Dose-related histopathology and bone remodeling characteristics of the knee articular cartilage and subchondral bone induced by glucocorticoids in rats.

The aim of the current study was to investigate histopathological changes and bone remodeling in the knee articular cartilage and subchondral bone in rats following treatment with glucocorticoids. A total of 30 3-month-old female Sprague-Dawley rats were randomly divided into either a vehicle control group or one of three experimental groups wherein dexamethasone (Dex) was administered at a dose of 1.0, 2.5 or 5.0 mg/kg (Dex1.0, Dex2.5 and Dex5.0, respectively), for 8 weeks. Articular cartilage and the epiphyseal subchondral bone of the proximal tibias were evaluated by histopathology or for bone remodeling using histomorphometry. No histological changes were identified in the knee articular cartilage but the bone formation rate of the subchondral bone was lower in the Dex1.0 group compared with that of the control group. Compared with the control and the Dex1.0 group, the width of the articular cartilage and the subchondral plate were larger, with abnormal morphology and increased apoptosis of chondrocytes, decreased cell/matrix volume ratio in the cartilage and fewer blood vessels in the subchondral plate in the Dex2.5 and Dex5.0 groups. A higher Dex dose resulted in more severe inhibition of bone formation, a greater number of apoptotic osteocytes and constrained bone resorption. All microstructure parameters indicated no significant changes in the Dex2.5 group but exhibited deterioration in the Dex5.0 group compared with the normal and Dex1.0 group. There were no significant differences in morphological changes, or in static and dynamic bone indices between the Dex2.5 and Dex5.0 groups. In conclusion, long-term glucocorticoid use induced dose-related histopathological changes in the knee articular cartilage, along with unbalanced bone remodeling and osteopenia in the subchondral bone. The degree of damage to the articular cartilage was milder and transformed from compensation to degeneration at higher doses.

The development of bone microstructure, metabolism and biomechanics in lumbar vertebra under short-term glucocorticoid exposure.

Abstracts Objective: This study investigated the characteristics of bone microstructure, metabolism, and biomechanics in rat's lumbar vertebra undergoing short-term glucocorticoid administration. Methods: Forty 4-month-old female Sprague-Dawley rats were treated with either vehicle (Cont) or prednisone acetate (Pre) at 3.5 mg/kg/day, respectively for periods of 7 days and 21 days. The lumbar vertebras were processed for MicroCT scan, histomorphometry analysis, mechanical compression test, in addition to Dual-Energy X-ray absorptiometry scan, respectively. Results: The connective density (Conn. D) along with trabecular connection nodes decreased while trabecular termini increased in Pre at day 21 when compared to Cont at day 21 as well as Pre at day 0. The mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR), osteoblast surfaces (Ob.S/BS) were lower in Pre at day 21 than that in Cont at day 21, Pre at day 0 and Pre at day 7. Only the bending stiffness of compression test decreased in Pre group at day 21 compared to age-matched control. Conclusion: The results suggested that excess prednisone significantly inhibited bone formation and slightly depressed bone resorption in the lumbar vertebra of intact rats for the duration of 21 days. Accordingly, the trabecular spatial microstructure made an adjustment yet failed to maintain the anti-compression mechanical property.

WISP1 silencing confers protection against epithelial-mesenchymal transition of renal tubular epithelial cells in rats via inactivation of the wnt/ß-catenin signaling pathway in uremia.

