| Background:Congenital pseudoarthrosis of tibia is a relatively rare skeletal dysplasia disease in pediatric surgery.It is characterized by repeated fractures and nonunion of the lower third of the tibia,the reason of which is still not clear.Many scholars have put forward a variety of hypotheses about the cause of this disease,such as intrauterine injury,metabolic disorders,and vascular malformations,but they have been invalidated.Some scholars believe that this disease is related to neurofibromatosis,but neurofibromatosis-like milk coffee spots are not seen on most children,and there is no neurofibroma tissue invasion in the pseudo-joint pathological tissue.But the periosteum tissues are embedded.Therefore,some scholars believe that the abnormal thickening of local pseudoarthrosis periosteum forms a constricted ring,which can grow aggressively,leading to local blood supply damage,bone atrophy,and fracture of the tibia and contributing to the formation of pseudoarthrosis.Although many scholars persisting in studying the etiology of congenital pseudoarthritis of the tibia,there is still no conclusion.The previous research of our research group found that not only abnormal pathological changes existed in the periosteum of the pseudo-joint,but the bone tissue also had abnormal bone metabolism.Vasoactive intestinal peptide was abnormally high expressed in osteoclasts in the pseudo-joint compared with the normal control group.This phenomenon suggests that neuropeptides are involved in the formation of c Congenital pseudoarthrosis of tibia.At the end of the 20th century,Kjaer first proposed the concept of"neuro-osteology".Scholars gradually began to pay attention to the innervation of bone growth,and peptidergic nerves are a research hotspot.Peptidergic nerve fibers are mainly distributed in metabolically active bone tissues,such as epiphyses,bone marrow,periosteum,etc.,which release neuropeptides and participate in local bone metabolism.These neuropeptides not only regulate normal bone tissues,but also regulate the proliferation and differentiation of tissue cells during the healing process of fractures.At present,peptidergic nerve fibers that release neuropeptides such as calcitonin gene-related peptide,neuropeptide Y,and vasoactive intestinal peptide have been found in bone tissues.Vasoactive intestinal peptide is composed of 28 amino acids.It was first isolated from the pig intestine and subsequently found in the central nervous system and peripheral nervous system.It is mainly released by enteric neurons,which are mainly distributed in the bone marrow and epiphysis.Vasoactive intestinal peptide has vasodilator effect,which can increase blood flow by expanding blood vessels in the periosteum and participate in the regulation of bone metabolism.At the same time,it can also directly or indirectly regulate the activity of osteoclasts by binding to the VIP-1,2 receptors on the surface of osteoclasts and osteoblasts,promote the proliferation of osteoclasts,enhance their activity and increase bone resorption.Based on the above findings,we infer that the abnormally high expression of vasoactive intestinal peptide in the osteoclasts of congenital tibial pseudoarthritis promotes local bone resorption,disrupts the balance between bone formation and bone resorption,and leads to nonunion of fractures.The regulation of bone metabolism by RANK/RANKL/OPG system has been a research hotspot in recent years,and it plays an important role in fracture healing and some bone metabolic diseases.As a member of the TNF superfamily,RANK(receptor activator of nuclear factor-κβ)is expressed on osteoclast precursor cells,mature osteoclasts and chondrocytes.RANK and its ligand(RANKL)interact in the outer layer of osteoclast precursor cells(OPC),causing these cells to mature and transform into osteoclasts.Osteoprotegerin(OPG)is a member of the tumor necrosis factor receptor(TNFR)family.It is expressed by bone stromal cells and osteoblasts.It acts as a decoy receptor for RANKL,preventing the binding of RANK and RANKL with its high affinity to RANKL and inhibiting the differentiation and maturation of osteoclasts.It can be explained that the interaction between the three factors affects the differentiation and activity expression of osteoclasts.We infer that VIP may be achieved through the RANK/RANKL/OPG system in the process of regulating osteoclast activity,and we further explored its mechanism through in vitro experimental studies.Objectives:Based on the findings of previous studies,the following questions were further clarified through experiments:What is the effect of VIP on the differentiation,proliferation and activity expression of osteoclasts in vitro?After VIP acts on bone metabolizing cells,does the expression of the RANK/RANKL/OPG system change?How does the downstream signaling pathway change?How does this change affect bone metabolism?What is the difference in the expression of RANK/RANKL/OPG system in CPT diseased tissues?To clarify the above problems through experiments and to clarify the role of VIP abnormal expression in the pathogenic mechanism of CPT.Methods:Firstly,marrow of long bone of mice was applied to induce and isolate mature osteoclasts under the action of inducers(M-CSF and RANKL).After HE staining,multinucleated giant cells(2 or more nuclei)can be seen and they are osteoclasts.They become burgundy positive after TRAP staining,confirming their bone resorption activity.After bone phagocytosis experiment,toluidine blue staining experiment and scanning electron microscope observation,bone lacuna can be seen,which further confirms that the induced culture is osteoclast with bone resorption activity.Different concentrations of VIP were applied to it,and the effect of VIP on the proliferation of osteoclasts was