| Diabetes mellitus is a type of metabolic disease characterized by higher glucose levels than the normal condition in the organism in clinics.There are mainly two types of diabetes mellitus in clinics?i.e.,type 1 diabetes and type 2 diabetes?.The prevalence of type 2 diabetes is more than 90% in the overall diabetic population,and type 2 diabetes is the most common senile disease in clinics.Type 2 diabetes is able to induce substantial complications,including diabetic cardiovascular diseases,diabetic neuropathy,diabetic nephropathy,diabetic retinopathy and diabetic foot ulcer,etc.Moreover,the skeletal and oral complications associated with type 2 diabetes have also gained extensive attention in the recent decades.Firstly,type 2 diabetes exhibited significant deterioration of bone architecture and decrease of bone quality,leading to the significant increase of bone fractures.Once the occurrence of fractures,the healing obstacle of bone fracture and soft tissue wound and the microcirculation disturbance in type 2 diabetic patients will increase the morbidity and mortality induced by the fracture.Secondly,type 2 diabetes exhibited many oral complications,including leukoplakia,periodontitis,alveolar and mandibular bone loss,which seriously influence the life quality of diabetic patients.Thus,it holds great clinically therapeutic significance of exploring effective strategy for the skeletal and oral complications associated with type 2 diabetes.In the past over five decades,substantial experimental and clinical studies have shown that pulsed electromagnetic fields?PEMF?have the capacity of generating positive efficacy on the treatment of various skeletal diseases,including delayed uniuon or nonunion of bone fracture,bone defect,osteoarthritis and osteoporosis.Furthermore,the beneficial effects of PEMF on oral diseases have also been verified by numerous studies,including acceleration of mandibular fracture healing,promotion of osseointegration of dental implants,acceleration of tooth movement during orthodontic treatment.However,it remains unknown whether PEMF stimulation is able to ameliorate the skeletal healthy status of the long bones and mandibles in type 2 diabetes,and it is also unknown the exact regulatory mechanism for PEMF in type 2 diabetic skeletons.In this study,we will systematically investigate the effects of PEMF on femoral and mandibular microstructure,biomechanical properties and the bone turnover in type 2 diabetic db/db mice.We will also study the potential mechanism by which PEMF regulate the cellular behaviors and functions in the skeleton of type 2 diabetes.This study not only may provide important cues for enriching the mechanisms by which PEMF regulate osteogenesis,but also may provide novel strategy for the clinical treatment of skeletal diseases associated with type 2 diabetes.The entire thesis includes the following three major parts.Part ?: The development of the programmable electromagnetic exposure system specific for the investigation of the biological effects of electromagnetic fields.Backgrounds.The basis for the experimental and clinical investigation of the biological effects of electromagnetic fields is the development of stable and scientific electromagnetic exposure system.The uneven distribution of the electromagnetic fields generated by the exposure system for the exposure target is the major factor restricting the reliability of experimental results for the biological effects study of electromagnetic fields.Therefore,the maximization of the evenness of the electromagnetic distribution is the key factor for increasing the repeatability and stability of the experimental results for the study of the biological effects of electromagnetic fields.Methods.We developed a novel programmable electromagnetic exposure system based on the 3-Helmholtz coils.This system is mainly based on the STC12C5410AD32 microcontroller,and the signal was filtered and amplified based on the chip OPA549 and then transferred to the novel the 3-Helmholtz coils model.Then,we systematically compared and analyzed the spatial distribution of the output electromagnetic fields generated the new 3-Helmholtz coils and traditional 2-Helmholtz coils.Results.This novel programmable electromagnetic exposure system has the advantages with compact dimensions,low weight,easy operation,excellent anti-interference,which is able to provide long-term stable output of all various electromagnetic signals,such as the triangle waveform,rectangle waveform,sinusoidal waveform,static waveform and PEMF,etc.Our theoretical analysis and Gaussmeter-based examination demonstrated that the 3-Helmholtz coils model was able to generate much higher evenness of the spatial distribution of the electromagnetic signals.Conclusion.This novel programmable electromagnetic exposure system based on the 3-Helmholtz coils can lay a solid foundation for the investigation of the biological effects of electromagnetic fields,and may improve the repeatability and stability of the experimental results for the study of the biological effects of electromagnetic fields.Part ?: The effects and mechanisms of low-intensity PEMF on bone loss in long bones and mandibles of type 2 diabetic db/db miceBackgrounds.Patients with type 2 diabetes can develop the skeletal and oral complications.Type 2 diabetes can induce the deterioration of bone architecture and decrease