Zhiqi Peng

Comparison of three-point bending test and peripheral quantitative computed tomography analysis in the evaluation of the strength of mouse femur and tibia.

We compared the mechanical and densitometric testing methods in evaluating the mechanical strength of mouse cortical bones. The femora and tibiae of 10 male mice (weight 32.8 +/- 4.0 g) were utilized. Volumetric cortical bone mineral density (vCtBMD), cross-sectional cortical area at midshaft (CSA), cross-sectional moment of inertia (CSMI), and strength strain index (SSI) were measured by peripheral quantitative computed tomography (pQCT). The precision of pQCT expressed as a coefficient of variation (CV) was 1.1%, 2.7%, and 6.4% for vCtBMD, CSA, and CSMI, respectively. The mechanical properties were measured by a three-point bending test. The method error measured from paired bones was 7.3%-10.1% for breaking bending force, 15.0%-15.2% for stiffness, 2.0%-2.4% for vCtBMD, 5.2%-6.4% for CSA, 13.5%-17.6% for CSMI, and 8.9%-18.1% for SSI. CSMI and CSA were found to be the best explanatory variables for the breaking force of femur and tibia, respectively, while CSA and CSMI were the best predictors for the elastic modulus of femur and tibia, respectively. CSA had a higher correlation with mechanical parameters than vCtBMD. On the basis of this study, the mechanical tests and the pQCT measurements are relevant in biomechanical studies on mouse bones and justify the use of the murine model. High-resolution pQCT gives better precision than the three-point bending test in studies of mouse bones.

The mechanical strength of bone in different rat models of experimental osteoporosis

In order to discover good parameters for experimental osteoporosis, we measured the failure load of the femoral neck and the bending strength of the tibia in orchidectomized (ORC) (20 rats for 4 weeks), ovariectomized (OVX) (28 rats for 6 weeks), and immobilized (IMM) (33 rats for 3 weeks) rats. Each of these operations led to a significant decrease in trabecular bone volume when compared with corresponding controls (p < 0.001). The ash weight of femurs was significantly decreased in ORC (p < 0.05) and IMM (p < 0.001) rats, but not in OVX rats. Growth of the femur was somewhat slower in ORC (p < 0.05) and IMM rats (p < 0.05), but not in OVX animals. All three osteoporosis models showed significant decreases in the maximal load of the femoral neck (ORC: 23.9%, p < 0.001; OVX: 15.8%, p < 0.001; IMM: 27.7%, p < 0.001), as well as in energy absorption (ORC: 43.9%, p < 0.001; OVX: 28.3%, p < 0.001; IMM: 45.3%, p < 0.001). In tibia orchidectomy reduced maximal strength and energy absorption significantly (10%, p < 0.01; 27.8%, p < 0.01), but ovariectomy decreased only maximal load (8.7%, p < 0.01) and immobilization only energy absorption (18.0%, p < 0.01). Our results suggest that the mechanical strength of the femoral neck is a sensitive indicator of bone loss in all three osteoporosis models.

Long-term effects of ovariectomy on the mechanical properties and chemical composition of rat bone

One hundred Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (Sham) at the age of 12 weeks. Eight or six animals from each group were sacrificed at the following time points: 0, 2, 4, 8, 16, 28, and 40 weeks. Mechanical testing of the bones showed that the maximal load of the femoral neck remained at the baseline level in OVX rats but clearly increased in Sham animals. However, after 28 weeks (at the age of 40 weeks) of ovariectomy, the femoral neck strength in Sham animals started to decrease, reaching the OVX level at the age of 1 year. The gain of torsional strength of the humerus was first slowed down after ovariectomy, but 16 weeks after operation this difference between the OVX and Sham animals disappeared. In OVX rats incorporation of 45 Ca2+ into the tibia and the number of osteoclasts on the trabecular bone surfaces were increased at 4-16 weeks and at 2-28 weeks, respectively, but they returned later to the levels observed in Sham animals. This indicates that the OVX-induced increase in bone turnover is transient. The trabecular bone volume at the distal femur decreased and the area of the marrow cavity in the distal third of the femur increased after OVX and did not show any recovery. Analysis of the inorganic composition of bone by energy-dispersion spectrometry showed that the percentage of magnesium was transiently decreased after OVX in both trabecular and cortical bone. Despite the OVX-induced loss of trabecular bone, increasing body weight or some metabolic changes may induce structural changes which may be responsible for maintaining the mechanical force of the femoral neck in aging OVX rats.

