| Blackgroud and ObjectiveOsteonecrosis of femoral head is a chronic disabling and crippling disorder. Without treatment,＞70%of femoral heads with osteonecrosis collapse and require prosthetic replacement within three to four years of diagnosis.Osteonecrosis of femoral head occurs often in the people aged in 30 to 50years and many of them need prosthetic replacement more than once.It is a heavy burden for the patients in body and spirit.Early therapy is vital for prognosis and prevention of cripple,the goal of early treatment for NOFH must be to reserve femoral head and prevente it from collapsing.Progression of collapse is greatly influenced by repair reaction,especially bone resorption in the necrotic bone.Necrotic bone retains load beating capacity.The death of bone cells per se does not cause structural failure.Rather,it is caused by the resorption of necrotic bone that occurs during or following the revascularization of the necrotic tissue.Excess osteoclast activity over osteoblast activity may decrease mechanical strength of the repair region and lead to collapse of the femoral head.If the bone resorption associated with osteonecreosis can be inhibited or delayed until sufficient new bone has formed,it would keep the structure of femoral head and delay or prevent it from collapsing. Bisphosphonates are the most important class of antiresorptive agents and areused in the treatment of ONFH and gained considerable attention recently.The biological basis for bisphosphonate therapy is derived from the relationship between the repair process maked by osteoclastic bone resorption and the development of deformithy.Studys show that at the tissue level,bisphosphonates treatment decreased the rate of bone remodelling,thereby contracting the remodelling spaces.Although the slower remodelling imperceptibly reduces bone tissue,the remaining bone tissue becomes more mineralized and thus denser.The slowly decreasing tissue mass thus becomes more densely laden with minerals and is better able to withstand the stress of weight-beating.Bisphosphonates thus seem to preserve bone architecture and also to increase bone mineral density.There are several limitations to the therapy of femoral head with bisphosphonates,including the disputation of influence on osteoblast,dosage and the ways of administer.Effects of bisphosphonates on the osteoclast reach a consensus. However,the effects on osteoblast are different because of the variety classes and different concentration of bisphosphonates.The currently dosage of therapy of femoral head consults the dosage for osteoporosis treatment.The optimal dosage for osteonecrosis of femoral head therapy remain unkown.Bisphosphonates are administered orally or systemically(subcutaneously or intravenously).Absorbability of oral administration is poor and there is complication of alimentary tract. Systemical administration maybe cause injury of kidney.Study of local bioavailability of systemically administered bisphosphonate in the infracted femoral head shows that bisphosphonate cannot access the infracted femoral unless revascularizaton of the femoral head is initiated.To get the therapeutic level and produce the pharmacological effect,bigger dosage has to be admininstered in the animal stydys.It will result in osteomalacia in adults and inhibition of long bone growth in children.In a local hip disorder caused by ischemic osteonecrosis,there is a need to explore the therapeutic potential of local administration of bisphosphonate to prevent the femoral head deformity.Bisphophonate is injected directly into the head and the dosage is smaller,its delivery and distribution does not depend on the vascular status of the infracted head.It also can avoid the complication.This study use zoledronic acid,a third-generation,nitrogen-containing bisphosphonate.To explore the influence of zoledronic acid on the proliferation,alkaline phosphatase activities,mineralization and OCN,OPG/OPGL mRNA expression of osteoblast isolated from the calvaria of neonatal SD rats within 24 hours and to investigate the possibility of therapy with zoledronic acid in osteonecrosis of femoral head.To establish and verify the rat model of traumatic osteonecrosis of femoral head through studying the pathological process.To achieve the effects on inhibition of bone resorption and promotion of new bone formation and to delay or prevent from deformity of femoral head by local injecton of zoledronic acid combined with osteoblast allotransplantation in the infracted femoral head after core depression.Methods1,Effects of zoledronic acid on the function of cultured rat osteoblast:Primary rat osteoblast cells were obtained by sequential enzyme digestion of excised calvarial bones from neonatal Sprague-Dawley rats.Cells was maintained in DMEM supplemented with 10%FCS at 37℃in 5%CO2.Cells was adherent after 24 hours and changed medium after 48 hours.Passage was made after 80%of the cells reaching confluence,3rd generation cells were used for the experiment.MTT colorimetric assay:Osteoblast were seeded at a density of 7×103 cells/200ul/well in 96-well plates.After overnight incubation,Zoledronic acid was added at concentrations ranging from 10-4M—10-11M in the medium and the control was changed medium without zoledronic acid.The cells were cultured with replacement of the culture medium