Analysis Of The Osteogenetic And Osteoclastic Activity With The Treatment Of Zoledronic Acid In The Rat

Objective1.To determine the vitro Morphology of the osteoclast.2.To determine the influence of ZOL(Zoledronic Acid)to the osteoblast,Macrophage and Bone Marrow Stromal Cell(BMSC).3.To explore the effect of osteogenic effect in the bone defect healing with the treatment of ZOL.Method1.OC(osteoclast)were isolated from 2-4 days rabbit with the method by washing the limb bone pieces.The OC of rat is cultured by induction of the rat bone marrow cell with the M-CSF(macrophage-stimulating factor)and RANKL(Receptor Activator of Nuclear Factor-κ B Ligand).with several metheods as HE staining and cell fluorescence,OC were compared with its shape and inner cellular organelle in this two method.2.The OB(osteoblast)were isolated from the neonatal rat by digesting the cutting pieces of its frontal bone with collagenase Ⅱ.BCIP/NBT(5-bromo-4-chloro-3-indolyl-phosphate/four azole nitro blue)staining was performed to detect the expression of ALP(alkaline phosphatase)in OBs qualitatively;p NPP(p-nitrophenyl phosphate disodium)was performed to detect the activity of ALP in OBs quantitatively.Using CCK-8(cell counting kit-8)to test the cell proliferation activity in the ZOL treatment.Bone marrow flushing method was used to isolate BMSCs from SD rats,and P3 cells were taken for CCK-8 detection.3.Micro-CT testing was performed to analyse to osteogenesis with the treatment of ZOL in three period as 2w,4w and 8w.histologic section was made with HE staining,Masson staining,Trap staining to figure out the chronological osteogenesis and activity of OCs with or without the ZOL.IH(immuno-histochem-ical)was also made several target as OP(osteopontin),RUNX2(runt-related transcription factor2).TEM(transmission electron microscopy)testing was made for the 2w section with local ZOL delivery to analyse to micro structure between the bone,cell and implant.Result1.Osteoclasts in the lamb bone of 2-4-day-old neonatal New Zealand white rabbits were successfully isolated.It can be seen that the typical characteristics of osteoclasts are large,multinucleated(5-100 cells),with closed rings at the boundary and it is also positive TRAP staining.The morphology of osteoclasts induced by primary bone marrow cell-macrophages is not as typical as that of isolated primary osteoclasts: irregular shape,multinucleated(2-10),cytoplasmic acidophilic,positive TRAP staining,and high lysosomal activity in some regions of the body.2.ZOL has an inhibitory effect on the growth of osteoblasts under long-term stimulation(7d)at a concentration of 0.5 μg/ml-2 μg/ml,and has no obvious effect in a short time(1d-3d).It has an inhibitory effect on its osteogenic activity at a concentration of 0.5 μg/ml-2 μg/ml(7d).ZOL has an inhibitory effect on the growth of macrophages raw264.7 under a longer time stimulation(day5)1.0 μg/ml-2.0 μg/ml,and the inhibitory effect increases as the concentration increases.Shorter time(3d)stimulation does not Obviously affect.ZOL has an inhibitory effect on the growth of BMSCs under a longer period of stimulation(day5)and at a concentration of 0.5 μg/ml-2 μg/ml,and has no obvious effect in a shorter period of time(1d).3.1.On the surface of the femoral defect at 4d-8d,there was still a ring defect with a clear edge.On 2 w,part of the defect was covered by fibrous tissue,and on4 w,the defect surface was completely repaired.2.Micro-CT showed that there was bone formation around the bone meal.at 2w,more bone formation at 4w,and the bone density was similar to that near the metaphysis,but no significant difference at 8w.Both the systemic administration group and the local administration group had more bone composition than the blank group,and the local administration group was more obvious.3.Masson trichromatic staining showed that early bone formation had occurred around the bone defect on day 14.HE staining showed that a large number of osteogenic interstitium(with abundant osteoblasts)existed in the early bone and bone meal at this time.Tissue section staining:(1)Timing change:Masson staining showed that a large number of mature bone had formed in the bone defect area at 4w,HE staining showed that there were no osteoblasts attached to the bone surface around the bone defect at this time,and TRAP staining showed that osteoclasts were rare at this time.8w Masson staining showed a slight decrease in central osteogenesis and a slight decrease in peripheral osteogenesis compared with 4w.There was little difference between HE and TRAP staining and4 w staining.(2)Different groups: Masson and HE staining showed that at 2w,the total and local administration of ZOL were more than that of the blank group,and the preosteogenesis interstitium was more than that of the blank group.Trap staining showed that osteoclasts in Zol treatment group(systemic and local administration)were more than those in blank group.IHC showed that Runx2(runt-related transcription factor 2)positive cells appeared more in early osteogenesis(2-4 weeks)and were mostly located around the new bone.There were still a large number of Runx2 positive cells in the bone marrow at 8 weeks,but fewer in the defect area.OP was mainly located on the site near the new bone.4.The typical morphology of osteoclasts under TEM(the experimental group):large,multinucleated,with well-developed mitochondria and lysosomes;The new bone on the surface of bone meal was directly bonded to the material.The osteoblasts were rich in endoplasmic reticulum and Golgi apparatus,and the cells were attached to the surface of bone to secrete bone matrix.Conclusion1.The morphology of osteoclasts induced by SD rat bone marrow cells was similar to that of rabbit primary osteoclasts showing multinucleate and TRAP positive characteristics.Morphologically,the induced osteoclasts were irregular and had fewer nuclei than the primary osteoclasts.2.ZOL has a strong inhibitory effect on the growth of osteoblasts,macrophages and bone marrow mesenchymal stem cells under prolonged stimulation in an in vitro environment.3.In the environment of local drug release and systemic administration,zoledronic acid increased both the number of osteoclasts during bone defect repair and induction of osteogenesis.