| Type 1 diabetes mellitus(T1DM)is a kind of serious endocrine and metabolic diseases.There are currently about 40 million T1DM patients in the world,and it is expected to exceed 60 million by 2035.A large number of studies have shown that T1DM has an adverse effect on bone health.In addition to severe bone loss,T1DM can also cause damage to osseointegration,delayed bone regeneration potential,and the increased risk of fragility fracture in patients with T1DM,which seriously affects the life quality of patients and imposes a huge social and economic burden.Due to the rapid increase of the diabetic population and the continuous prolonged survival of T1DM patients,understanding the mechanism of bone damage caused by T1DM and exploring effective methods to resist such bone damage are of important clinical significance.As a novel,non-invasive and safe physical therapy strategy,pulsed electromagnetic field(PEMF)stimulation has become a research focus in promoting the healing of fractures,osteoporosis and femoral head necrosis in recent years.However,whether PEMF can repair bone damage associated with T1DM,and its specific signaling pathways and conduction mechanism remain unclear.In this study,we systematically explored the effect of PEMF stimulation on bone injury repair and bone metabolism regulation in patients with T1DM and the related molecular mechanism based on the treatment requirements of T1DM bone disease and the research concept of interdisciplinary combination of medicine and engineering.First of all,this study constructed a T1DM animal model with bone defect and porous titanium alloy(pTi)implants with low elastic modulus,and explored the effects of PEMF stimulation on the repair of bone defect and inhibition of bone loss of T1DM animals through in vivo experiments.Secondly,the ulna of T1DM animals was subjected to periodical stimulation with fatigue loading using the small animal axial compression loading device developed by our group,and the occurrence and development of T1DM animal bone microdamage under fatigue loading as well as the related mechanism of PEMF simulation on bone microdamage repair in T1DM rats were systematically studied.Thirdly,the effects of PEMF stimulation on the behavioral function of in vitro primary osteoblast(OB)in high glucose(HG)environment were systematically studied.Finally,the molecular mechanism by which PEMF stimulation regulates the behavior and function of OB in the normal and HG environment was clarified by gene silencing.This study proposed the new idea and new approach to study the effect of PEMF on bone integration and bone damage repair in patients with T1DM,aiming to systematically explore the effect of PEMF stimulation on bone anabolism in patients with T1DM and the related molecular mechanism and to provide experimental basis for the clinical application of PEMF stimulation in the treatment of bone diseases,such as bone integration and bone damage repair in patients with T1DM.The whole research is divided into the following four parts:Part Ⅰ:The effect of PEMF on bone loss and bone defect in T1DM rabbitsBackground:Patients with T1DM have severe bone system injury,thus it is of great significance to systematically study the related mechanisms of T1DM-induced osteopenia,osteoporosis and delayed bone defect healing,and explore effective ways to improve the repair efficiency of T1DM bone defect.As an economic,whether PEMF,a safe and noninvasive physical method,can improve T1DM-related osteopenia and osteoporosis and promote bone defect repair has not been reported thus far.Methods:A PEMF signal generation system was designed with high performance data acquisition and signal generation module as the core.The rabbit model of T1DM femoral defect was established by injecting alloxan combined with surgery.The porous titanium alloy Ti2448 implants were established by the electron beam melting technique.The rabbits in the experimental group were continuously stimulated with PEMF for 8 weeks.The ELISA assay,Micro-CT scanning,fluorescence double labeling assay,immunostaining,three-point bending testing,nanoindentation testing and qRT-PCR assays were used to detect the serum markers of bone turnover,the microstructure of right femoral trabecular bone and cortical bone,the formation rate of femoral trabecular bone,the bone ingrowth and trabecular bone appearance of the pTi implants,the biomechanical properties of femora and the expression levels of osteogenesis related genes in T1DM bone.Results:The concentrations of serum OCN and P1NP in T1DM rabbits were decreased significantly.The microstructure of trabecular bone and cortical bone in distal femur of T1DM rabbits was significantly compromised and the bone mass was also significantly reduced as compared with the Control rabbits.The mineral apposition rate(MAR)of trabecular bone around the implant and in the area without bone defect in T1DM rabbits,revealing that the pTi osseointegration was significantly destroyed.Besides,the