observed and analyzed by the MTT method,and the effect of VIP on the activity of osteoclasts was analyzed by the changes in the number of bone lacunae in the bone phagocytic experiment.Secondly,VIP was applied to the osteoclast cell line,the osteoblast cell line,and the co-cultured cell line of the two,and the fluorescence quantitative PCR technology and Western-Blot technology were used to detect the RANK/RANKL/OPG system and the protein expression differences of downstream signaling pathway in the three cell lines.Different concentrations of VIP(10-9,10-8,10-7,10-6mol/L)were used on three cell lines respectively for 48 hours.In addition,VIP(-)was set as a control group and real-time fluorescent quantitative PCR was conducted to analyze mRNA expression levels of RANK,RANKL and OPG.Then Western blot was used to detect the protein expression levels of RANK,RANKL and OPG.NF-κβ signaling pathway and ERK signaling pathway play important roles in bone metabolism,the effect factors IL-6 and CAII of which are important substances involved in bone resorption.Then,the expression changes of NF-κβ signaling pathway and ERK signaling pathway after VIP action in the three cell lines were detected.Thirdly,detect the difference in protein expression of RANK/RANKL/OPG system and its downstream signaling pathways in CPT diseased tissues.The bone tissues of the lesions of 5 children with CPT were selected as the experimental group and the ipsilateral normal fibula was used as the control group.The pathological tissues were made by wax blocks for immunohistochemical staining,and the staining results were collected using Image-Pro Plus 6.0 software.Statistical analysis was performed to compare the expression differences of RANK,RANKL,OPG,NF-κβand ERK.After three parts of experiments,cytology experiments and pathological tissue experiments were used to verify the changes of RANK/RANKL/OPG system and its downstream signal pathway protein expression under the action of VIP.Results:According to the analysis of the MTT test results,the proliferation ability of OC cells in each concentration group has no significant difference in the same VIP treating time;meanwhile,on any level of VIP concentration,the proliferation ability of OC cells is also shows no obvious change with the prolonged time(6h-48h).According to the results of toluidine blue staining and scanning electron microscopy,with same VIP treating time,high-concentration VIP(10-6mol/L)and VIP(10-7mol/L)are compared with low-concentration VIP(10-8mol/L)group,the bone resorption activity of OC cells(10-9mol/L)group is inhibited,and the number of lacunas formed decreases;With same VIP concentration and prolonged time(6h-48h),the bone resorption activity of OC cells also gradually decreased.In osteoclast cell lines,the mRNA levels and protein expression levels of RANKL and RANK decreased with the increase of VIP concentration,while OPG expression increased,and the expression of NF-κβ and ERK signaling pathways also decreased.In osteoblast cell lines,the expressions of RANKL and IL-6 all increased with the increase of VIP concentration,but the expression of OPG decreased.In the co-culture system of osteoclasts and osteoblasts,the expressions of RANKL and RANK increased with the increase of VIP concentration,OPG showed a downward trend,while NF-κβ,IL-6,ERK and CAII all showed an increasing trend.In CPT diseased tissues,the expression of RANKL,RANK,NF-κB,IL-6,ERK,and CAII increased compared with normal bone tissues,while OPG decreased.This expression trend is consistent with the results of in vitro cell line experiments.Conclusion:The bone marrow cell induction method can effectively induce the differentiation of mature osteoclasts and shows bone resorption activity with the advantages of high purity and large quantity.In the simple osteoclast cell line,VIP has no effect on the differentiation and proliferation of osteoclasts,but has inhibitory effect on the bone resorption activity of osteoclasts.It can be seen from the bone phagocytic experiment that with the increase of VIP concentration,the number of bone lacunas decreased.Subsequent PCR and Westen-Blot detection revealed that VIP decreased the expression of RANKL and RANK,and the expression of NF-κβ and ERK signaling pathways also decreased,while OPG increased;The ratio of RANKL/OPG protein expression decreases in a dose-dependent manner,and the activity of osteoclasts is inhibited.VIP can enhance the bone resorption activity of osteoclasts in a co-culture system of osteoclasts and osteoblasts.The expression of RANKL,RANK and NF-κβ,IL-6,ERK and CAII were all enhanced under the action of VIP,while the expression of OPG was inhibited.In the osteoblast cell line experiment,we found that VIP up-regulated the expression of RANKL,but decreased the expression of OPG.When osteoclasts and osteoblasts coexist in one environment,osteoblasts have a synergistic effect on the differentiation and maturation of osteoclasts,and VIP inhibits OPG expression by stimulating the expression of RANK and RANKL,resulting in an increasing ratio of RANKL/OPG protein expression and osteoclast activity is activated and amplified.In CPT diseased tissues,VIP expression increased,and the trend of changes in the RANK/RANKL/OPG system and its downstream signaling pathways was the same as that in the co-culture system of osteoclasts and osteoblasts under the action of VIP.Moreover,the above-mentioned expression differences were statistically analyzed with significant differences(P<0.05).It can be seen that the effect of VIP on osteoclast activity is achieved through the RANK/RANKL/OPG system,which participates in the pathological process of CPT bone metabolism by regulating the expression of RANK/RANKL/OPG. |
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