Uremia can affect hepatic metabolism of drugs by regulating the clearance of drugs, but it has not been clarified whether gene silencing could modulate the epithelial-mesenchymal transition (EMT) process in uremia. Hence, we investigated the effect of WISP1 gene silencing on the renal tubular EMT in uremia through the wnt/ß-catenin signaling pathway. Initially, microarray-based gene expression profiling of uremia was used to identify differentially expressed genes. Following the establishment of uremia rat model, serum creatinine, and urea nitrogen of rats were detected. Renal tubular epithelial cells (TECs) were transfected with shRNA-WISP1 lentivirus interference vectors and LiCI (the wnt/ß-catenin signaling pathway activator) to explore the regulatory mechanism of WISP1 in uremia in relation to the wnt/ß-catenin signaling pathway. Then, expression of WISP1, wnt2b, E-cadherin, α-SMA, c-myc, Cyclin D1, MMP-2, and MMP-9 was determined. Furthermore, TEC migration and invasion were evaluated. Results suggested that WISP1 and the wnt/ß-catenin signaling pathway were associated with uremia. Uremic rats exhibited increased serum creatinine and urea nitrogen levels, upregulated WISPl, and activated wnt/ß-catenin signaling pathway. Subsequently, WISP1 silencing decreased wnt2b, c-myc, Cyclin D1, α-SMA, MMP-2, and MMP-9 expression but increased E-cadherin expression, whereas LiCI treatment exhibited the opposite trends. In addition, WISP1 silencing suppressed TEC migration and invasion, whereas LiCI treatment promoted TEC migration and invasion. The findings indicate that WISP1 gene silencing suppresses the activation of the wnt/ß-catenin signaling pathway, thus reducing EMT of renal TECs in uremic rats.

Effects of stanozolol on bone mineral density and bone biomechanical properties of osteoporotic rats.

OBJECTIVE: To evaluate the effects of stanozolol on the bone mineral density (BMD) and bone biomechanical properties of rats with glucocorticoid (GC)-induced osteoporosis (OP). METHODS: Twenty-eight male Sprague-Dawley rats of 3-month old were randomly divided into Group A (the basal control group), Group B (the age-matched control group), Group C (GC-induced OP group) and Group D (stanozolol-administrated group), 7 in each group. The rats in Group A were killed when experiment commenced, and those in Group B were given normal saline ig., while those in Groups C and D received the prednisone acetate (4.5 mg/kg, twice a week) alone and in combination with stanozolol (0.5 mg/kg, 6 times a week), respectively. Ninety days later, the bilateral femur and the 5th lumbar vertebra of the rats were isolated for BMD test using dual-energy X-ray absorptiometry scanner, and the torsion test, three-point bending test and compression test using electronic testing device. RESULTS: Compared with Group B, the mean BMD of the femur and the 5th lumbar vertebra in Group C decreased by 14.64% (P<0.01), the BMD of the bilateral distal femoral segment and the 5th lumbar vertebra decreased by 21.42% (P<0.01), 19.62% (P<0.05) and 23.48% (P<0.01) respectively. The load that the femur withstood in three-point bending test decreased by 17.1% (P<0.05), and the other biomechanical parameters also declined. When compared with Group C, the BMD in Group D increased, the torsional angle of the femur increased by 72.5% (P<0.05) and the other biomechanical parameters also tended to increase. CONCLUSIONS: BMD and biomechanical properties of the rat femur and the 5th lumbar vertebra decrease in response to a long-term GC administration, which can be prevented by stanozolol.

[Effects of stilbestrol on bone growth and metabolism in ovariectomized rats].

OBJECTIVE: To study the effects of stilbestrol on bone growth and metabolism in ovariectomized rats. METHODS: Twenty-seven 3-month-old female SD rats were randomly divided into 3 equal groups, namely the control group, ovariectomized group (OEG), and OEG+estrogen (OEG+E) group. After ovariectomy, the rats in the third group were given stilbestrol 0.022 5 mg/d x kg x b x w by intragastric gavage for 90 d. A digital image analysis system was used for the measurement of histomorphometric parameters in the proximal tibial metaphysis of the rats. The three-dimensional structure of the femur was observed with scanning electron microscope, with the blood cholesterol level determined and the main viscera weighted. RESULTS: In comparison with the control rats, the trabecular bone area (TbAr) of ovariectomized rats was significantly reduced (-65%), trabecular bone space (TbSp) enlarged (+189%), and the bone formation rate (BFR) accelerated (+91%). The cancellous bone of the femurs manifested obvious signs of thinning, roughening and resorption lacunae to cause trabecular bone discontinuance, and osteoporosis occurred. The rats treated with stilbestrol, in contrast, had increased TbAr (by 128%), reduced TbSp (by 63%), lowered BFR (by 49%), and reduced osteoclasts (by 32%). The cancellous bone of the femurs was well arranged in close connection with each other, and the high cholesterol level and endocrinic changes in response to ovariectomy was corrected by stilbestrol, which also caused the increase of the uterine weight. CONCLUSION: Stilbestrol may help prevent primary osteoporosis but may increase the risk of female genital cancer.

