| Osteoporosis is a chronic, degenerative disease of the skeleton characterized by reduced bone mass and micro-architectural deterioration, leading to enhanced bone fragility and a consequent increase in fracture risk. Osteoporosis is a systemic disease. The mortality and morbidity of this disease is high all over the world. Decreased hormone levels in postmenopausal women and the long-term use of glucocorticoids is a common cause of osteoporosis. Glucocorticoids (GCs) are often used for treating chronic diseases such as rheumatoid arthritis and asthma by some postmenopausal women, but there is little research on the interaction between these two factors. Calcitonin is one of effective drugs for postmenopausal osteoporosis (PMO), but the clinic studies on the effect of calcitonin on glucocorticoid-induced osteoporosis (GIO) are contradictory and the effect of calcitonin on postmenopausal-glucocorticoid induced osteoporosis is still unknown. The aim of our study was to establish three animal model of osteoporosis, including ovariectomized rat model of osteoporosis, rat model of glucocorticoid-induced osteoporosis and ovariectomized-glucocorticoid-induced osteoporosis of rat model, to compare the difference among these three kinds of rat model, to evaluate the effects of calcitonin, and to explore the bone remodeling features and mechanism in these three kinds of rat model. It was expected to provide experimental evidence for the clinic use of calcitonin. This study was consisted of three tests: Chapter III:Establishment and evaluation the osteoporosis modelTest I:Evaluation of rat bone mass measurement by dual-energy X-ray absorptiometry.Objective:To prove the precision of bone mineral density (BMD), the accuracy of bone mineral content (BMC).Methods:The precision of variation coefficient (CV) in measurement of BMD at various skeletal regions was repeatedly determined in 20 SD rats by DXA. The accuracy of BMC by DXA measurements was assessed in comparison with the ash weight at different skeletal sites in 18 SD rats.Results:The CV s for BMD measured by DXA were as follows, (0.74±0.40)% for whole skeleton,(1.47±0.57)% for L4,(1.38±0.54)% for L5,(1.38±0.48)% for L6,(0.54±0.28)% for femur(0.62±0.21)% for tibia. The CV s for BMC measured by DXA were as follows, (0.84±0.32% for whole skeleton, (1.37±0.67)% for L4, (1.48±0.54)% forL5, (1.44±0.39)% for L6, (0.72±0.34)% for femur, (0.57±0.32)% for tibia. There were highly significant correlations between in vitro DXA measurements and ash weights of the femur, tibia and lumbar spine. DXA-measured BMC at all skeletal sites were significantly lower than those of ash weight.Conclusion The rat bone mass measurement by dual-energy X-ray absorptiometry is precise and accuracyTestⅡDetection of sensitive regions of bone loss by Dual X-ray absorptiometry in rat models of osteoporosisObjective:To determine the sensitive regions of bone loss in rat osteoporotic models by dual X-ray absorptiometry (DXA).Methods:Thirty female Sprague-Dawley rats,44-week-old, were randomized into sham-operation (SHAM), ovariectomy (OVX), methylprednisolone injection (PRED) at 2.5mg-kg-1·d-1 and observed for 12 weeks post-surgery. The whole skeleton BMD, BMC, and bone area in vivo were measured by DXA (QDR-4500A, Hologic) every 4 weeks. After sacrifice of the animals, their lumbar spine and femur and tibia were resected and freed from soft tissue, and their BMD, BMC, and projectional area, including 7 equalized zones in the femurs and in the tibias, were measured using DXA.Results:①The body weight was significantly increased in OVX than that in SHAM since 8 weeks post-surgery (P<0.05), and markedly decreased in PRED than in SHAM since 4 weeks post-surgery (P<0.05).②The whole body BMC was significantly higher in OVX than that in SHAM at 12 weeks post-surgery (P<0.05) and in PRED at 8 and 12 weeks post-surgery (P<0.05) in vivo. ③The BMD of L5 and L6, and the BMC of L6 in OVX was significantly decreased at 12 weeks post-surgery (p＜0.05) in vitro, compared to that in SHAM and PRED. There was no significant difference in BMD, BMC, and bone area of the lumbar spine (L4, L5, L6) in vitro between PRED and SHAM.