The Study On Mechanism Of Mandible Mesenchymal Stem Cell-Derived Exosomes In Regulation Of Hoxa11 And Free Bone Graft Healing Through MiR-126a-5p

Objective:Clinically,large area bone defects are a challenge for oral soft and hard tissue repair.Among approximately 13 million patients with bone defects each year,only 10% of bone defects larger than 25 mm have a chance of self-healing.Autotransplantation of free limb bone graft is the best method for repairing large area defects of the mandible.However,5%-10% of patients still suffer delayed healing of the bone defect site after surgery.To develop new therapies for transplantation between mandible and limb bones,it is necessary to analyse the healing mechanism of autologous free bone transplantation.From the perspective of embryology,maxillofacial bones and limb bones originate from two different germ layers: neural crest and mesoderm;From the perspective of cytology,bone marrow mesenchymal stem cells(BMSCs)of limbs express homeobox a11 gene(Hoxa11),while mandible BMSCs(M-MSCs)almost do not express Hoxa11;From the perspective of differentiation direction,the osteogenic differentiation ability of M-MSCs is higher than that of limb bone BMSCs.However,in the mandible-femur free bone transplantation of this study,how the mandile recipient bed adjusts the activities of BMSCs in the femur bone graft is unknown.This paper’s aim is exploring the influence of M-MSCs derived exosomes(M-MSC-exos)on bone healing during free bone transplantation and evaluating the effects of micro RNA(miRNA)cargo loaded by M-MSC-exos in the recipient bed on the differentiation and healing of limb bone grafts by regulating the microenvironment.The aim of this study is to initially reveal the mechanism of communication and regulation between exosomes of BMSCs with different embryonic origins,and to open up a new idea for free bone transplantation healing.Methods:1.The rat model with autologous mandible-femur free transplantation was developed.Paraffin sections were stained with Masson,Goldner and H&E.The situation of defect filling,new bone structure,relative content of osteoid,relative content of mineralized bone and the osteogenic activity in the graft area were analysed using a bone morphometric analysis system.2.A Transwell co-culture system of M-MSCs and femur mesenchymal stem cells(F-MSCs)was developed.The quantity of the cells in the upper and lower compartments is 1:5.Real-time quantitative polymerase chain reaction(RT-q PCR)is utilized to check the effect of M-MSCs on F-MSCs’ osteogenic activity.3.By using ultracentrifugation,Mandible mesenchymal stem cell derived exosomes(M-MSC-exos)were collected.Then,nanoparticle tracking analysis(NTA)and transmission electron microscopy(TEM)feature them.4.By adding the classical exosome inhibitor GW4869 to the Transwell co-culture system of M-MSCs and F-MSCs,it is verified that exosomes play a signal communication role in it through RT-q PCR.5.The sequencing of small RNA showed the differences in the expression of small RNA in the contents of M-MSC-exos and femur mesenchymal stem cell-derived exosomes(F-MSC-exos)are observed,and miR-126a-5p is a statistically significant miRNA and is involved in regulating bone formation.6.Using lentivirus transfection technology,the effect of miR-126a-5p-knock down M-MSCs on the osteogenic ability of F-MSCs was tested.Then,the dual luciferase reporter gene experiment was used to testify the binding ability of miR-126a-5p and Hoxa11.Results:1.When the same femoral bone graft is transplanted to different implant beds(mandible or femur),there are differences in osteogenic healing in the graft area.After repairing mandibular bone defects with femoral bone graft,the new bone trabeculae were evenly interwoven with each other,and abundant new blood vessels were visible.The osteogenesis effect reflected by Runx2,OSX,OCN and Col-1 is enhanced.After free transplantation of femur bone grafts to repair femoral defects,there are more irregular collagen arrangements between the bone graft and the recipient bed,with disordered bone trabeculae,relatively low mineral/organic matrix ratios,and relatively low expression of osteogenic markers such as Runx2,OSX,OCN and Col-1.2.By establishing a co-culture system of M-MSCs and F-MSCs,RT-q PCR results showed that under the exposure of M-MSCs microenvironment,osteogenic indicators of F-MSCs such as Runx2,OSX,OCN and Col-1 were upregulated.3.After using the exosome inhibitor GW4869 in the co-culture system of M-MSCs and F-MSCs,RT-q PCR showed that when the exosomes of the jaw microenvironment was blocked,the previously upregulated Runx2,OSX,OCN and Col-1 in the F-MSCs were reversed,which suggests the exosomes played a pivotal role in it.4.The exosomes of M-MSCs were successfully extracted and identified,and their structures and particle sizes were shown with TEM and NTA.Furthermore,the sequence of small RNA in M-MSC-exos and F-MSC-exos was analyzed.When the log2(FC)was set to greater than 1,it was found that there was a difference in the expression of miR-126a-5p in the contents between M-MSC-exos and F-MSC-exos.5.The osteogenic influence on F-MSCs was weakened when miR-126a-5p expression was knockdown in M-MSCs.It is suggested that exosomal miR-126a-5p is an important substance regulating the healing of free bone transplantation.6.The miR-126a-5p in M-MSC-exos promotes the osteogenic healing effect of femoral bone grafts in the mandible recipient bed by targeting the Hoxa11 gene.Conclusion:During the process of autologous mandibular bone block were swapped for femoral bone block to repair the mandible defect,the M-MSCs in the recipient bed can transmit miR-126a-5p to the F-MSCs of the bone grafting block through exosomes.Exosomal miR-126a-5p is in charge of regulating the expression of Hoxa11 and osteogenic effect of the F-MSCs,and therefore facilitates the reconstruction of the femur grafting block with the mandible recipient bed.