Expression And Mechanism Of PDGF-BB Couples With TGF-beta 1 In The Process Of Fracture Repair

PrefaceWith the development of social economy and transportation,the incidence of fracture is gradually increasing.Bone is one of the important organs in human body.It has its own development process,metabolism,repair and reconstruction.With the increase of fracture patients,the proportion of fracture nonunion is increasing,which is mainly caused by trauma,congenital malformation,malignant tumor,operation and infection.The occurrence of fractures is accompanied by an increase in the proportion of poor bone healing,which may be caused by various factors such as trauma,congenital malformations,malignant tumors,surgery and infection.There are many factors that affect fracture healing,such as the location of the fracture,the type,blood supply,and the age and health of the patient.Fractures have a significant adverse effect on the lives and society of patients.The effective treatment of fractures and the limitations of other bone reconstruction techniques are two important reasons for our increased fracture studies.Although bones have considerable repair capabilities,clinical interventions are needed to promote bone regeneration when adverse regeneration conditions,such as infections,inadequate blood supply to surrounding tissues,systemic diseases,and the like.Repair of fractures remains a major challenge in orthopedics.At present,most of the clinical methods for repairing bone damage include autologous bone transplantation,allogeneic transplantation,xenograft bone transplantation and implantation of artificial materials.Current treatments have significant limitations in clinical applications,including inability to fuse with surrounding host tissues,limited donor tissue available,iatrogenic injury,and postoperative pain.Bone is a dynamic and highly vascularized tissue in which there is a close spatial and temporal relationship between blood vessels and bone cells to ensure bone integrity.Fracture repair involves the coordination of multiple events,such as osteogenesis and angiogenesis.Recent advances in stem cell biomedical research indicate that stem cell therapy has important application prospects in tissue regeneration and organ repair.Stem cells are primitive cells that are highly proliferative and self-renewing and that can differentiate into a variety of cells.Among them,adult stem cells can differentiate into multiple layers across the germ layer.During embryogenesis,mesoderm differentiates into a variety of mesenchymal tissues such as bone,cartilage,muscle,tendon,medullary matrix,and fat.These precursor cells are called mesenchymal stem cells(MSCs)because of their stem cell characteristics.Mesenchymal stem cells have the potential to shorten healing time and treat nonunion in fracture patients because these cells are not only diverse,but also capable of differentiating into osteoblasts,and also have immunoregulatory functions.Therefore,the key to the success of bone tissue regeneration is to prepare enough MSCs.MSCs have the main features of self-renewal and pluripotency,as well as the potential for use in regenerative medicine,which was originally found in many postnatal tissues such as bone marrow,adipose tissue,skin,umbilical cord and placenta.In theory,MSCs from different sources may differentiate into osteogenesis and chondrocyte lineages.Osteogenesis is also essential for steady-state renewal of bone and healing of fracture regeneration.Shaping the bone structure and ensuring the integrity of the bones is a dynamic bone remodeling process throughout life.A variety of cell proliferation,differentiation,and synthesis and calcification of extracellular matrices are involved in this process.Bone remodeling relies on the precise coordination of bone resorption and bone formation.During this process,progenitor cells and mesenchymal stem cells differentiate into functional osteoblasts.The extracellular matrix of these osteoblasts is ossified to form new bone.Genetic testing has been found.Some molecules that regulate the lineage of these cells during bone remodeling reveal their potential for counteracting bone loss and repairing damaged bone tissue.There is no bone without the vascular system.Angiogenesis plays a crucial role in skeletal development and fracture repair.Angiogenesis combined with osteogenesis occurs primarily in angiogenesis and mediates the transport of nutrients,oxygen,minerals and metabolic waste,which is essential for maintaining proper bone matrix synthesis and mineralization.During angiogenesis,endothelial cells are recruited and proliferated,involved in the formation of capillaries.It is worth noting that newly formed blood vessels require MSCs to be stable,and the interaction of MSCs with endothelial cells further regulates angiogenesis by secreting angiogenic growth factors,cytokines and other signaling molecules.Based on recent advances in vivo and in vitro,we have used bone formation and bone repair models to provide a basis for better understanding of the complementary nature of the vascular system during skeletal development and fracture repair.An active vascular network is a necessary prerequisite for tissue engineered bone survival and integration with existing host tissues.A growing body of evidence suggests that under the influence of various hormones,growth factors and mechanical stress,cytokines promote bone growth and healing around the implant by directing cell proliferation,migration and differentiation.Transforming growth factor-?1(TGF-?1)is the most abundant cytokine in the bone matrix and can be released to induce migration and differentiation of MSCs into osteoblasts to produce