Ovariectomy-Induced Bone Loss Can be Affected by Different Intensities of Treadmill Running Exercise in Rats

Abstract. Fifty-six Sprague-Dawley rats were either ovariectomized (OVX, n= 24), sham-operated (Sham, n= 24), or sacrificed (n= 8) at the beginning of the experiment to serve as a baseline group. The OVX and Sham groups were further randomly divided into control (CTRL), slow running (R10), and faster running (R18) groups. R10 and R18 groups ran for 2 × 30 min/day for 8 weeks at speeds of 10 m/min and 18 m/min, respectively. Exercise did not affect the mechanical or histomorphometric parameters of bone in the sham-operated rats. There was no effect of exercise on body weight gain in the OVX-R10 group, but in OVX-R18 it decreased the gain of body weight. In the OVX–CTRL group the maximal load and energy absorption of the femoral neck were 16.7% (P < 0.001) and 30.0% (P < 0.001) lower than in the Sham–CTRL group, respectively. In OVX animals, slow running had a positive effect on the maximal load of the femoral neck (86.5 N) when compared with OVX–CTRL rats (77.1 N, P < 0.07). 51.7% of the trabecular bone was lost in the distal femur as a result of OVX and exercise reduced this loss to 30.2% (R10) and 39.9% (R18). Ovariectomy increased the bone formation rate (BFR) and the mineral apposition rate (MAR) on the periosteum of the femoral shaft. Exercise decreased the periosteal BFR and MAR in OVX rats, but increased it at the endosteum. Osteoclast numbers in the femoral metaphysis were increased after OVX and running exercise inhibited this effect significantly. The maximal bending load of the humerus increased after OVX by 12.1% (P < 0.05). Exercise enhanced this effect, the slow running being more effective. These results suggest that bone in OVX rats is either more sensitive to exercise than in sham-operated rats or that the higher body weight with slow running induces optimal loading and strengthens the bones.

A Novel Immunoassay for The Determination of Tartrate-Resistant Acid Phosphatase 5b From Rat Serum

Osteoclasts secrete tartrate‐resistant acid phosphatase 5b (TRACP 5b) into the circulation. We have developed an immunoassay for the determination of rat TRACP 5b activity. Intra‐assay variation of the immunoassay was 4.5%, interassay variation was 3.8%, dilution linearity was 104.6 ± 7.6%, and recovery of recombinant rat TRACP was 99.1 ± 5.8%. We studied serum TRACP 5b as a marker of bone resorption using orchidectomized (ORC) rats as a model for osteoporosis and age‐matched sham‐operated rats as controls in a 6‐month study. After the operation, trabecular bone mineral density decreased significantly more in the ORC group than in the sham group, whereas cortical bone mineral density increased similarly in both groups. Serum TRACP 5b activity was significantly elevated within the first week after ORC, returned to the control level in the third week, and was not increased above the sham level at any of the later time points. At 6 months, trabecular bone volume was 80% lower in ORC rats than in controls. Osteoclast number per trabecular bone perimeter was slightly increased, but the absolute number of osteoclasts in trabecular bone was significantly decreased. These results suggest that absolute bone resorption is increased within the first week after ORC. Later, it is decreased because there is less bone to be resorbed. However, relative bone resorption (compared with the amount of remaining bone) is still increased, leading to further bone loss. We conclude that serum TRACP 5b is a useful marker for monitoring changes in the bone resorption rate in rat ORC model.

Effect of running exercise on the bone loss induced by orchidectomy in the rat

The effect of exercise on castration-induced osteoporosis in 3-month-old male rats weighing 264±4 g at the beginning of the experiment was studied. A testosterone deficiency was induced by orchidectomy (ORC), and the exercise group ran 10 m/minute for 1 hour a day on a treadmill at 0% grade. There were seven groups of eight rats (n=56) randomized into a control group killed at time 0, and sham, ORC and ORC and exercise groups killed at 4 and 8 weeks. ORC reduced body weight gain (with analysis of variance (ANOVA) P<0.001), and at 4 weeks the body weight was 343±14 g in ORC group and 301±4 g in the ORC and exercise group (P<0.01). The increase in femoral length was slower in the ORC+exercise groups. The ash weight of the tibia did not decrease significantly after ORC or ORC+ exercise. ORC did not affect 45Ca incorporation, but exercise slightly increased it in the whole tibia 8 weeks after ORC (with ANOVA P=0.057). ORC had significantly lowered the trabecular bone volume in the secondary spongiosa of the distal femur at 4 and 8 weeks, and exercise did not prevent this. This is an opposite finding to our previous study with ovariectomized female rats [12]. ORC also significantly had reduced the osteoblast-lined trabecular bone surface and the number of osteoclasts by 8 weeks after the operation. Exercise increased the osteoblast-lined surface and the number of osteoclasts. The mechanical strength of the femoral neck also was reduced after ORC and this was not prevented by exercise either. In conclusion, ORC reduces bone growth and turnover which leads to osteopenia in growing rats. Moderate treadmill exercise does not reverse the ORC-induced loss of trabecular bone and the reduced mechanical strength of the femoral neck, although it has a positive effect on the osteoblast and osteoclast indices and on calcium incorporation into bone.

High Dietary Phosphate Intake Reduces Bone Strength in the Growing Rat Skeleton

Nutrition influences peak bone mass development in early adulthood. The effect of high dietary phosphate intake on the growing skeleton of 1‐month‐old male rats (n = 30) was assessed in an 8‐week intervention. High dietary phosphate intake increased bone remodeling and impaired bone material properties, diminishing bone mechanical strength.