④Compared with SHAM, a significant decrease in BMD was revealed at whole femur (-7.45%), distal femur (-11.97%), proximal femur (-7.28%), and proximal tibia (-11.97%) in OVX at 12 weeks post-surgery, and a significant decrease in BMC was observed at these anatomical sites except the proximal femur.⑤There was no significant difference in BMD, BMC, and bone area of whole femur and all regions of interest of the femur, and BMD of whole tibia and all regions of interest of the tibia between SHAM and PRED at 12 weeks post-surgery.⑥Compared with SHAM, OVX group and PRED group showed a significant increase in bone mass at distal tibia in vitro.ConclusionThe sixth lumbar spine, distal femur, proximal femur, and proximal tibia, which are predominantly consisted of cancellous bone structure, are the most sensitive regions for detecting bone loss in mature ovariectomized rats by DXA. The findings are similar to bone loss in postmenopausal osteoporotic women. There is no significant change in cortical and cancellous bone mineral and projectional areas of the mature female rats after methylprednisolone injections, as assessed by DXA.Test III:Establishment and evaluation the osteoporosis model Objective:To establish three animal model of osteoporosis, including ovariectomized rat model of osteoporosis, rat model of glucocorticoid-induced osteoporosis and ovariectomized-glucocorticoid-induced osteoporosis of rat model, to compare the difference among these three kinds of rat modelMethods:Seventy female Sprague-Dawley(SD) rats age 44 weeks were divided into seven group, each group contain 10 rats. Rats in Group OVX were ovariectomized, rats in Group Sham were removed some fat tissue, rats in Group PRED received methylprednisolone(2.5mg/kg/d,sc),and rats in Group OVX+PRED were ovariectomized and received methylprednisolone. The rest would be used in Chapter IV.The total BMD of whole body would be examined by DXA every 4 weeks. After 12 weeks, these rats were anesthetized. Blood samples were withdrawn to assess biochemical markers of bone metabolism and the level of Ca and P in Chapter V. At sacrifice, the fourth lumbar vertebra, the fifth lumbar vertebra, the sixth lumbar vertebra, the femora and tibia were separated, cleaned of soft tissues. DXA, Micro CT scanning, bone histomorphometric analysis, biomechanical testing was progressed gradually. The data were statistically analyzed by SPSS 16.0.Results:The operations were made successfully. After 12 weeks, the body weight of Group OVX is heavier than Group Sham significantly, the body weight of Group Sham+M reduced significantly than Group Sham, the body weight of Group OVX+M is not different from Group Sham. The weight of uterus and coefficient of uterus were significantly reduced in Group OVX and Group OVX+M than Group Sham (P<0.05). The total BMD of whole body and the ash weight of right tibia is not different among these group (P>0.05)1、Results of DXA:The image of femur and tibia were divided into seven areas (R1-R7), called Region of interest (ROI). Compared with Group Sham, the BMD of lumbar vertebra, total femora, distal femur (FROI-1、2), proximal femur (FROI-7), total tibia and proximal tibia (TROI-1～3) was significantly decreased in Group OVX. Compared with Group Sham, the bone mass in Group PRED was not significantly changed. In Group OVX+PRED, bone lost was more severe.2、Bone histomorphometric analysisCompared with Group Sham, BV/TV and Tb.N were significantly decreased and Tb.Sp was significantly increased in Group OVX, Group PRED and Group OVX+PRED. In Group OVX, Tb.Th was not different from Group SHAM, BFR and MAR increased by 118.94% and 74.07%. Compared with Group SHAM, Tb.Th, BFR and MAR were significantly decreased by 15.96%,28.46% and 24.69% respectively in Group PRED. Compared with Group Sham, Tb.N was significantly decreased by 51.48%; BFR was significantly increased by 29.67%.3、High-resolution X-ray micro-computed tomography (Micro CT) The micro-architecture of cortical bone in proximal tibia changed tinily (P>0.05).After Ovariectomy, the connection of bone trabecula deteriorated and the thickness of bone trabecula kept normal. After the injection of methylprednisolone, the thickness of bone trabecula thinned. When the two factor came together, all kinds of parameters deteriorated rapidly. 