bone.Platelet-derived growth factor-BB(PDGF-BB)is also involved in angiogenesis.Known as a powerful mitogen,it is also used as a source of mesenchymal cells.PDGF-BB is thought to activate these cells,stabilize newly formed blood vessels,coordinate cellular components,and promote osteoblast differentiation.PDGF-BB is important in the multi-component cascade kinetics of vascular-peripheral cell-MSC-osteoblasts,suggesting that PDGF-BB coupled TGF-?1 can serve as a central signal between cellular components and osteogenic factors.This study is mainly to explore:(1)the expression and function of PDGF-BB during the repair of bone tissue damage;(2)between PDGF-BB and TGF-?1 Expression changes in mesenchymal stem cells;(3)Expression and role of bone repair related genes in MSCs in vitro;(4)Proliferation and differentiation of MSCs by PDGF-BB and TGF-?1 The role and further explore the role of PDGF-BB and TGF-?1 in the repair of fractures.In this study,we selected bone marrow of 6-week-old mice,made fracture model,isolated bone marrow mesenchymal stem cells,through PDGF-BB and TGF-?1 intervention,to detect the two factors and bone marrow mesenchymal stem cells(MSCs)migration and proliferation,vascular endothelial cells and related factors expression changes,so as to study the mechanism of PDGF-BB and TGF-?1 in bone repair.Expression and mechanism of PDGF-BB couples with TGF-beta 1 in the process of fracture repairObjective:Fracture repair is still a huge challenge for clinicians.How to effectively prevent,treat and recover is a problem that needs to be solved urgently.However,tissue engineering provides promising therapies for generating new bone.Bone formation is a cellular process that is important for skeletal development throughout life and involves the coupling of osteogenesis and angiogenesis.Here,we studied the expression of PDGF-BB and TGF-?1 during fracture repair,and examined the migration and proliferation of these two factors and bone marrow mesenchymal stem cells(MSCs),and changes in the expression of vascular endothelial cells and related factors.Further study of the molecular pathways of the two in fracture repair mechanisms provides clues.It is helpful to further understand the molecular mechanism of fracture repair and the role of related factors,and provide new ideas for the prevention and treatment of fractures.Method:1.In the animal tissue specimens,the PDGF-BB,TGF-?1 and VEGF were detected by real-time quantitative PCR technology and western-blot after processing the fracture repair tissues taken at the 1st,3rd,6th,and 9th weeks.Correlation analysis of the three mRNAs and protein expressions using Pearson;by establishing an implant graft model,bone tissue and species were collected at the 2nd,4th,6th,8th,and 10th weeks.Plant specimens,the time of plant and plant action were measured,and the content of alkaline phosphatase and calcium salt in bone tissue were detected by AP and Von Kossa staining.2.MSCs cells and EPCs cells were cultured in vitro.the migration and proliferation andability of MSCs cells under different conditions was detected by Transwell experiment;the calcium content of MSCs cells under different conditions was observed by ARS staining;osteoblast-related protein Runx2 was detected by real-time quantitative PCR technology,Alkaline phosphase(AP),Collagen type I alpha 1(Coll ? 1)and Osteocalcin(Ocn)gene expression levels;flow cytometry was used to detect endothelial progenitor cell surface markers;ELISA was used to detect EPCs cells under different treatment conditions The expression of VEGF in the supernatant and the mechanism of fracture repair response at the molecular level.Results:1.Compared with the bone tissue of normal mice,the mRNA expression and protein expression of PDGF-BB,TGF-?1 and VEGF in fracture repair mice were higher than those in the control group;while at the gene expression level,TGF-?1 and PDGF-BB and VEGF were positively correlated;by measuring the implant graft,it was found that the effect time was longer than 4 weeks,and the alkaline phosphatase content and calcium salt content were higher than those of the control group.2.Transwell experiments found that PDGF-BB or TGF-?1 can significantly promote MSC migration;not only that,we also found that TGF-?1 shows greater potential than PDGF-BB;ARS staining observed MSCs treated with PDGF-BB Calcium is abundant in cells and Calcium is reduced in MSCs cells treated with TGF-?1;by detecting osteoblast-related proteins,we found that its expression was increased in the PDGF-BB group and decreased in TGF-13 1.During the action,the expression levels of related protein genes Runx2?AP and Coll a 1 increased,while the expressions of and Ocn decreased;PDGF-BB can induce capillary formation of EPCs,and the addition of MSCs further enhanced EPCs tube formation;VEGF was detected by ELISA in PDGF-EPCs treated with BB and MSCs are highly expressed.Conclusions:1.PDGF-BB and TGF-?1 can recruit MSCs and promote their migration and proliferation,but TGF-?1 is more significant;on the contrary,PDGF-BB can promote osteogenesis,but TGF-?1 can inhibit osteogenesis.2.The combined administration of PDGF-BB and TGF-?1 can significantly induce new bone formation in vivo.3.We found that co-culture of PDGF-BB and MSCs can enhance the angiogenesis induced by EPCs,which may be because the important angiogenesis growth factor(VEGF)is secreted by MSCs.4.PDGF-BB and TGF-?1 play an important role in fracture healing and repair.Therefore,appropriate regulation of the expression of both of them in MSCs may be a new way to regulate the repair of fractures after fracture injury,and also become a research to promote bone repair after fracture A new target.