Clodronate Prevents Osteopenia and Loss of Trabecular Connectivity in Estrogen-Deficient Rats†

Daily oral clodronate treatment was evaluated in Sprague‐Dawley rats for its ability to inhibit estrogen‐deficiency–induced changes in femoral neck, femoral diaphysis, and lumbar vertebrae (L4–L5). Six‐month‐old ovariectomized (OVX) rats were administered by gavage a vehicle (Veh) or clodronate (100 or 500 mg/kg/day). Sham‐operated (SHAM) control rats received the vehicle (n = 15/group). Treatment was started on the day of operation and continued for 3 months. Trabecular bone volume (BV/TV) and structural variables (trabecular number, Tb.N; thickness, Tb.Th; separation, Tb.Sp; and trabecular bone pattern factor, Tb.Pf) were assessed on secondary spongiosa of the right femoral neck. Furthermore, cantilever bending test of the left femoral neck and compression test of L4, ash weight of L5, and morphometric studies of femoral diaphysis were carried out, and serum and urinary markers of bone turnover were determined. The OVX/Veh group had higher levels of serum osteocalcin and alkaline phosphatase and higher urinary excretion of deoxypyridinoline/creatinine than the SHAM/Veh group at 3 months postsurgery, and clodronate reduced these changes. BV/TV of femoral neck, bone mass of L5, and the maximum loads of the femoral neck and L4 were lower after OVX than SHAM operation. Although clodronate prevented trabecular bone loss in the femoral neck and preserved Tb.Pf at the SHAM control level, it failed to preserve the mechanical strength at the femoral neck. However, in lumbar vertebrae, clodronate prevented the loss of bone mass and mechanical properties. Furthermore, there was a good positive correlation between maximum load of L4 and the ash weight of L5 (n = 58, r = 0.69, p < 0.001). In the femoral neck (n = 55), Tb.Pf correlated negatively with BV/TV and Tb.N (r = −0.59 and r = −0.55; p < 0.001, respectively) and positively with Tb.Sp (r = 0.61, p < 0.001). In femoral mid‐diaphysis, there were no significant changes in cortical bone geometry in any of the groups. We conclude that orally administered clodronate suppresses the enhanced bone turnover in adult OVX rats and preserves trabecular bone volume and connectivity in the femoral neck. In the axial skeleton, clodronate has a beneficial effect on lumbar vertebral bone mass and strength.

Effect of exercise on osteoporosis induced by ovariectomy in rats

Immobilization and inactivity result in bone loss, but weightbearing exercise results in bone mineral density being maintained or increased. Although postmenopausal osteoporosis has many causes, estrogen deficiency is considered a major one. It has been suggested that postmenopausal bone loss is related to levels of physical activity [ 1 ]. In the study reported here, we induced osteoporosis in rats by ovariectomy and assessed whether or not bone loss was prevented by exercise.

Alteration in the mechanical competence and structural properties in the femoral neck and vertebrae of ovariectomized rats.

The structural and mechanical properties of bone in the femoral neck and various other sites were investigated in intact (INT), sham‐operated (Sham), and ovariectomized (OVX) rats. Six weeks after operation, the maximal load and energy absorption of the femoral neck were significantly lower in the OVX than in the INT or Sham groups, being 73.2 ± 1.4 (SE) N, 86.3 ± 4.1 N, and 87.1 ± 3.2 N, respectively (p < 0.01) for load. The total cross‐sectional area of the femoral neck did not change after OVX, but the marrow cavity area was enlarged, leading to a reduced bone area (including both cortical and trabecular bone) (p < 0.01). Histomorphometric analysis showed that new bone formation could not be detected at the periosteum of the femoral neck below the femoral head, but at the endocortical surfaces the double tetracycline labeling revealed an increased mineral apposition rate (MAR) and bone formation rate (BFR) in OVX animals (p < 0.001). In contrast, MAR and BFR were significantly increased in both periosteal and endocortical surfaces of the tibia, humerus, and femoral shaft, thus preventing a decrease in cortical bone area. The maximal bending loads of the tibia and humerus were not different in the various groups of animals. The correlation coefficient between maximal load and bone area revealed positive relationships in the femoral neck (r = 0.54, p < 0.01), tibia (r = 0.46, p < 0.01), and humerus (r = 0.51, p < 0.01). Ovariectomy resulted in a decreased trabecular bone volume of lumbar vertebra VI (L6) decreased compressive loads of lumbar vertebrae I, III, and IV. These lumbar bone loads were positively related to their L6 bone area (L4/L6: r = 0.66, p < 0.001). Element analyses (energy dispersion spectrometer) from trabecular and cortical areas of bone showed some changes related to aging but not to OVX. These results indicate that ovariectomy influences the biomechanical properties of rat bone by changing structural properties rather than material ones.