| BackgroundWith the development of trafic and industry in the society,the common clinical damage of fracture is increasing year by year.Fractures caused by high-energy injuries often suffer from bone defects and severe soft tissue injuries.Therefore,fractures often occur delayed union and nonunion.Delayed union and nonunion are common complications of fracture and about 5%-10%patients will to be this diease which affect the quality in patients'life.The treatment of nonunion is a difficult clinical problem.The commonly used methods are conservative and surgical treatment.Conservative treatment includes physical therapy(electromagnetic,ultrasound,shock wave,etc.),molecular biological therapy and traditional Chinese medicine treatment.However,the actual application is poor.Surgical treatment includes intramedullary nailing(for aseptic long bone nonunion),plate fixation(to stable bone defect)and autologous bone graft operation.In general,foucs to the causes of nonunion,combination of the above methods to treat.Compared with conservative treatment,surgical treatment is effective,but the cost is high,the trauma is great,the patient is generally unacceptable.However,whether conservative treatment or surgical treatment,there are still some nonunions which can not be cured,which brings great pain and burden to patients and their families.Therefore,the search for a safe,effective and minimally invasive treatment is a hot and difficult point in the study of current trauma department of orthopedics.Stem cell transplantation is an advanced medical technique.It used the stem cells'multidirectional differentiation potential and self replicating ability to repair tissue damage It has always been a hot topic in trauma treatment.Mesenchymal stem cells(MSCs)are multipotent stem cells derived from mesoderm with multiple differentiation potential.Bone marrow mesenchymal stem cells(BMSC)are generally considered to be significant in bone healing.However,BMSC transplantation in nounion are still have many limitations,such as poor differentiation or tumorigenesis and the treatment of period always long.The use of allogeneic BMSC is also limited by lack of donation,shortage of resources and the safety factors such as allograft rejection are not clearly.These factors have greatly influenced the application of BMSC in the clinical study of nonunion.And the current domestic stem cell treatment control policy is also difficult to ensure that stem cell transplantation strategies are approved in the near future.Therefore,it is a hot and difficult point in the field of medical research to find a way to replace the treatment of MSC.In the past MSC were often considered to perform repair processes at the injured site by differentiating into the needed tissue cells.However,recent studies have shown that MSC from the nest site to the injury tissue,which number and proportion are very limited.The time of BMSC differentiation into tissue cells is also very short,usually will soon end after differentiation apoptosis.More and more studies have suggested that MSC can act on the injured part through the paracrine mechanism,so as to achieve the purpose of repair.In the substances secreted by these MSC,exosome is considered to have an important role.30 years ago,exosomes had been founded when scientists in the study of sheep reticulocyte maturation.In the beginning,the researchers only used the tiny particles with 40-100nm diameter,lipid bilayers and vesicles as a trash can for cells to clean up cytoplasm waste.However,with the depth study of the mechanism of cell secretion,by the gradually found exosomes with abundant expression of cell specific protein,lipids,nucleic acids and other substances,it can be through fusion with the plasma membrane and cell,transfer of material and information in different cells,play an important biological function.Through literature review and some previous studies,we found that exosome plays an important role in cell proliferation,migration and differentiation.Exosome can repair various kinds of injuries,such as cardiovascular system,respiratory system nervous system and urinary system,and exosome body has some applications in the diagnosis of some diseases.Relative to stem cells,exosome has many advantages,such as a large amount of production,storage and low immunity.It has been reported that the exosomes derived from mesenchymal stem cells promotes the proliferation of other cells.It suggests that the exosomes derived from MSCs may contain key substances which could regulating MSCs proliferation.ObjectiveThe present study found that exosomes derived from MSC in cardiovascular disease,acute kidney injury,immune system diseases,nervous system diseases and some tumors can play a positive and effective role in the diagnosis and treatment but whether the body can repair bone injury has not seen reports.The purpose of this study is to explore the effictive of exosome derived form MSC in bone injury,and lay a foundation for subsequent research on molecular biology and cytology mechanism.In this study,we first established the preparation process of exosome from human umbilical cord mesenchymal stem cells(uMSC).The exosome from uMSC was extracted by ultracentrifugation.Then we used BMSC cell sheet and exosomes to treat fracture of rat animal models.Than we compard the treatment effectives.Preliminary make clear the effective of exosomes derived from MSC in bone repair,discussed the feasibility of exosomes used in the treatment of nonunion.Part ?:Preparation and identification of uMSC exosomeMethods:(1)uMSCs was isolated and cultured in the original generation,and the exosome secreted by uMSCs in the early generation(P2 and P3)were collected by gradient centrifugation.(2)Direct observation by electron microscope,nanoparticle particle analysis system and microfluidic imaging system to identify the external secreting body.Results:The overall shape of exosomes by electron microscopy,exosomes size and specific surface markers by nanoparticle size analysis system and micro flow imaging system has been confirmed:the exosome obtained with gradient ultracentrifugation which in all aspects accords with the generally accepted description of the exosome.Conclusion:the gradient ultracentrifugation is a simple and effective method to extract the exosome.The exosome prepared by this method is reliable.Part ?:The isolation and culture of BMSC in SD rats and the establishment of animal fracture modelMethods:(1)The range of SPF SD rats were sacrificed and their femur and tibia bone were removed.Bone marrow and bone fragments were used to isolate and culture primary BMSC,and their passages were amplified to fourth generation(P4).(2)Direct observation the BMSC of PO and P4 by microscope,and the identification of rat BMSC was carried out by flow cytometry.(3)The establishment of a SD rat fracture model:using a saws to cross the tibia of the rat and use 0.88mm Kirschner pin to fix it.Results:(1)After the passage of BMSC,the morphology was observed as spindle like,whirlpool or radiation.The:flow cytometry showed that the BMSC was high expression of CD29,CD44 and low expression of CD34.(2)After internal fixation of tibial fracture in SD rats,the state was stable and there was no shedding sample.Conclusion:(1)The cells isolated from bone marrow and bone slices were consistent with the morphology and phenotype of BMSC,indicating that we obtained a relatively homogeneous BMSC.(2)The fracture model of SD rats was established successfully,and further experimental study could be carried out.Part ?:Study on the repair of bone injury by uMSC-exosomeMethods:12 SD rats of tibial fracture model fixation by intramedullary nail,were randomly divided into 4 groups,control group,PBS treatment;uMSC-exosomes group,exosomes treatment;uMSC-exosome supernatant group,uMSC-exosome supernatant treatment;BM.SC cell sheet group,BMSC cell sheet treatment.Three methods were used to evaluate the healing of 3 weeks after operation by micro-CT,tissue sections staining and qRT-PCR.Results:By micro-CT,tissue sections staining and qRT-PCR show that,uMSC-exosome group and BMSC cell sheet group were faster than the blank control group and uMSC supernatant group in the fracture healing.uMSC-exosome group and BMSC cell sheet group had the smilar healing efficiency.Conclusion:The exosome secreted by uMSC plays a positive role in the repair of bone tissue,and its effect is similar to the BMSC. |
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