| BACKGROUNDDistraction osteogenesis(DO)is a surgical technique which involves subperiosteal osteotomy,slowly separating bone by using one kind of fixing device,and allowing the bone healing process to fill in the gap,so the bones will be lengthened and widened,and craniofacial deformity or defect will finally be corrected.DO has been proved to be an extremely effective therapy in clinic.Distraction osteogenesis is highly efficient,it could produce new bone by 1mm in the distraction gap per day and there is no limit being found to bone production.The mechanism of distraction osteogenesis is very complex,and its high efficiency of osteogenesis is beyond all previous knowledge of bone formation.Whether the molecular mechanism of DO is similar to bone fracture healing,or to another form of bone regeneration? It's still not fully known.OBJECTIVESDistraction osteogenesis efficiency is very high,which could reach the growth rate of 4 to 6 times compared to infants and young children.Therefore,we propose the hypothesis that mechanical force or minimally invasive injury inDO may activate some of the factors or signal pathways which are only active during embryonic or neonatal development,thus leading to a bone formation process which is different from bone repair and even bone regeneration.This may be a similar atavistic biological process.To prove the mechanism,in this study,the canine model of mandibular distraction osteogenesis and canine model of pregnant dogs were established.Using high-throughput sequencing technology,bone tissues from mandibular of distraction gap,Fracture,embryo,new born canine,puppies and adult canine were been tested to show dynamic mRNA and miRNA of whole genome expression profiles.By analysising the difference and relevance of gene such as mRNA and miRNA's between the DO group and other groups,there would be a more comprehensive understanding of the similarities and differences between DO,embryonic development and fracture healing.We would explore the mRNA,miRNA and signaling pathways which associated with the start factors of DO,the unique formation of new bone,vascularization,selected the key gene for subsequent validation studies.METHODS1.Animal experimental center of Guangxi Medical University provides a total of 33 Chinese pastoral dogs.All the canine would be divided into 11 groups:(1)DO immediate(DO-0)group,n = 3;(2)DO 2-week consolidation(DO-2)group,n = 3;(3)new born(P-0)group,n = 3;(4)2 weeks old(P-2)group,n = 3;(5)4 weeks old(P-4)group,n = 3;(6)embryo of 30 days gestation(E-1)group,n = 3;(7)embryo of 35 days gestation(E-1)group,n = 3;(8)embryo of 50 days gestation(E-1)group,n = 3;(9)normal adult canine(AC)group,n = 3;(10)bone fracture group contrast to DO-1 group(BF-1),n = 3;(11)bone fracture group contrast to DO-2 group(BF-2),n = 3.Unilateral mandibular DO wasperformed on dogs of both DO-0 group and DO-2 group,and they were sacrificed immediately after the completion of distraction and after 2 weeks of consolidation period respectively.Bone tissue samples in and around distraction gap were collected,and assessment of distraction effect was made by mandible bone gross observation,preoperative CBCT and HE staining.Before the operation,ultrasound examinations were performed to observe the embryo of pregnant dogs(E1\E2\E3).Then Caesarean would be performed to induced the embryo,and embryo gross observation and HE staining were made.Pregnant dogs would be taken back to the cage feeding after cesarean section.Fracture operations were performed to the experimental dogs of fracture groups(BF1/BF2),which the two groups were corresponded to distraction group as control group.Fracture group canine underwent the fracture surgeries,and the bone fracture gap was maintained to 7mm by titanium plate.BF1 group maintained 14 days,and BF2 group maintained 28 days(corresponding to the latency phase of 7 days,the distraction phase of 7 days and fixed period).Mandibular bone tissue samples of P-0 group,P-2 group,P-4 group,P-8 group and NC group were also collected.All of the animals except the pregnant dogs were no limitation with sex,and the age of the adult dog was 1.5-2.0 years old,and the average weight was 12.5kg + 2.5kg(mean 13.5kg).2.TotalRNAs of all 11 groups were extracted,and c DNA libraries of mRNA and miRNA were then built.Illumina Hiseq4000 high throughput sequencing platform was applied to achieve the mRNA gene expression profile and miRNA gene expression profile.3.After mRNA and miRNA sequencing and standardized statistics the data of the gene expression,55 1-vs-1 paired comparison groups and 36 1-vs-1&1paired comparison groups were established based on 11 different groups ofsamples.Then the differentially expressed mRNA and miRNA genes were screened out and annotated by gene ontology(GO)and KEGG signaling pathways with specific softwares and databases.Screening the differentially expressed genes in the gene base,and exploring the similarities and differences of the biological similarities and between the DO group,the embryo group,the fracture group and the normal control group.RESULTS1.All subjects of DO-0 group and DO-2 group had successfully undergone DO and the subsequent follow-up observation.There were no severe complications observed in all the cases.Obvious mesial advancement of right