of mechanical strength,which can induce much higher fracture risk than the nondiabetic populations.Moreover,the patients with type 2 diabetes also have the nonunion or delayed union issues once the fracture occurs.In addition,type 2 diabetes can also develop several oral complications,such as the alveolar and mandibular bone loss and periodontitis.However,the effective,economic and safe approaches for the treatment of skeletal and oral complications associated with type 2 diabetes are still lacking.Methods.The 12-week-old type 2 diabetic db/db mice?n=9?were exposed to whole-body PEMF exposure with 2 days per day for consecutive 12 weeks.The wild-type mice and the mice in the db/db group were not subjected to the PEMF exposure.After sacrifice,the serum samples were collected for the determination of serum biomarkers of bone formation and bone resorption.The left femora were extracted for the biomechanical 3-point bending and nanoindentation examinations.The right femora were extracted for the Micro CT and bone histomorphometric analyses.The bilateral tibiae were collected for the PCR and Western blotting analyses.The left mandibles were extracted for Micro CT,histomorphometric and biomechanical examinations.The right mandibles were used for the gene expression analysis.Results: Our Micro CT results showed that PEMF significantly improved the femoral cancellous bone and cortical bone architecture,and also significantly improved the mandibular bone architecture.The biomechanical 3-point bending and nanoindentation results demonstrated that PEMF significantly increased the whole-bone mechanical strength in the femora of db/db mice,and also improved the biomechanical material properties in the femora and mandibles of db/db mice.The serum biochemical and bone dynamic histomorphometry revealed that PEMF significantly increased the bone formation rate in the femora and mandibles of db/db mice,but exerted few impacts on bone resorption in the skeletons of db/db mice.Our PCR and Western blotting results showed that PEMF significantly upregulated the gene and protein expression associated with osteogenesis in tibiae and mandibles of db/db mice?e.g.,OCN,Runx2 and BMP2?,PEMF also upregulated the gene and protein expression in the canonical Wnt signaling,including Wnt3 a,Lrp6 and ?-catenin.Conclusion: PEMF significantly improved the bone microstructure and mechanical strength in type 2 diabetic db/db mice.This efficacy was mainly associated with the canonical Wnt signaling-mediated promotion of bone formation under the stimulation of PEMF.This study can provide important experimental evidence for the clinical treatment of skeletal complications associated with type 2 diabetes.Part ?: The effects of low-intensity PEMF on the biological activities and functions of in vitro osteoblasts in type 2 diabetic db/db mice and its molecular mechanismsBackgrounds.Although our in vivo animal studies have shown that PEMF-induced improvement of bone quality was associated with the promotion of bone formation by PEMF,it remains not fully identified for the regulatory effects and mechanisms of PEMF on the biological activities and functions of in vitro osteoblasts in db/db mice.Thus,in the experiments of this part,we will systematically identify the regulatory mechanism of PEMF on osteoblast functions in type 2 diabetes based on the in vitro primary osteoblast isolation and cell culture from the skeletons of db/db mice.Methods.The primary osteoblasts were isolated from the mandibles of 12-week-old WT and db/db mice based on the combined approached of enzyme digestion and tissue explants.Then,the primary osteoblasts from the db/db mice were exposed to PEMF with 2 hours per day.Then,the cellular proliferation,cytoskeletal microstructure,alkaline phosphatase activity,mineralization rate,and gene and protein expression of canonical Wnt signaling were determined.Then,the in vitro osteoblasts were pretreated with the antagonist for Wnt signaling,and the changes of biological activities and functions of PEMF-exposed osteoblasts were evaluated.Results: PEMF stimulation was able to significantly improve the cytoskeletal microstructure of primary osteobalsts in db/db mice,and also promote the proliferation,differentiation and mineralization of primary osteobalsts in db/db mice.PEMF also significantly promoted osteogenesis-associated gene and protein expression of primary osteobalsts in db/db mice,including OCN,BMP2,Runx2 and COL-1.PEMF also upregulated the gene and protein expression in the canonical Wnt signaling,including Wnt3 a,Lrp6 and ?-catenin.The promotion on osteoblastic proliferation,differentiation and mineralization induced by PEMF also disappeared after the canonical Wnt signaling was inhibited,indicating that the canonical Wnt signaling played a critical role in regulating PEMF-induced promotion of osteoblast activities and functions.Conclusion: Our in vitro studies demonstrate that PEMF has the capacity of promoting osteoblast activities and functions,and the potential mechanism is associated with the PEMF-induced activation of the canonical Wnt signaling.This study can provide important cues for identifying the mechanism by which PEMF regulate bone metabolism,and also provide important experimental evidence for more scientific and reasonable clinical treatment of skeletal diseases. |
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