1 2 3 4 5 6 7 8 9 10 11 In vitro 12 13 in vitro In this study, we investigated whether treatment of postmenopausal women with raloxifene for 2 years would change osteocyte survival in trabecular bone and whether the level of osteocyte apoptosis would be associated with the level of bone remodeling. Materials and Methods Study Outline 10 In this study, bone biopsies were obtained at baseline and after 2 years from 26 women who were enrolled in the European centers of the MORE trial. These women were part of the bone histomorphometry substudy of the MORE trial that included 65 women from two centers in the United States and two centers in Europe. Bone biopsies from the other 39 women were not available for sectioning. All women had given their informed consent, and the study was approved by the institutional ethics review boards. Markers of bone turnover that were measured were serum osteocalcin, bone-specific alkaline phosphatase (BSAP), and urinary type 1 collagen C-telopeptide corrected for creatinine (CTX-I). Data are from the baseline and 24-month evaluations. Bone Biopsies N,N 14 15 16 17 18 Immunohistochemistry l 4 2 2 2 Cell Counting The identity of the sections was blinded, and they were randomly numbered. In each section, the total trabecular bone area was measured using Osteomeasure software (Osteometrics, Atlanta, GA). In the entire trabecular bone area, the total number of osteocytes, the number of cleaved caspase-3-positive osteocytes, and the number of empty lacunae were counted with x200 magnification. Artefacts, such as areas where bone marrow obscured the trabeculae or where trabeculae were crossed, were avoided. All sections were analyzed by the same investigator. percentage of positive osteocytes per total osteocytes (Pos.Ot.N/Tt.Ot.N) percentage of empty lacunae per total lacunae (EL.N/Tt.L.N) empty lacunae per bone area (EL.N/B.Ar) total lacunae per bone area (Tt.L.N/B.Ar) \documentclass[12pt]{minimal}
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\begin{document}$$\overline {x_j } $$\end{document} j Statistics t t Results 16 1 1 1 1 Fig. 1 a b Arrows a b c d Arrows  Table 1 Results of baseline and follow-up biopsies for the calculated parameters a Time point n b  n Pos.Ot.N/Tt.Ot.N (%) (1.28) Baseline 6.66 ± 5.28 5.30 ± 3.51 Follow-up 6.82 ± 5.27 4.99 ± 2.29 EL.N/Tt.L.N (%) (2.43) Baseline 9.00 ± 2.25 9.11 ± 2.88 Follow-up c 9.74 ± 1.91 2 Baseline 21.2 ± 6.13 21.0 ± 6.49 Follow-up 24.5 ± 3.68 22.5 ± 4.67 2 Baseline 233.9 ± 28.2 231.5 ± 22.9 Follow-up 219.5 ± 27.3 231.1 ± 19.6 a b c P 2 Fig. 2 A B P  2 3 Table 2 Association of histomorphometric indices and biochemical indices of bone remodeling with osteocyte apoptosis (Pos.Ot.N/Tt.Ot.N) and empty lacunae (E.L.N/Tt.L.N)  Histomorphometric indices OS/BS BFR/BV ES/BS Ocl.N/B.Ar % Positive osteocytes (Pos.Ot.N/Tt.Ot.N) r r r r P a P a P a P a % Empty lacunae (E.L.N/Tt.L.N) r r r r P P P P a  Biochemical indices Osteocalcin BSAP CTX % Positive osteocytes (Pos.Ot.N/Tt.Ot.N) r r r P P P % Empty lacunae (E.L.N/Tt.L.N) r r r P P P a P Fig. 3 A P B P  3 Table 3 Results of baseline and follow-up biopsies for histomorphometric indices and biochemical markers of bone remodeling Parameter Time point % change n a  n OS/BS (%) Baseline 10.2 ± 6.8 9.9 ± 5.4 Follow-up 7.5 ± 3.2 11.6 ± 4.6 BFR/BV (%/year) Baseline 28.8 ± 20.3 35.3 ± 18.6 Follow-up 19.8 ± 8.7 23.3 ± 12.6 ES/BS (%) Baseline 5.3 ± 2.6 6.3 ± 2.9 Follow-up 5.3 ± 2.4 6.3 ± 2.4 n 2 Baseline 0.6 ± 0.3 0.8 ± 0.4 Follow-up 0.6 ± 0.3 0.6 ± 0.3 BSAP (μg/L) Baseline 14.9 ± 7.3 14.4 ± 4.6 Follow-up 12.2 ± 5.6 b Osteocalcin (ng/mL) Baseline 22.9 ± 10.6 25.5 ± 9.1 Follow-up 18.2 ± 3.8 b CTX-1 (μg/mmol creatinine) Baseline 283.9 ± 267.0 277.4 ± 198.2 Follow-up 137.7 ± 63.8 b a b P Discussion We investigated the effect of treatment with raloxifene for 2 years of postmenopausal osteoporotic women on osteocyte apoptosis as measured by activated caspase-3 immunohistochemistry in iliac crest bone biopsies. Direct comparison of follow-up with baseline in the placebo and raloxifene groups did not show differences in the percent positive osteocytes. This suggests that raloxifene has little or no influence on osteocyte apoptosis. We did find a significant increase in the percentage of empty lacunae at 2 years in the placebo group, while there was no change in the raloxifene group. This lack of accumulation of empty lacunae in the raloxifene group could be the consequence of an inhibitory effect of raloxifene on osteocyte apoptosis. At baseline, histomorphometric indices of bone remodeling were inversely correlated with apoptotic osteocytes but not with empty lacunae. Biochemical markers of bone remodeling were not correlated with apoptotic osteocytes or empty lacunae. in vitro 13 19 in vitro 20 21 22 23 24 25 5 26 27 28 Biochemical markers of bone formation and resorption were not correlated with osteocyte apoptosis or with empty lacunae. Biochemical markers reflect bone remodeling in the whole skeleton, both in trabecular bone and in cortical bone. Correlations between such general markers and parameters measured locally in trabecular bone of the iliac crest are possibly more difficult to find. The major limitation of our study is the small number of patients. The percentage of apoptotic osteocytes varied considerably between individuals, and this makes it difficult to find significant differences between such small groups. The variation was not caused by variation in the method, as shown by the low average standard deviation, indicating that the percentage of apoptotic osteocytes has to be an individual characteristic. 29 30 31 32 8 The number of empty lacunae that we observed could be an overestimation caused by sectioning artefacts or the decalcification step in the immunohistochemistry method. We do not know to what extent this occurs, but this would be equal in all biopsies. Goldner-stained sections showed more sectioning artefacts, and therefore, empty lacunae in these sections were not counted. Inconsistency between the change in percent empty lacunae and empty lacunar density in the placebo group is probably also related to the small number of patients studied. 10 33 34 35 In conclusion, we did not find evidence for an effect of raloxifene treatment on osteocyte apoptosis in postmenopausal women, but small effects of raloxifene treatment on osteocyte apoptosis cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual.