Influences Of Alendronate On Osteoporotic Fraeture Healing In A Rat Model

PART1ESTABLISHMENT AND EVALUATION OF ANOSTEOPOROSIS RAT MODELObjective：To investigate the influences of ovariectomy(OVX) onbone mineral density(BMD) and biomechanics in a femaleSprague-Dawley (SD) rat to provide stable and reliable osteoporosis modelfor follow-up experiment.Methods：One hundred four-month-old adult female SD rats of250to300grams were randomly divided into two groups: blank group (n=5) andovariectomized group(n=95). To confirm bone loss of ovariectomised mice,we performed Micro-CT analysis8weeks after ovariectomy (OVX). Thebone mineral density (BMD) measurement of rats of blank group andovariectomized group were detected by DEXA (dual energy X-rayabsorptiometry, DEXA) of the operation and three-point-bending test wasused to evaluate the mechanical properties of bone (femur shaft). Theresults of the two groups were compared and statistically analyzed.Results:8weeks later, Micro-CT scanning results revealed thattrabecular bone thicknesss, cortical wall thickness and trabecular numberwere significantly lower in the ovariectomized group than those in the blank control group. The BMD detected by DEXA in ovariectomized groupand blank group were0.219±0.039（g/cm2）and0.249±0.063（g/cm2）respectively. The BMD of ovariectomized group had significantlydifference compared with blank group (p<0.05). In3-point bending test, theproperties of bone decreased significantly in ovariectomized groupcompared by blank group at8weeks and there was difference between thetwo groups.Conclusion: An osteoporosis rat model can be established byovariectomy after8weeks. It is an ideal osteoporosis model to understandthe pathogenesis and develop new drugs of osteoporosis.PART2THE STUDY ON OSTEOPOROTIC FRAETUREHEALING IN AN RAT MODEL WITH ALENDRONATEObjective：In this study, we mainly focused on castration SD rat modelof osteoporotic fraeture by assessing the change of biomechanicsperformance and using other experimental methods including histologicalobservation, immunohistochemistry and molecular biology. The aim of thisstudy was to investigate the effects and mechanisms of alendronate on thebiological characteristics of the relevant factors functioning osteoporoticfracture healing at different time in Rat Femora and to provide proofs and anew way for therapy strategies of alendronate to prevent and treatosteoporotic fracture. Methods：After these models were confirmed, middle open fracturemodel was established in all60model rats on right side femur and eachfracture was fixed with a Kirschner pin. The other30rats were used assham group of a puncture with Kirschner pins into femur but without bonefracture. Fracture model rats were randomly divided into control andtreatment groups of30rats each. Treatment group rats were given bygavage once a week with suspensions of alendronat at does of1.5mg/kg.The sham operation group and control group were given an equivalentvolume and the same quantity of normal saline. Drawing blood fromeyeball of randomly selected five rats in each group after operation and allthe blood were centrifuged to get serum. The changes of expression ofblood ALP, BMP-2, bFGF and IGF-1were detected. Fracture healing of thefemur in rats were observed and compared with DEXA. After killing therats, callus specimens from fracture ends were fixed in4%paraformaldehyde solution overnight. Paraffin slices decalcified by5%nitric acid were monitored by hematoxylin and eosin (H&E) stain,tartrate-resistant acid phosphatase (TRAP) stain and immunohistochemistryof BMP-2, bFGF and IGF-1. Micro-CT, scanning electron microscopy andthree-point bending test were used to observe healing of fracture in threegroups at4and12weeks.Results: The DEXA and histology observation of callus showed,compared with control group, alendronate could accelerate the fracture healing process after treantment for2weeks. HE staining of tissue sliceshowed callus formation is increased and endochondral ossification ofalendronate group is speeded up compared with control group.DEXA observation showed fracture lines of alendronate group were fuzzywhile the control group were still with visible fracture lines, whichprompted bone union came into being more quickly after treatmentwith alendronate for6weeks. The TRAP staining results showed thenumber of osteoclast in alendronate group decreased gradully and wassignificantly lower than that of control group after treatmentwith alendronate. The changes of ALP, BMP-2, bFGF and IGF-1of serummeasured by ELISA have no significant differences in all time periods insham group. The difference of the values of serum ALP betweenalendronate and control group at2,4and6weeks have statistical sense.The values of serum BMP-2, bFGF and IGF-1in each group both reachedpeak after alendronate treatment for2weeks and then decreased to nomallevel at8,12weeks. The values of serum BMP-2in control group werelower than that of alendronate group before8weeks and there weresignificant differences in values of serum BMP-2between alendronateand control group with statistical meanings after alendronate treatment for2and6weeks. The values of serum bFGF in control group were muchlower than that of alendronate group at4and8weeks and the differenceshave statistics meaning. The values of serum IGF-1in alendronate group were much higer than that of control group and there were significantdifferences in values of serum IGF-1between alendronate and controlgroup with statistical meanings at2and4weeks. There were no differencesbetween two groups in the cellular localization of the expression of BMP-2,bFGF and IGF-1detected by immunohistochemical staining during fracturehealing process. The mean optic densitys of BMP-2detected byimmunohistochemical staining of alendronate group were higher than thoseof control group at4weeks，but lower at8weeks. These differences werestatistically significant. The mean optic densitys of bFGF of alendronategroup were higher than those of control group at4and8weeks.The mean optic densitysof bFGF of alendronate group were higher thanthose of control group at2and4weeks. At4weeks posttreatment, callusvolume and the percentage of callus volume mineralization quantifiedusing Micro-CT in alendronate group were higher than those of controlgroup while had not changed significantly at12weeks. The callus wereobserved with scanning electronic microscope and the numbers of boneresorption pits of alendronate group were lower than those of control groupat12weeks posttreatment. The result of biological mechanics showedmaximum load value of alendronate group were higher than those ofcontrol group at both4and12weeks and these differences werestatistically significant.Conclusion: alendronate could promote the healing of rat’s osteoporotic fracture because its effects on increasing the concentration of ALP, BMP-2,bFGF and IGF-1cytokines which can accelerate bone formation, inhibitedthe activity of osteoclasts, increased new bone callus, the activityof osteoblasts, bone density of callus and bone biomechanical.