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, Shiga, Japan). Serum levels of C-terminal telopeptide of type I collagen (CTX-I) was measured by an enzyme-linked immunosorbent [https://en.wikipedia.org/wiki/Sclareol Sclareol] assay (RatLaps EIA; Immunodiagnostic Systems Ltd., Boldon, UK). BMD assay The BMD of femurs was measured by X-ray computed tomography (Latheta LCT-200; Hitachi Aloka Medical, Ltd., Tokyo, Japan). BMD of diaphysis was measured at the mid-diaphysis position of femurs and BMD of metaphysis was measured at 1.5 mm proximal to the growth plate in the condyle of femurs. Biomechanical testing Biomechanical properties were measured by a three-point bending test at mid-diaphysis and a compression test at metaphysis using a servo-hydraulic fatigue-testing instrument (Servopulser; Shimadzu Co., Kyoto, [http://www.selleckchem.com/products/PLX-4032.html Raf inhibitor] Japan). The mid-diaphysis was placed on the two supports that were 20 mm apart. The load of a three-point bending test was applied in the anteroposterior direction midway between the two supports. Load displacement curves were recorded at a crosshead speed of 6 mm/sec. Alternatively, metaphysis was placed on the test apparatus with the lateral side up. Compression load was applied on the specimens from the lateral aspect to the medial aspect. Load displacement curves were recorded at a crosshead speed of 6 mm/sec. The maximum load, breaking energy and stiffness were calculated using a load-displacement curve. Histomorphometry The left femurs were fixed and embedded in glycol methacrylate. Coronal sections of distal femur were cut at a thickness of 20 ?m and stained with toluidine blue for histomorphometric analyses. Measurements were performed in the secondary cancellous bone region from 0.2 to 2.25 mm proximal to the growth plate using a semi-automatic image analysis system (OsteoMetrics, Inc., Decatur, GA, USA) and the primary parameters, such as tissue volume (TV), bone volume (BV), bone surface (BS), osteoblast surface (Ob.S) and osteoclast surface (Oc.S), were determined. These data were used to calculate the following parameters: BV/TV, Ob.S/BS and Oc.S/BS. Statistical analysis All the data are expressed as mean �� standard deviation. Multiple comparisons of data among the groups were performed by analysis of variance [http://www.selleckchem.com/products/AZD2281(Olaparib).html www.selleckchem.com/products/AZD2281(Olaparib).html] with Tukey's post hoc test. P