mandibular canine was observed of all dogs,and osteoid like material existed in mandibular distraction space,all of which indicated that the right mandible had been successfully elongated by DO.All embryos of pregnant dogs group were induced by caesarean section,and the operation process was successful,and the embryos was complete,and the morphology of the embryos was in accordance with the developmental morphology of each pregnancy.After the induction of labor,no adverse reaction was found in the pregnant dogs.All pregnant dogs kept survival.Surgeries of the fracture groups were successful,and obvious mesial advancement of right mandibular canine was observed of all dogs.Fibrous tissue hyperplasia was seen at both ends of the fracture in the BF group,and no osteoid like substance could be identified.The Sample collection process of the new puppy group and the normal control group was successful.2.Adequate totalRNAs was extracted from all 11 groups of samples,and c DNA libraries of mRNA and miRNA for subsequent sequencing were successfully built respectively.High-throughput sequencing of bone tissues fromdistraction gap and from normal mandibles was completed successfully.3.There were 22523 expressed mRNA genes been found out by High-throughput sequencing.Based on the results of all 55 1-vs-1 paired comparison groups and 36 1-vs-1&1 paired comparison groups,We found that mRNA which both over-expressed in the DO groups and embryos groups,and also down regulation in AC group counted a total of 50.The most characteristic genes are CXCL12,MINOS1,OGN,PTN,SFRP2,MFAP5,IGFBP5,POSTN,HSPB6,and so on.The gene MINOS1,MFAP5 and HSPB6 were almost no expression in normal adult dogs(AC group).The functions of these genes include EMT regulation,vascularization regulation mobilization of mesenchymal stem cells and osteoblasts.There were 1921 genes collective expressing in the DO groups and fracture groups.The most characteristic genes are ATF4,CD99L2,IGFBP5 and RPL15,these genes have characteristic expression of different trends in DO groups and BF groups,mainly for the EMT control,and compared with the normal group,their expression was significantly changed(increased or decreased),all of which represented the different characteristics of DO and fracture healing.4.There were 354 expressed miRNA genes been found out by High-throughput sequencing.Based on the results of all 55 1-vs-1 paired comparison groups and 36 1-vs-1&1 paired comparison groups,We found that miRNA which both over-expressed in the DO groups and embryos groups,and also down regulation in AC group counted a total of 46.The most characteristic genes are mi R-136?mi R-299?mi R-369?mi R-376a?mi R-380?mi R-382?mi R-410?mi R-432?mi R-503?mi R-20a?mi R-23a?mi R-126?mi R-370?mi R-433.mi R-370 and mi R-433 were almost no expression in normal adult dogs(AC group).The functions of these genes include the regulation ofvascularization and angiogenesis;embryonic stem cells,endothelial progenitor cells and mesenchymal stem cells mobilization;vascular endothelial cell migration;osteoblast and osteoclast differentiation regulation.There were 26 expressed miRNA genes collective expressing in the DO groups and fracture groups.Excluding the genes which also significant express in AC group,we got mi R-203?mi R-204?mi R-205?mi R-338?mi R-9 ? mi R-34 a.These genes have characteristic expression of different trends in DO groups and BF groups,mainly for the EMT control,and compared with the normal group.Their expression was significantly changed(increased or decreased),all of which represented the different characteristics of DO and fracture healing on vascularization and ossification.CONCLUSIONIn the process of distraction osteogenesis,the following biological processes are induced by the initial ischemic injury,inflammatory response,mechanical stretch stimulation or static stretch stimulation: After the embryonic stage of development,a large number of genes second time significantly express and regulate vascularization mainly in the regeneration of blood tubular(vasculogenesis),at the same time,the mechanism of angiogenesis(Angiogenesis)in the early stage of distraction osteogenesis was inhibited,and there are many genes involved in the control of the vascular network with overload development in the late stage;immune mononuclear cells can be induced to differentiate into vascular endothelial cells;epithelial mesenchymal transition(EMT)was significantly inhibited,and a large number of mesenchymal cells are transformed into epithelial and endothelial cells to form new vascular networks;heat shock proteins(HSPs)are activated to maintain thestability of the cytoskeleton to enhance vascularization and ossification;a large number of bone marrow mesenchymal stem cells regulated by multiple genes proliferate rapidly to differentiate into osteoblasts,existing osteoblasts recruit,and muscle tissue specific protein secretes and promotes bone metabolism,maintain a rapid regeneration of new bone.By contrast,gene expression in the fracture healing process is significantly different from DO,part of the gene promoter is slower than DO,EMT process is significantly more active,and the healing process lacks the characteristics of rapid vascularization. |
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