structural and material properties of T1DM rabbit tibiae were significantly decreased,and the gene expression levels related to bone formation were significantly decreased in T1DM bone.However,the concentrations of serum OCN and P1NP were increased significantly after PEMF stimulation.The microstructure and parameters of trabecular bone and cortical bone were significantly improved after PEMF stimulation.PEMF stimulation could promote the ingrowth of trabecular bone into the pore space of pTi,and also significantly increase the MAR.The structural and material properties of tibiae were significantly improved after PEMF stimulation.Besides,the expression levels of bone-formation-related genes were significantly increased after PEMF stimulation.Conclusion:PEMF can significantly improve the microstructure and mechanical properties of trabecular bone and cortical bone of T1DM rabbits induced by alloxan,and promote the bone growth and integration of new Ti2448 pTi in T1DM bone defects by enhancing bone anabolism.This part of research enriches the understanding of the response of T1DM skeleton to external PEMF signals,and provides a convenient and efficient treatment for TIDM-related bone diseases such as T1DM induced osteopenia,osteoporosis and bone defect.Part Ⅱ:The repair effect of PEMF on bone microdamage in T1DM ratsBackground:Daily physiological loading can initiate microdamage in bone tissue,and the accumulation of microdamage is an important factor leading to significant decrease in the biomechanical properties of bone tissues.However,it is still unclear whether PEMF can prevent the occurrence and development of bone microdamage in T1DM skeleton.Based on this question,this part of the study aimed to study the occurrence and development of bone microdamage in T1DM rats under fatigue loading so as to enrich the understanding of the regulatory effects of PEMF on T1DM bone quality.Methods:The right ulna of normal,T1DM and PEMF-treated T1DM rats were subjected to cyclic axial compressive loading for 14 days using the small-animal axial compression loading device developed by our group.The accumulation of ulnar microdamage in T1DM rats was detected by SPECT scanning.The linear microcracks and diffuse microdamage of ulna in T1DM rats were observed and quantitatively analyzed by basic fuchsin staining.The expression of Runx2,an important bone morphogenetic protein,was detected by the immunohistochemistry technique.Results:The results of SPECT scanning showed that the accumulation of bone microdamage in the T1DM rats was significantly higher than that in the Control group under fatigue loading,while the number of T1DM rats under fatigue loading was significantly reduced after PEMF stimulation.The basic fuchsin staining showed that PEMF stimulation significantly reduced the accumulation of linear microcracks and diffuse microdamage in bone tissues of the T1DM rats under fatigue loading.The results of immunohistochemistry showed that the number of Runx2-positive cells on the bone surface and in the bone matrix near the linear microcrack in the middle of ulnar shaft in the T1DM group was significantly decreased,while the number of Runx2 positive cells in ulnar samples of T1DM rats was significantly increased after PEMF stimulation.Conclusion:PEMF can significantly reduce the accumulation of linear microcracks and diffuse microdamage in bone tissues of T1DM rats,which is associated with the promotion of bone formation.This part of study reveals that PEMF improves the bone strength and bone quality by resisting the accumulation of microdamage in the bone tissue of T1DM rats,and provides an experimental basis for enriching the scientific understanding of the regulation effects of PEMF on T1DM bone quality.Part Ⅲ:The regulatory effects of PEMF on the behavior and function of in vitro OB in the HG environment and the preliminary mechanism studyBackground:In the first two parts of the study,we have confirmed that PEMF can significantly resist bone loss and accelerate the repair of bone defects in T1DM animals,whereas the potential regulatory mechanism remains unclear.It has been shown that the primary cilia,locating on the cell membrane,act as "sensors" in detecting the stimulation of external physical factors in bone cells.Based on the in vitro primary OB model,this part of the research aimed to systematically study the regulatory effects of PEMF on the biological activity and function of OB in the HG environment.Moreover,the potential mechanism of PEMF on the regulation of OB based on primary cilia was systematically investigated.Methods:The primary OB was extracted from rats,and the MTT and BrdU approaches were used to study the effect of PEMF on the proliferation of primary OB in the HG environment.The ALP activity analysis and ALP staining assays were used to detect the differentiation ability of primary OB.The Alizarin Red S staining approach was used to detect the mineralization ability of primary OB.The qRT-PCR