| ObjectiveThe traumatic ONFH model in rats was improved,and based on the animal model,the effects of zoledronic acid and human umbilical cord mesenchymal stem cells(hUCMSCs)on the prevention and treatment of early ONFH in rats were explored through imaging,histology,pathology and other means,so as to provide support for clinical application.Methodsthe Existing rat model with traumatic ONFH was modified appropriately,according to the rat femoral anatomic characteristics,the main blood supply is located on the head and neck bone in circumflex artery anastomoses,we used silk thread to round the femoral head and neck,after the two end cross,slow to pull both sides with standard unified operation process and pulled the same number and strength,which ensure the homogeneity of the animal model.4 weeks after surgery,imaging,pathology and other means were used to verify that the animal model was successful or not.2.The effect of zoledronic acid on the prevention and treatment of ONFH in rats was evaluated by micro-CT,HE,toluidine blue,Masson,TRAP staining and other methods based on local injection of femoral head on the basis of stable animal model 3.On the basis of the stability of ONFH model,micro-CT,HE,Masson,saffron O solid green staining and immunofluorescence staining were used to evaluate the prevention and treatment effect of hUCMSCs on ONFH.Results1.Four weeks after surgery,the morphology of the femoral head on the side of the ONFH model was significantly changed compared with that of the healthy side in micro-CT images.The continuity of the subchondral bone tissue trabecula was interrupted and disorganized and the local trabecula was sparse,and even the bone resorption was obvious.X-ray scanning of the bilateral hip joints of rats showed that the shape of the femoral head on the model side was significantly smaller and abnormal compared with that on the healthy side,the femoral neck was thinner and the metaphyseal height was significantly smaller.He staining analysis of the femoral head coronal surface at different magnification times:the bone trabecula in the healthy side of the femoral head was compact,continuous and strong,and almost no empty bone lacunae or osteocytes died.In the femoral head of the side of the model,bone trabeculae were sparse,broken,continuity was interrupted,and a large number of bone cells died.The results showed that the femoral head necrosis was observed in all rats,and the traumatic ONFH model was stable in rats.2.4 weeks after local injection of zoledronic acid,according to the results of Micro-CT scan,groups of femoral head internal structure were different,model group and normal saline treatment femoral head external morphological changed obviously,the femoral neck became thinner and femoral head became smaller,the internal trabecular bone was thinning,interruption,uneven distribution and even had severe bone resorption.On the contrary,femoral head shape in zoledronic acid group was good,the femoral neck was even thickness,internal bone trabecular was coarser,trabecular bone fracture was less and had good continuity,there were not obvious bone resorption area,structure is relatively complete,close to the normal group,BMD,Tb.Th and BS/BV were significantly different among the normal group,the model group,the saline group and the zoledronic acid treatment group,and the difference was statistically significant(P<0.05),while there was no significant difference in BV,BV/TV and Tb.SP among the four groups(P>0.05);He,Masson and toluidine blue staining results showed that zoledronic acid treatment group was better than model group and normal saline treatment group.TRAP staining analysis showed that there were significantly less multinucleated cells on the surface of bone trabecular in the normal group and the zoledronic acid treatment group with red cytoplasm and light blue nuclei(osteoclasts)than in the model group and the normal saline treatment group.Therefore,zoledronic acid can significantly improve the morphology and bone structure of femoral head in rats.3.4 weeks after local injection of hUCMSCs,according to the results of Micro-after a CT scan,groups of femoral head internal structure were different,in 4 week model group,4 week NS group,8 week model group,8 week NS group and 8 week hUCMSCs group femoral head external morphological changed obviously,the femoral neck became thinner obviously,trabecular bone was thinning,interruption,uneven distribution and had severe bone resorption.On the contrary,in 4-week hUCMSCs group,femoral head shape was good,the femoral neck was even thickness,internal bone trabecular was coarser,trabecular bone fracture was less and had good continuity,There was no obvious bone resorption,and the structure was more complete,closer to the normal group.BMD,BV/TV,Tb.Th and BS/BV were significantly different among the normal group,the 4-week model group,the 4-week normal saline group and the 4-week hUCMSCs group,and the differences were statistically significant(P<0.05);HE staining,Masson staining,and saffine O solid green staining showed that the 4-week hUCMSCs group was significantly better than the model group and 8-week hUCMSCs group.Immunofluorescence test,found that in 4-week hUCMSCs group,there were a large number of hUCMSCs in femoral head,but in 8-week hUCMSCs group,hUCMSCs were not observed,in 4-week hUCMSCs group,the expression of cartilage growth active protein SOX9 and SP7/Osterix responsing osteogenesis ability were significantly better than that of 4-week model group,4-week normal saline group and 8-week hUCMSCs;Serum suspension microarray(Luminex)showed that human derived cytokines,such as IL-1β,IL-8,IL-4,IL-6,IL-10,MCP-1,TNF-α,RANTES and VEGF were present in serum of the 4-week hUCMSCs group and the 8-week hUCMSCs group.ConclusionThe femoral head shape of modified rat ONFH changed,trabecular bone fracture,Bone trabeculae was sparse,integrity was broken,there are many empty bone trabecular on the bone surface,osteonecrosis was obvious,animal model was stable,on the basis of the stable ONFH model,we verified that zoledronic acid could inhibit the pathological process of early traumatic ONFH,and indicated that hUCMSCs had a positive outcome in early traumatic ONFH. |
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