4、Biomechanics testsIn the three-point bend test, the max load, max stress, elastic load and elastic stress did not change in Group OVX compare with Group Sham (P >0.05), however these parameters was decreased significantly in Group PRED and Group OVX+PRED(P<0.05).In the compression test, the maximum loading and elastic modulus was decreased significantly in Group OVX and Group OVX+PRED than Group Sham (P<0.01). The maximum loading in Group PRED is higher than it in Group Sham (P<0.05)Conclusion:It is successful to establish three kinds of rat model of osteoporosis. After 12 weeks, the rats in model group are in osteoporotic status. Body weight changes significantly. Bone mass is reduced. Trabecular micro-architecture has changed,and bone biomechanical performance is worsening. Chapter IV Experimental study of calcitonin effects on rat model of osteoporosisObjective:To evaluate the effects of calcitonin in different rat model of osteoporosisMethods:Thirty rats which were from the rest in ChapterⅢTest 3 were divided into three groups:Group OVX+C, Group PRED+C and Group OVX+PRED+C. These rats would receive calcitonin (4mg/kg/d, sc).The rest methods are the same which mentioned in ChapterⅢTest 3.Results:Compared with the corresponding non-intervention group, the body weight decreased significantly in intervention group (Group OVX vs. Group OVX+C, Group Sham+M vs. Group Sham+M+C, Group OVX+M vs. Group OVX+M+C). Compared with the corresponding non-intervention group, the weight of uterus and coefficient of uterus did not change.1、DXA measurements:The bone mass in lumbar was not significantly changed between the intervention group and non-intervention group. Compared with Group OVX, the BMD and BMC of total femur and FROI-1, and the BMD of FROI-2, and the BMC of FROI-5,6 were significantly decreased in Group OVX+C. The BMD of FROI-1,2 were significantly lower in Group PRED+C than Group PRED and SHAM. The BMD of total femur and FROI-1,2 in Group OVX+PRED+C were significantly increased than Group OVX+PRED The BMD, BMC of total tibia and TROI-1 in Group OVX+C were significantly lower than Group SHAM. The BMC of total tibia was significantly lower than Group OVX. The bone mass in all skeleton area was not changed significantly in Group PRED+C. The BMD of total tibia and FROI-1,2 in Group OVX+PRED+C were significantly increased than Group OVX+PRED.2、Bone histomorphometric analysisAfter the intervention of calcitonin, the parameters of trabecular bone were inproved to some extent. Compared with Group OVX, BV/TV was increased by 111.95%, Tb.N was increased by 62.20%, Tb.Sp was decreased by 55.41% in Group OVX+C.BV/TV was better in Group OVX+C than Group Sham. Compared with Group Sham+M, BV/TV, Tb.Th, Tb.N was increased by 70.60%,27.23%,39.30% respectively in Group Sham+M+C, and Tb.Sp was decreased by 39.70%. Compared with Group OVX+M, BV/TV, Tb.Th, Tb.N was increased by 140.91%,20.59%,99.06% respectively in Group OVX+M+C. All parameters in Group OVX+M+C were restored to normal3、High-resolution X-ray micro-computed tomography (Micro CT)After the intervention of calcitonin, the parameters of trabecular bone which were examined by Micro CT were improved to some extent4、Biomechanics testsIn the three-point bend test, the max load, max stress, elastic load and elastic stress did not change in Group OVX+C compare with Group Sham and Group OVX (P>0.05).In the compression test, the maximum loading and elastic modulus were improved in Group OVX+C but the maximum loading were not restored to normal. In Group SHAM+M+C, the maximum loading of shaft of femur and second lumbar vertebra were improved to normal(P<0.01,vs.Group Sham+M; P> 0.05,vs.Group Sham),and other parameters were insignificantly improved.Compared with Group OVX+M, elastic modulus of second lumbar vertebra and elastic stress