Recruitment Of Mesenchymal Stem Cells In Mandibular Distraction Osteogenesis Through The SDF-1/CXCR4Pathway

Distraction osteogenesis (DO) has been a widely applied technique inorthopedics and craniofacial surgery. However, the exact mechanism of DO,especially for the origin and differentiation of osteoblasts, is still poor understood.During the process of DO, the intramembranous bone formation is thepredominant mechanism of ossification in which neo-callus formation occursthrough the direct differentiation of mesenchymal stem cells (MSCs) intoosteoblast lineages. Evidences suggested that MSCs can home to injured orischemic tissues, involving migration across endothelial cell layers. It is likelythat injured tissue expresses specific receptors or ligands to facilitate trafficking,adhesion, and infiltration of MSCs to the site of injury. These ligands include akey stem cell homing factor, stromal cell-derived factor-1(SDF-1or CXCL12),which belongs to the CXC subfamily of chemokine and is widely expressed inmany tissues, especially in bone marrow. In this study, we tested the hypothesisthat in response to the gradual distraction stresses, there is a systemicmobilization of MSCs to the neo-callus in distraction gap via the SDF-1/CXCR4 axis. The aims of this study were to explore the mechanism of DO and providenew strategy for shortening the treatment time of DO. Five parts were included inthis study:1Rat unilateral mandibular DO modelObjective: To establish a rat unilateral mandibular DO model using aself-developed distractor. Methods:15male Sprague-Dawley (SD) ratsunderwent the right mandibular distractor implantation surgery and the leftmandibular fracture rigid internal fixation surgery. After a latency period of5days, gradual distraction was performed at a rate of0.2mm twice a day for10days. Animals were sacrificed with an overdose of pentobarbital sodium on the0,2,4weeks of consolidation time, and the callus from the distraction gap and thefracture site was harvested. Results: The right mandibles of all15rats werelengthened successfully, and the distractors were fairly stable. The average widthof the distraction gap was3.5mm, which was87.5%of the expected gap width.HE staining showed that the regenerated bone was close to normal bone tissue atthe four weeks of consolidation time. Conclusion: Our self-designed externalmandibular distractor was easy to operate and meet the requirements of the ratunilateral mandibular distraction osteogenesis. This rat mandibular DO model hassuperb feasibility and reproducibility, and can provide a favorable platform forfurther molecular and genetic study on DO.2In vitro isolation and culture of rat bone marrow mesenchymal stem cellsObjective: To isolate, purify and culture rat bone marrow mesenchymal stemcells and identify the expression of chemokine receptor CXCR4. Methods: Therat MSCs were cultured by using the whole bone marrow adherence method. The morphological and growth characteristics were observed, and osteogenic,adipogenic differentiations were conducted. Cell surface markers and the CXCR4expression were identified by flow cytometry. Results: The cultured cells canproliferate rapidly in vitro, and can differentiate into osteoblasts and adipocytesunder special conditions. Flow cytometry identified that cell surface markersCD29(+), CD44(+), CD90(+), CD34(-) and CD45(-). The cell surface CXCR4expression was16.5%, and intracellular CXCR4expression was83.2%.Conclusion: The cultivated cells are multipotential mesenchymal stem cells andthey can express CXCR4both intracellularly and extracellularly.3The expression of SDF-1in rat mandibular DO and bone fractureObjective: To examine the changes of SDF-1expression in the callus during DOand fracture healing in a rat model. Methods: Forty male SD rats underwent theright mandibular distractor implantation surgery and the left mandibular fracturerigid internal fixation surgery. After a latency period of5days, gradualdistraction was performed at a rate of0.2mm twice a day for10days. Animalswere killed on days0,3,6,9,12,15,18, and21after surgery (n=5). Tissueextracts from both DO and fracture callus were prepared. ELISA andImmunohistochemistry were used for