPTD is synergistic with mechanical loading in the human skeleton. Such an effect of PTH and loading to increase bone mass has been described previously in animal studies [23–25,50]. It has been suggested that PTH may augment bone formation by sensitizing osteoblasts to mechanical forces [51]. Various in vitro and in vivo studies show similar increased cell proliferation, activation of cell signaling, and transcriptional activation of a number of genes following either PTH treatment or mechanical loading. Both stimulants appear to activate common second-messenger systems and gene expression, including increases in PGE2 levels [24,52,53]. Additionally, there is increased cyclooxygenase-2 (COX-2), IGF-1, transforming growth factor-β (TGFβ), nitric oxide, collagen, osteocalcin and connexin-43 [50,54],which are involved in signaling pathways. Finally, similar to PTH treatment, mechanical loading results in local suppression of osteocytic sclerostin levels and stimulates the canonical Wnt/β-catenin-signaling pathway [55]. Therefore, bone anabolic pathways activated by PTH signaling and mechanical loading might partially overlap.

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In contrast to the TPTD-treated group, there were no differences between any of the cortices for any bone forming indices in the SrRtreated group, probably indicating negligible bone forming activity at the cortical-endosteal and -periosteal surfaces. This is the first histomorphometric human bone biopsy analysis to compare the effects of TPTD and SrR on the periosteal surface, and the first report of the effects of SrR on the human periosteum. Limitations of this post hoc analysis include the lack of a placebo control group, and the fact that analyses were based on samples derived from a cross-sectional design (i.e., biopsies before treatment were not available). The potential bias of the open-label study design was expected to be negligible because the evaluation of the histomorphometric parameters was carried out by readers blinded to treatment assignment. Additionally, a double-dummy design reduces compliance. The quantification of only one section was another limitation of this study. However, unlike cancellous bone where there are differences in trabecular sections taken 300 μm apart, the morphology of cortical sections taken this distance apart are less dissimilar. Additionally, when the first unstained section did not show tetracycline fluorescence or double-label, a second section was used for fluorochrome-label measurement. In the majority of cases the incidence of fluorochrome-label was the same for both sections on either the periosteal or endocortical surfaces. Our analyses were based solely on samples derived from the iliac crest that may not be representative of bone status at other locations. In conclusion, compared to SrR, six months treatment with TPTD of postmenopausal women with osteoporosis was associated with higher dynamic bone formation indices at both the periosteal and endosteal combined cortices for most parameters. The response to TPTD for dynamic bone formation variables was higher in the thicker than the thinner cortices; this difference was not apparent in SrRtreated patients. These results provide details of changes in bone quality that may underlie the synergistic effects of mechanical loading and TPTD, and the reduction for fracture risk in osteoporotic patients treated with TPTD. Conflicts of interest statement J.J. Stepan and P. de la Peña have received travel grants, consultancy and/or speaker's fees from Lilly. S. Ish-Shalom has received travel grants, consulting and lecture fees from Aventis, Chemical and Technical Supplies, Lilly, Merck Sharp & Dome, Meditec, Transpharma, NPS Allelix Corp and Novartis. R.R. Recker has been a paid consultant/speaker for Merck, Lilly, Wyeth, Procter & Gamble, Amgen, Roche, Glaxo Smith Kline and Novartis. L.Y. Ma, F. Marin, X. Wan and Q.Q. Zeng are employees of Eli Lilly and Company Inc. No other authors have declared any conflicts of interest. Author contributions Dr. R.R. Recker and Dr. Y. L. Ma (principal investigators) had full access to all data in the study and take responsibility for the integrity of the data. Conception and design: R.R. Recker, F. Marin, L.Y. Ma. Acquisition of data: S. Ish-Shalom, R.Möricke, F. Hawkins, G. Martínez, G. Kapetanos, M.P. de la Peña, J. Kekow, J. Malouf, J. Stepan, G. de la Peña, Q.Q. Zeng. Analysis and interpretation of data: L.Y. Ma, X. Wan, F. Marin, R.R. Recker. Drafting of the article: L.Y. Ma, X. Wan, F. Marin, R.R. Recker. Critical revision of the article for intellectual content: L.Y. Ma, F. Marin, J. Stepan, H. Ish-Shalom, R.Möricke, F. Hawkins, G. Kapetanos, M.P. de la Peña, J. Kekow, G. Martínez, J. Malouf, Q.Q. Zeng, X. Wan, R.R. Recker. Acknowledgments We thank all the patients who participated in the study. The authors thank Laura Briones, Beatriz Sanz and Nadine Baker, and Eli Lilly and Company, for their support in the coordination of the study,

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