| Objective: The effective reconstruction of craniomaxillofacial bone defects is a major challenge in plastic surgery.In clinical practice,autologous and allogenic bone grafts can be a “ gold standard ” in bone defect treatment,but there are great limitations.Patients with autogenous bone transplantation need to suffer the pain of multiple operations.20%-30% of patients will have scars,injuries,deformities and even disability in the donor area,and this operation is not suitable for the treatment of large-area bone defects.While the incidence of complications in allogeneic bone transplantation patients exceeds 30%,including fractures,nonunion,infection,etc.Additionally,the high incidence of craniomaxillofacial bone defects and high medical costs have placed a heavy burden on individuals,families and society.Therefore,how to effectively treat bone defects is related to the living standards of our people and the social and economic development.Bone tissue engineering is mainly composed of three elements: seed cells,inducing factors,and scaffold materials,whose effective and reasonable choices have become the foundation to promote the clinical application of bone tissue engineering,providing new ideas and directions for the treatment of bone defects.Adipose-derived stem cells(ADSCs)are a population of adult pluripotent cells extracted from adipose tissue that have the property of self-renewal and can differentiate into multiple lineages.It has become a popular "seed cells" and has been widely studied due to the advantages of abundant storage in vivo,easy acquisition(through liposuction,etc.),and expansion.Achieving the directional osteogenic differentiation of stem cells is the key to bone regeneration,and "inducing factors" play a key role in this process.The focus of this study is to find an appropriate and efficient "inducing factors" to induce osteogenic differentiation and explore the specific mechanism.Exosomes are a type of sphere-or dish-shaped extracellular vesicle whose diameter is between 30 and 150 nm.Exosomes are found in abundance in endosome-derived components,which are considered to play important roles in intercellular communication due to their ability to transfer “cargos”.Exosomes have been widely proved to play an important role in tissue regeneration,including promoting bone regeneration and repair.Therefore,ADSCs-derived exosomes are expected to be used as a novel "inducing factor" in the construction of bone tissue engineering.However,seed cells and inducing factors cannot act on local bone defects alone.The scaffold material provides a "matrix" support effect.Magnesium alloys have been widely studied as biomaterials,but there are still problems such as excessive degradation rates.The study found that it can be adjusted by adding appropriate alloy elements.This current experiment explores new Mg-2Zn-0.5Zr-0.5Nd alloy The mechanical properties,corrosion resistance and biocompatibility of the materials are intended to provide an experimental basis for the clinical application of bone defect repair.Methods:1.ADSCs were isolated to demonstrate their ability to differentiate into adipocytes,osteoblasts,and chondrocytes.The expression of the surface markers including CD10,CD13,CD49 d,CD34,CD31,CD45,CD44,CD73,CD105 were detected by flow cytometry.2.ADSCs-derived exosomes were obtained by Differential Ultracentrifugation.In order to define the exosomes,transmission electron microscopy(TEM)was used to observe their structures.The characteristic markers including TSG,Calnexin and CD9 were analyzed by western blot.To identify uptake of exosomes by ADSCs,exosomes were labeled with Dil and nuclei of ADSCs were stained with Hoechst33258 After co-culture,exosome uptake was observed by fluorescence microscopy.3.In order to establish more accurate results,we set-up 4 comparative groups: a negative control(NC)group,a positive control(PC)group,an Exos_D0 group,and an Exos_D14 group.ADSCs in the PC group were induced with osteogenic differentiation medium,while the NC group remained untreated.The expression level of osteogenic related genes and proteins was detected by q PCR and Western blot respectively.The activity of alkaline phosphatase(ALP)was detected by ALP activity assay,and calcium deposits were detected by Alizarin Red S(ARS).4.The expression of mi RNAs in Exos_D0 and Exos_D14 was detected by microarray,and the data were analyzed by bioinformatics.5.We selected the miR-130a-3p with the highest fold change,and used Targetscan online tool to predict the downstream target genes and related pathways. Dual-luciferase reporter gene assay was performed to verify the targeted binding of mi R-130a-3p and SIRT7.6.We transfected ADSCs with lentiviral overexpression particles(Overexpression group),lentiviral knockdown particles(Knockdown group)and lentiviral GFP particles(Lenti-control