SDF-1detection according to themanufacturer’s protocol. Results: The expression of SDF-1was mainly aroundthe osteoblasts and vessels, and there were more intensive expression of SDF-1inDO zones than in bone fracture zones. ELISA showed that SDF-1increased by3times after osteotomy both in DO and in bone fracture. In bone fracture site,SDF-1began to decrease1week later, and in DO site, SDF-1increased againafter the starting of distraction by5times of baseline until1week ofconsolidation time. Conclusion: The expression of SDF-1reached a greater peak and maintained a longer up-regulating duration in DO than in fracture healing.4In vitro chemotaxis assay of MSCs to SDF-1Objective: To investigate the migration of MSCs in response to a gradient ofSDF-1and CXCR4inhibition in a Transwell chamber assay. Methods: In aTranswell chamber assay with8-μm pores, the upper chambers were loaded with1×105MSCs in500μL DMEM, and dilutions of0,100,200,300and400ng/mLof recombinant SDF-1in1.5mL of media were placed to the lower side of themembrane. For neutralization studies cells were pretreated with a potent andspecific antagonist of CXCR4(AMD3100,5μg/mL) for30minutes at37°C.After24hours of incubation at37°C in5%CO2, the migrated cells were stainedand counted under a light microscope. Results: The migration of MSCs increasedaccording to the different doses of SDF-1in a dose-dependent manner (P<0.05).Blocking of CXCR4using5μg/mL AMD3100reduced the migrated cell numbermarkedly (P<0.05). Conclusion: SDF-1facilitated the migration of MSCs invitro, and this can be inhibited by treatment of MSCs with CXCR4antagonistAMD3100.5Homing of ex vivo-expanded MSCs to DO regeneration site is mediatedby SDF-1/CXCR4pathwayObjective: To investigate whether ex vivo-expanded MSCs can home to DO siteafter infusion via tail vein, and to determine the influence of SDF-1/CXCR4pathway on this migration. Methods:32male SD rats underwent the rightmandibular distractor implantation surgery and were divided into4groups (n=8):group A received systemic administration of GFP-MSCs; group B receivedsystemic administration of AMD3100(5μg/mL, Sigma) pretreated GFP-MSCs; group C received local injection of PBS in the distraction gap1hour beforeadministration of GFP-MSCs; group D received local injection of100ngrecombinant SDF-1in the distraction gap1hour before administration ofGFP-MSCs. The GFP-MSCs were infused into each rat through the tail vein on5days after starting of distraction. Half of animals were sacrificed at24hourspost-transplantation and the soft callus were harvested and cut into frozensections for fluorescence microscopy examination. The left animals weresacrificed at2weeks of consolidation time and HE staining was performed forhistomorphometric analysis. Results: GFP-positive osteoblasts which originatedfrom injected cells made a significant contribution to DO new bone formation.Local injection of SDF-1into the distraction gap (group D) increased the hominglevels of MSCs in the regenerated bone (P<0.05), whereas blockage of theSDF-1/CXCR4interaction with AMD3100(group B) reduced homing markedly(P<0.05). Histomorphometric analysis showed that group D had greater BV/TVthan other3groups, while group B had least BV/TV in4groups (P<0.05).Conclusion: The MSCs from intravenous infusion can migrate to the distractiongap, and this can be promoted by local infusion of exogenous SDF-1indistraction gap and inhibited by treatment of MSCs with CXCR4antagonist. Thematurity of the new bone was positively correlated with the number of migratedcells in distraction gap.Summaries: This study firstly found that SDF-1was up-regulated duringpost-osteotomy and the distraction period in a rat unilateral mandibulardistraction osteogenesis model. We testified firstly that MSCs can migrate todistraction gap from peripheral circulation during DO. We also firstly putforward that SDF-1/CXCR4axis can recruit MSCs to distraction gap during DO. This study further explored the molecular mechanism of DO, and provided atheoretical basis for not only the strategy of systemic administration of stem cellsin DO, but also the clinical application of SDF-1to promote new bone formationand shorten the treatment time of DO.