[Piperazinyl estrone prevents bone loss in ovariectomized rats].

AIM: To determine the effect of piperazinyl estrone, a new estrogen derivative, on bone turnover, bone mass and uteri in ovariectomized rats. METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) or sham operated (sham) at the age of 3 months and treated with estrone (E) at 0.75 mg.kg-1.d-1, or with piperazinyl estrone (P-E) at 1 or 10 mg.kg-1.d-1, orally, for 3 months. At the time of death, the uterine weight was measured. Bone histomorphometric analysis of proximal tibial metaphyses (PTM) was performed in undecalcified sections. RESULTS: Bone histomorphometric data showed that the percent trabecular area (% Tb.Ar) of OVX rats with bone high turnover was significantly decreased. The uteri were atrophied. The percent trabecular area (% Tb.Ar) of estrone treated group was increased in decreasing bone turnover manner. But the size and weight of uteri in this group were increased vs OVX group. The bone loss induced by OVX was preserved by P-E treatment, but the mechanism of maintaining bone is different from that of E-treated rats. P-E treatment in low dose did not decrease any bone formation indices, such as percent labeling perimeter, bone formation rate per bone volume (BFR/BV), except bone mineral apposition rate (MAR) compared with E-treated group, and maintained them at OVX level. The uteri were found to be in atrophy compared with the match dose (0.75 mg) of E-treated OVX rats. But rats treated with high dose of P-E showed the same change like E-treated group. CONCLUSION: The finding of this study shows that lower dosage of piperazinyl estrone has effect on preventing the bone losses in OVX rats, while the bone formation and the uterus are not affected, thus supporting the hypothesis that piperazinyl estrone has the potential to prevent postmenopausal bone loss in women with less side effects.

Effects of low doses of hydrochloride tetracycline on bone metabolism and uterus in ovariectomized rats.

AIM: To study the effects of low doses of hydrochloride tetracycline (Tc) on bone metabolism and uterus in the ovariectomized (Ova) rats. METHODS: Forty 3-month-old rats were randomly divided into 5 groups: sham group, Ova group, Tc1 group (1.2 mg/kg/d), Tc2 group (4.8 mg/kg/d), and estrone group (1.48 mg/kg/d), oral fed for 3 months. The proximal tibia metaphyses were processed undecalcified for quantitative bone histomorphometry and the soft tissues were processed in paraffin for pathological observation. RESULTS: Placebo-treated (lactose) Ova rats were characterized by trabecular area (TA) decreasing and their architecture worsening compared with sham controls, and bone resorption was over formation with high bone turnover. The uteri were atrophy. (2) In estrone-treated group, TA and trabecular numbers were significantly increased and the trabecular separation decreased vs Ova group. Estrone slowed down Ova-inducing bone high turnover. But the size, weight, and the endometrium of the uteri in this group were increased vs Ova group. (3) TA was increased in both Tc1 and Tc2 groups compared with Ova rats. Tc maintained bone formation indices almost at Ova level, and only decreased mineral apposition rate (MAR) in Tc1 group, and declined bone resorption perimeter. The uteri and the cell of liver and kidney almost maintained at Ova level; Tc2 decreased labeling perimeter and increased MAR in comparison with Tc1 group. The uteri were atrophy, whose size maintained at Ova level; yellow labeling was not found in bone with these doses of Tc, while yellow labeling could be seen with the doses of 30 mg/kg/d of Tc for bone marker. CONCLUSION: The two doses of Tc have similar effects on preventing bone loss in Ova rats while the bone formation and uterus are not affected. However, Tc2 does not have more effects on increasing bone mass, Tc2 causes less mild damages to the liver and kidneys.

Effects of prednisone on bone mineral density and biomechanical characteristics of the femora and lumbar vertebras in rats.

OBJECTIVE: To explore effects of prednisone on the bone mineral density (BMD) and biomechanics of the femora and lumbar vertebras in rats. METHODS: Twenty one 3-month-old male Sprague-Dawley rats weighing 226+/-12 g were randomly divided into basal control, age-matched and hormone groups. The rats in basal control group were killed at the beginning of the experiment without any treatment, and those in age-matched group were given oral normal saline (5 ml x kg(-1) x d(-1)) while the rats in hormone group received oral prednisone acetate (4.5 ml x kg(-1) x d(-1) twice a week) to establish osteoporotic models. The treatment for the latter 2 groups of rats lasted for 90 days, after which the BMD and mechanical measurements of the femurs and L5 vertebra were carried out by way of torsion, three-point bending and compression tests. The measurements were also conducted in the basal control group at the time indicated above. RESULTS: In hormone group, the total BMD of the femora and L5 vertebra was decreased by 14.64%(P<0.01), and the BMD in the right and left distal femoral segments and the vertebra decreased by 21.42% (P<0.01), 19.62% (P<0.05) and 23.48%(P<0.01), respectively, in comparison with the control group. In the meantime, the loads of three-point bending test in hormone group was decreased by 17.1%(P<0.05), whereas the rest parameters tended to decrease as compared with the control group. CONCLUSIONS: Chronic use of corticosteroid is more liable to cause bone mass loss in rat cancellous bone than in the cortical bone, and mechanical properties of the cortical and cancellous bone, especially those of the latter, will also decline, to give rise to easy bone fracture at the trabecular bone in osteoporotic conditions.

Effect of intermittent injection of recombinant human parathyroid hormone on bone histomorphometry of ovariectomized rats.

AIM: To observe the effect of intermittent parathyroid hormone (PTH) administration on bone histomorphology of relatively old ovariectomized rats. METHODS: The 6-month-old female SD rats were randomly divided into 5 groups: (1) sham-operated for baseline (ShamB, n=5), (2)ovariectomized for baseline (OVXB, n=6), (3) Sham-operated for end point (ShamE, n=6), (4) ovariectomized for end point (OVXE, n=6), (5) ovariectomized for PTH treatment (OVXEP, n=6). ShamB and OVXB rats were sacrificed 3 months after operation, ShamE, OVXE and OVXEP rats were sacrificed 4.5 months after operation. During 3-4.5 months after operation, OVXEP rats received daily subcutaneous injection of rhPTH1-84, while ShamE and OVXE received vehicle injection. The proximal tibiae of all rats were processed without decalcification for quantitative bone histomorphometry. RESULTS: The percent trabecular area (TbAr) of OVXEP was significantly greater than that of OVXE (P <0.05), and was similar to that of OVXB (P >0.05), but was smaller than that of ShamE (P <0.05); the trabecular thickness (TbTh) of OVXEP was thicker than any other group (all, P <0.05); the trabecular number (TbN) of OVXEP was only slightly higher than that of OVXE; the percent labeled perimeter (LPm), mineral apposition rate (MAR) and bone formation rate with bone area as referent (BFR/BV) of OVXEP were all higher than those of ShamE and OVXE respectively (P <0.01), whereas the osteoclast number (N of Oc) of OVXEP was similar to those of ShamE and OVXE (P >0.05). CONCLUSION: Short-term intermittent injection of rhPTH1-84 can prevent further bone loss in 9-month-old rats 3 months after ovariectomy, the mechanisms of this therapy are that PTH could increase TbTh while not alter TbN, and promote bone-forming activity while not influence bone-resorptive activity.