and Western blotting were used to determine the differentiation ability of primary OB.The effects of PEMF on the expression of important genes and proteins in primary OB in the HG environment were examined by the Western blotting assay.The effects of PEMF on the expression of primary cilia and the downstream signaling proteins of primary OB in the HG environment were determined using the immunofluorescence assay and Western blotting assay.Results:PEMF significantly promoted the proliferation and significantly increased the ALP staining area and ALP activity of primary OB in the HG environment.Besides,PEMF stimulation also significantly increased the number and proportion of mineralized nodules of primary OB in the HG environment.In addition,PEMF significantly also increased the gene and protein expression levels of Runx2,OSX,OCN and COL-1 of primary OB in the HG environment.In addition,the HG environment significantly reduced the number of OB with primary cilia and the expression levels of primary cilia,p-GSK-3β and β-catenin in primary OB were significantly up-regulated after exposure to HG.Conclusion:PEMF stimulation can significantly improve the damage of the proliferation,osteogenic differentiation and mineralization induced by the HG environment in OB.Moreover,PEMF increased the expression levels of primary cilia in OB and activated the downstream β-catenin pathway.This study suggests that PEMF-induced improvement of bone quality is associated with its regulation of the behavior and function of OB.In addition,primary cilia and β-catenin pathway may play a key role in the regulation of OB under HG by PEMF.Part Ⅳ:The mechanism study of the regulatory effects of PEMF on the behavior of OB in the HG environment involving primary cilia/β-catenin signaling cascadeBackground:The previous part of our study revealed that PEMF stimulation can increase the expression of primary cilia in primary OB in the HG environment,and then activate the downstream β-catenin pathway.However,the role of the activation of this pathway in the regulation of the behavioral function of primary OB by PEMF in the HG environment as well as the signaling cascade of primary cilia detecting the PEMF signals in the HG environment remains unclear.This part of the research aims to study the potential mechanism by which PEMF regulates the behavioral function of OB in the HG environment involving the primary cilia/β-catenin signaling cascade by blocking the expression of the primary cilia and their downstream key molecules of OB through gene silencing/inhibitors.Methods:Firstly,the gene silencing technique was used to suppress the expression of primary cilia in OB under both normal and HG environment,and then the cell proliferation was quantified using the MTT and BrdU tests,and osteogenic differentiation was determined using the ALP activity analysis,ALP staining and Runx2,Osx,OCN and Col-1 gene expression examination.Then,the mineralization ability was analyzed by Alizarin Red S staining.The expression levels of calcium channel proteins Piezol,PC2,TRPV4 on the primary cilia as well as the expression of PKA,p-GSK-3β and β-catenin proteins were examined using Western blotting assays.Secondly,the expression of genes associated with osteogenic differentiation and the expression of the downstream proteins of TRPV4(PKA,p-GSK-3β and β-catenin)was detected after TRPV4 was inhibited by gene silencing technology.Thirdly,the expression of the downstream protein of PKA(p-GSK-3β andβ-catenin)was detected after the PKA expression was blocked using the inhibitor.Results:After silencing the IFT88 gene of primary cilia,PEMF-induced increase in the proliferation,ALP activity,formation of mineralized nodules,and Runx2,Osx,OCN and Col-1 gene expression levels of OB under both normal and HG environment was significantly blocked.PEMF significantly increased the expression of calcium channel protein TRPV4 on the plasma membrane of OB,whereas PEMF did not significantly change the expression of Piezo1 and PC2.However,PEMF-induced increase in the TRPV4 expression and PKA and GSK-3β phosphorylation is significantly inhibited after the blockade of IFT88.PEMF-induced increase in Runx2,Osx,OCN and Col-1 gene expression as well as phosphorylation levels of PKA and GSK-3β in OB under both normal and HG environment was significantly suppressed after TRPV4 was silenced.Finally,PEMF-induced increase in β-catenin and GSK-3β phosphorylation levels in normal and HG environment OBs were also inhibited after the blockade of PKA.Conclusion:Primary cilia play an important role in detecting and transducing external PEMF stimulation in OB under normal/high glucose environment.PEMF stimulation can activate the downstream signaling cascade of primary cilia(TRPV/PKA/GSK-3β)in OB,so as to regulate the behavioral function of OB under normal and high glucose environment,and eventually accelerate bone repair and improve bone quality in T1DM. |
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