every 3 days.MTT colorimetric assay was measured at 1,2,3, 5 and 7 days.In continuation of the protocol,the medium was aspirated and 20μl of MTT was added to each well and cultures continued for 4 hours at 37℃.The cells were then aspirated and 150μl of DMSO was added to each well.Colorimetric changes were quantified in a microplate reader at the wavelength of 490nm.Alkaline phosphatase assay:Osteoblasts were cultured in the flask,zoledronic acid was added at concentrations of 10-7 M,10-9 M,10-11 M in the medium the next day.Cells of control were changed blank medium.After 5 days,cells were digested with trypsinization and washed with PBS.1%SDS lysis buffer were added after centrifugalization.Alkaline phosphatase activity was assessed by PNPP approach with 50ul/well cell lysate in the 96-well plate.Cell lysate were repeated in six wells. Mineralization assay:Osteoblasts cultured in the flask with blank medium or zoledronic acid in the medium at concentrations of 10-7 M,10-9 M,10-11 M.The medium with or without zoledronic acid supplemented with 10mMβ-glycerol phosphate and 50ug/ml L-ascorbic acid.The cells were cultured for 21 days,with replacement of the culture medium every 3 days and daily replacement of L-ascorbic acid.At the end of the cultures,mineralized nodules were detected by von kossa staining.The areas of the mineralized nodules were examined by IPP6.0 image analysis soft.Real time-polymerase chain reaction:Osteoblasts cultured in the flask with blank medium or zoledronic acid in the medium at concentrations of 10-7 M,10-9 M,10-11 M.After 3days and 7days post-treatment,total RNA was isolated from cells using TRIzol reagent.Reverse transcription and real time fluorescent quantitation PCR was run to test the expression of OCN,OPG,OPGL mRNA after assessment of RNA quality.β-actin was utilized as a housekeeping gene.Outcome was analysised by Comparative Delta-delta Ct method.2,Establishment on Animal Model of Traumatic Osteonecrosis of the Femoral Head:32 SD rats about 6 mouths old were divided randomly into experimental and control groups.The animal models of femoral head necrosis were established in 32 SD rats according the method of Norman's:The animals were anaesthetized with Chloral Hydrateby.After skin shaving,local antisepsis and draping,a proximal longitudinal incision was made over the large trochanter.The gluteus maximus muscle was spilt in the direction of its bundles and the antenor two thirds of the gluteus medius muscle were detached from the bone.The antero-lateral join capsule insertion was transected along the trochanteric ridge.The ligamentum teres was cut and the femoral head dislocated using a number 11 blade,the periosteum at the base of the neck was incised together with reflected fibers of the capsue.Following relocation of the femoral head,the joint capsue and gluteal muscles were sutured and the skin was closed.The rats were placed in spacious cages so that their perambulation was unhampered and encouraged the rats to stand erect while feeding. 32 animals were sacrificed at 1th,2th,4th and 6th week after operation respectively. The specimens were examined through gross anatomy,X-ray plate and histological observation under light microscope.Other side was served as normal control group without any operation.The comparison of fat tissue with hematopoietic tissue,fat cells morphologic parameters(diameter,area,cycle) in the cavity of bone marrow of femoral head were performed,followed by CMIAS computer-assisted image and statistical analysis.The percentage of empty lacuna in the femoral heads was demonstrated.Immunohistochemitry were used to determine the expression of OPG/OPGL protein in ONFH.3,Experimental study on treatment of osteonecrosis of femoral head with local injection of Zoledronic acid combined with osteoblast allotransplantation:The animal models of femoral head necrosis were established in SD rats about 6 mouths old according the method of Norman's.The ligamentum teres was cut and the femoral head dislocated,the periosteum at the base of the neck was incised together with reflected fibers of the capsue.SD rats were divided randomly into experimental group from B to I.and control group A.A:modeling without therapy.One week after modeling,another operation was conducted with rats of group B,D-I.Incision was made over the large trochanter,the approach was made with the help of C-arm radiography.B treat with core depression;C treat with 0.1mg/kg zoledronic acid subcutaneous after modeling 1 and 4w;D-G treat with core depression and local infection of zoledronic acid at 2.5%,5%,7.5%,10%of the total cumulative subcutameous dose;H treat with core depression and osteoblast allotransplantation in the infarcted femoral head;I treat with core depression and local infection of zoledronic acid at 7.5%of the total cumulative dose combinated with osteoblast allotransplantation.Osteoblasts were obtained by sequential enzyme digestion of excised calvarial bones from neonatal Sprague-Dawley rats.Before allotrasnsplantation the osteoblasts were treated with profound hypothermia freezing, resuscitation and passage.The density was 107/0.5ml.Drilling holes were closed with bone wax,sutured the skin.The end of therapy was the day after modeling 6weeks.Rats were sacrificed and the specimens were examined through gross anatomy,X-ray and