group),respectively.To verify the effect of mi R-130a-3p on the osteogenic differentiation of ADSCs,the expression level of osteogenic related genes and proteins was detected by q PCR and Western blot respectively.Calcium deposits were detected by Alizarin Red S(ARS).The effect of mi R-130a-3p on proliferation was detected by Cell Counting Kit-8(CCK-8).7.To further clarify the specific mechanism by which mi R-130a-3p regulated osteogenic differentiation of ADSCs,the Wnt signaling pathway Knockdown groups was added based on the original experimental grouping,namely Control + DKK1,Lenti-control +DKK1,Overexpression + DKK1 and Knockdown + DKK1.The expression of osteogenic related genes and protein was detected by q PCR and western blot,respectively.8.Use chemical analysis,scanning electron microscope,universal electronic testing machine,etc.to test the properties of Mg-2Zn-0.5Zr-0.5Nd alloy materials.9.Cell proliferation was detected by CCK-8,and morphology of cytoskeleton was observed by phalloidin;rabbit mandibular defect model was surgically constructed,and Mg-2Zn-0.5Zr-0.5Nd alloy material was implanted and analyzed by micro CT;thus in vivo and in vitro The biological properties of Mg-2Zn-0.5Zr-0.5Nd alloy materials were verified.Results:1.We detected the characteristic surface markers of ADSCs using flow cytometry and obtained the following results: the expressions of CD10,CD13,CD49 d,CD44,CD73,CD105 are positive,while the expressions of CD34,CD31,CD45 are negative.Importantly,ADSCs can differentiate into adipocytes,osteoblasts,and chondrocytes,confirming multi-lineage potential of ADSCs.2.TEM revealed that vesicles with particle sizes between 30 nm and 150 nm in diameter exhibited spherical morphology,proving the presence of exosomes.In addition,the western blot results showed that the exosome-associated proteins TSG101 and CD9 were expressed while the endoplasmic reticulum protein calnexin was hardly expressed in exosomes.Uptake assay showed that many exosomes were observed in the cytoplasm of their homotypic cells—ADSCs at only 6 h post incubation by fluorescence microscopy.3.Compared with NC group(conventional culture medium + ADSCs),the expression levels of Alp,Runx2 m RNA and protein in Exos_D14 group were significantly increased,but there was no significant change in Exos_D0 group.Additionally,Alizarin Red S and ALP activity assay showed that Exos_D14 group had better osteogenic effect.4.Compared with the Exos_D0 group,201 mi RNAs were up-regulated and 33 mi RNAs were down regulated,which was statistically significant(p < 0.05,| Fold change| ? 2).The results of bioinformatics showed that axon guidance,Wnt signaling pathway and MAPK signaling pathway were the main signaling pathways,and the main biological functions and processes were enzyme binding,cell projection,transcription factor activity,regulation of gene expression,and cell metabolism.5.Targetscan tool predicted that mi R-130a-3p can bind to the 3'UTR of SIRT7,which was confirmed by dual-luciferase reporter genes assay.6.Compared with the control and lenti-control groups,the expression of Alp,Runx2 m RNA and proteins in Overexpression group was increased significantly,while that in Knockdown group was decreased,but the results were not dramatically.CCK-8 results showed that the cell proliferation rate of Knockdown group was the highest and that of Overexpression group was the lowest.Through further study,it was found that the expression of ?-catenin,Axin2 proteins in Overexpression group was significantly increased.Compared with groups without DKK1 treatment,the expression of osteogenic related genes and proteins in DKK1 treatment gourps decreased,and the expression of Wnt signaling pathway related proteins was also down regulated.7.Mg-2Zn-0.5Zr-0.5Nd alloy material has more excellent mechanical properties and corrosion resistance,and has good biocompatibilityConclusion:1.ADSC-derived exosomes were more easily ingested by homotypic cells,namely ADSCs.Exosomes derived from osteogenically differentiated promoted osteogenic differentiation of ADSCs,but not exosomes derived from undifferentiated ADSCs.2.The changes of mi RNAs in exosomes played an important role in the osteogenic differentiation of adipose stem cells.3.The overexpression of miR-130a-3p promoted osteogenic differentiation of ADSCs while inhibited proliferation.The knockdown of mi R-130a-3p promoted the proliferation of ADSCs,but had no significant effect on osteogenic differentiation of ADSCs.Furthermore,mi R-130a-3p can bind with SIRT7.The results revealed that mi R-130a-3p promoted osteogenic differentiation of ADSCs by activating Wnt signaling pathway.4.Mg-2Zn-0.5Zr-0.5Nd alloy material has more excellent mechanical properties and corrosion resistance,and has good biocompatibility... |
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