of shaft of femur were improved significantly in Group OVX+M+C,),and other parameters were still insignificantly improved.Conclusion:1:Calcitonin had effect on body weight.2:Calcitonin had different effect on BMD and BMC in different skeleton area and group.3:It can reverse the effects of OVX, methylprednisolone on bone strength to same extent, at the same time calcitonin has effect on bone remodeling.3:The cancellous bone is more sensitive to calcitonin. Calcitonin has the effect on reducing the fracture rate through improvement of bone micro-structure Chapter V Study on the mechanism of protective effect on bone by calcitoninObjectiveTo explore the bone remodeling features and mechanism in these three kinds of rat model and to evaluate the mechanism of protective effect on bone by calcitonin.MethodsAfter 12 weeks, these rats were anesthetized. Blood samples were withdrawn to assess biochemical markers of bone metabolism and the level of Ca and P. After the examination of DXA, the distal left femur would be fixed, decalcified, and then embedded in paraffin. Serial sections were collected on charged, precleaned microscope slides. These slides would be used for TUNEL stain.Results1、TUNEL assay:Compared with Group Sham, positive expression rate of apoptosis cells was significantly increased in the osteoblast in the rest group. There is no difference between the corresponding non-intervention group and intervention group in positive expression rate of apoptosis cells in the osteoblast (P>0.05).Compared with Group Sham, positive expression rate of apoptosis cells was significantly increased in the osteoclast in Group OVX, Group OVX+M, Group OVX+C and Group OVX+M+C. There is no difference between the corresponding non-intervention group and intervention group in positive expression rate of apoptosis cells in the osteoclast (P>0.05). There is no difference among Group Sham, Group Sham+M and Group Sham+M+C2、Biochemical markers of bone metabolism and the level of Ca and PCompared with Group Sham, the level of Ca was significantly decreased in Group OVX, Group OVX+M, Group OVX+C and Group OVX+M+C. Compared with Group Sham+M, Ca was significantly decreased in Group OVX+M. Compared with the corresponding non-intervention group, and Ca was significantly decreased in intervention group. Compared with Group Sham, the level of P was significantly decreased in Group OVX, Group OVX+M, Group OVX+C, Group Sham+M+C and Group OVX+M+C.The level of P in Group OVX+M was lower than Group Sham+M and higher than Group OVX. After the intervention, P was decreased in Group OVX+C and Group OVX+M+C in comparison with Group OVX and Group OVX+M respectively. Compared with Group Sham+M, P was increased in Group Sham+M+C.Compared with Group Sham, ALP-B was increased significantly in those groups which received Ovariectomy and was decreased significantly in Group Sham+M+C. There was no difference between Group Sham and Group Sham+M. Compared with the corresponding non-intervention group, ALP-B was significantly decreased in intervention group.Compared with Group Sham, Osteocalcin was increased significantly in those groups which received Ovariectomy and was decreased significantly in Group Sham+M and Group Sham+M+C. Compared with Group OVX+M, Osteocalcin was decreased significantly in Group OVX+M+C. There was no difference between Group Sham+M+C and Group Sham+M, and Group OVX+C and Group OVX. Compared with Group Sham, CTX-I was significantly increased in Group OVX, Group OVX+M, Group OVX+C, Group Sham+M+C and Group OVX+M+C. Compared with Group OVX, CTX-I was significantly increased in Group OVX+C. Compared with Group Sham+M+C, CTX-I was significantly increased in Group Sham+M.Conclusion1:Encountered with any factor, the number of osteoblast or osteoclast not was not proportional to its activity.2:Calcitonin had no effect on the interaction which was between osteoblast and osteoclast, the number and function of osteoblast.3:Calcitonin only depressed the function of osteoclast and did not induce osteoclast apoptosis... |
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