histological observation under light microscope,Bone histomorphometric parameters(including percent trabecular area,BV/TV;Trabecular number,Tb.N;Trabecular thickness Tb.Th;Trabecular separation,Tb.Sp) was measured by undecalcified slices staining.Immunohistochemistry were used to determine the expression of OPG/OPGL protein in ONFH.Results1,Effects of zoledronic acid on the function of cultured rat osteoblast:Zoledronic acid inhibited proliferation of osteoblast at concentration of 10-5M and greater,had no effect on proliferation of osteoblast at concentation between 10-6M to 10-11 M.There was no effect of the zoledronic acid on alkaline phosphatase activity at 10-7TM—10-11 M concentrations and mineralized bone nodules formation at concentration of 10-9M,but zoledronic acid inhibited mineralized bone nodules formation at concentration of 10-7 M(P＜0.05),promoted mineralization at concentration of 10-11M(P＜0.05).The expression of OPG mRNA and OCN mRNA was increased(P＜0.05) but expression of OPGL mRNA was inhibited(P＜0.05).2,Establishment on Animal Model of Traumatic Osteonecrosis of the Femoral Head: ①Osteonecrosis of the femoral head was confirmed in experimental group: Cartilage stripped and the head of femoral was deformed after modeling.There was different density and collapse of femoral head through radiographic assessment. ONFH model in progressive stage was duplicated in the typical pathological changes appeared after modeling.②Compared with normal control group,the percentage of empty lacuna remarkable increase was found in experimental groups at different periods(P＜0.05).The ratio of fat tissue of ONFH to hematopoietic tissue,fat cells diameter,area,cycle in the cavity of bone marrow of femoral head were significantly higher than that control groups(P＜0.05).③The expression of OPG/OPGL protein of ONFH at different periods were found in the cytoplasm of osteoblasts,bone matrix, chondrocytes,osteoclasts,fibroblasts,vascular endothelial cells and in ONFH group. The expression of OPG protein of ONFH was significantly lower than that normal control groups(P＜0.05),and the expression of OPGL was significantly higher control groups(P＜0.05).3,Experimental study on treatment of osteonecrosis of femoral head with local injection of Zoledronic acid combined with osteoblast allotransplantation:There was no statistical difference between group A and group B but significant difference comparing group A,B with group C-G at gross observation,X-ray, epiphyseal quotient,histological observation,number of osteoclasts,the expression of OPG/OPGL protein and bone histomorphometric parameters.There was no statistical difference between group C and D,too.A dose-dependent effect on preseravation of the femoral head structure was abserved in group D to F according to the outcome from gross observation,X-ray,epiphyseal quotient,histological observation,number of osteoclasts,the expression of OPG/OPGL protein and bone histomorphometric parameters.Group G wasn't better than group F.Osteoblasts allotransplantation can improve bone formation.Zoledronic acid administered intraosseously combination with osteoblast allotransplantation in the infarcted femoral head can preserve femoral head structure best in this study. Conclusion1,There is no effect on proliferation and Alkaline phosphatase activity of zoledronic acid at lower concentrations(≤10-9M).But the expression of OCN mRNA was promoted and zoledronic acid inhibited the differentiation of osteoclasts through increasing the rate of OPG/RANKL mRNA.It is possible for zoledronic acid to treat Osteonecrosis of the Femoral Head.2,①Norman's traumatic model showed progressive stage and typical pathological changes of ONFH.It can be used in the experiment of therapy of ONFH.②The expressional level of OPG/OPGL protein in ONFH is obvious correlation with pathological change.The expression of OPG protein decreased gradually at different periods,while OPGL protein is opposite to OPG.The overexpression OPG protein has been identified to inhibit osteoclastic bone resorption effect,but low expression might promote bone resorption.③The increasing concentration of OPG in the field of ONFH may inhibit bone loss and have beneficial repairing process of ONFH.It is possible to offer the new research pathway in the early- stage therapy of ONFH.3,It doesn't depend on the vascular status of the infarcted head when zoledronic acid administered intraosseously.The dose that Zoledronic acid injected into the head can be controlled and the complication that zoledronic acid inhibit bone growing when it was administered in a systemic way can be avoid.It will be a new method about osteonecrosis of femoral head.A dose-dependent effect on preseravation of the femoral head structure was abserved when the dose was lower than the percentage 7.5 of the total cumulative systemic dose.It was presumed 7.5%of the total cumulative systemic dose is suitable for zoledronic acid to administer intraosseously into the infarcted femoral head.Therapy of zoledronic acid administered intraosseously combination with osteoblast allotransplantation took a better effects on the osteonecrosis of femoral head than zoledronic acid or osteoblasts alone injected into the infracted head by inhibiting the absorbed caused by osteoclasts and improving the bone formation.
|
PDF Full Text Request