Sympathetic Nervous System Regulates Mesenchymal Stem Cell Mobilization In Rat Mandibular Distraction Osteogenesis

Distraction osteogenesis (DO) has been widely used to regenerate bone tissues fordeformities, defects and fractures in long bones and maxillofacial bones.However, itremains largely unknown on the osteogenic mechanisms during DO and the efficiency ofDO awaits improvement. In order to differentiate into osteoblasts, mesenchymal stemcells (MSCs) are needed to migrate from their stem cell niches to bone-forming sites.Therefore, MSC mobilization is prerequisite to the bone formation in DO. It has beendemonstrated that MSCs are a subset of perivascular cells, and sympathetic nervoussystem (SNS) profusely innervate skeletal system and regulate the bone formation andresorption. Therefore, it is very likely that sympathetic nerves contribute to the perivascular stem cell niche for MSCs to reside in the bone marrow. The aim of thisstudy was to investigate the mechanism of effect of sympathetic denervation on MSCsmobilization and migration in rat mandibular DO. Six parts were included in this study:1Sympathic innervation and MSCs distribution in SD rat mandibleObjective: To determine the sympathic innervation and MSCs ditribution in SD ratmandible. Methods: Normal mandibles of2adult male Sprague-dawley (SD) rats wereused in Immunofluorescence assay for staining of sympathetic fiber marker TH and MSCsmarker Nestin. Results: Nestin+cells were abundant in perivascular regions andsympathetic fiber marker TH staining was intense along blood vessels. Conclusion:Mandibular bone morrow MSCs were confined by sympathetic nerves under normalcircumstance. This provide the foundation for following study.2Rat unilateral mandibular DO with transection of cervical sympathetictrunk (TCST) modelObjective: Establishment of rat unilateral mandibular DO combined with transectionof cervical sympathetic trunk model. Methods:6adult male SD rats were devided into2equal groups(n=3) as DO group and DO+TCST group. DO group only received the rightmandibular distractor implantation surgery. DO+TCST group underwent the rightmandibular distractor implantation surgery and bilateral TCST operation. Rats cervicalsympathetic ganglia of DO+TCST group were resected and underwent histologicalobservation. Sympathetic denervation was confirmed by observation of Horner’ssyndrome and HE stain of ganglia. Rats were sacrificed at consolidation time of4weeks,the new regenerated tissues were harvested for general observation and histomorphometricassay. Results: After surgery, DO+TCST group rats showed classic Horner’s syndrome,and the ganglia HE staining was consistent with typical neurohistology character. Theright mandibles of all6rats were lengthened successfully. The average length of disractionwas3.4mm, which was85%of the prospective length. HE staining indicated that theregenerated tissue was similar with normal bone at the4weeks of consolidation time in both groups, but the DO+TCST group showed more volume and quality bone trabeculae.Conclusion: This rat model is reliable for further molecular study.3Effect of sympathetic denervation on alternation of NE/adrb3expre-ssion and MSCs distribution in rat DO medolObjective: To determine the alternation of NE/adrb3, Nestin expression in rat medol.Methods: According to intervention and time point,20adult male SD rats were devidedinto4equal groups(n=5) respectively. The right mandibles and the distraction zonespecimens were harvested for immunohistochemistry assays at specific time point.Results: The expression of NE was hardly found, and very little adrb3expression wasobserved around the blood vessel in TCST group, but the expression of NE and adrb3wasabundant in the control group; the Nestin+MSCs were abundant and mainly distrubuted incallus matrix in TCST group, whereas that Nestin+MSCs were mainly located adjacentto perivascular region in the control group. Conclusion: TCST depleted sympatheticmediator NE and its receptor adrb3in distraction zone; Nestin+MSCs have highertendency of migration from perivascular region to callus matrix under distraction stressafter TCST in vivo.4The expression of SDF-1/CXCR4in rat mandibular DO+TCST modelObjective: To examine the alternation of SDF-1/CXCR4expression in theregenerated tissue during DO after TCST. Methods: According to intervention and timepoint,30adult male SD rats were devided into6equal groups(n=5) respectively. The rightmandibles and the distraction zone specimens were harvested for immunohistochemistryassays at specific time point. Results: The expression of SDF-1in osteogenic zone wassignificantly higher than that in perivas-cular region in DO+TCST groups. The percentageof perivascular region SDF-1expression in DO+TCST groups were apparently lower thanthat in DO groups. Conclusion: SDF-1emerged low-to-high concentration gradient fromperivascular region to callus matrix after TCST. 5Isolation, culture and identification of jaw bone marrow mesenchymalstem cells(MSCs) in vitroObjective: To isolate and characterize rat jaw bone marrow MSCs and verify theexpression of Nestin, adrb3and CXCR4. Methods: The rat jaw bone marrow MSCs werecultured by tissue enzymatic digestion-adherent explants method. The morphological andgrowth characteristics of cells were observed, cell surface markers expression wereidentified by flow cytometry, and osteogenic, adipogenic differentiations were induced.Nestin, adrb3and CXCR4expression were detected by immunohistochemistry. Results:The cultured cells can proliferate rapidly in vitro, and can differentiate into osteoblasts andadipocytes under special conditions. Flow cytometry confirmed that cell surface markersCD34(-), CD45(-), CD29(+), CD44(+) and CD90(+). The cell surface expression Nestin,adrb3and CXCR4. Conclusion: The cells obtained from jaw bone are multipotentialMSCs and target cells of NE and SDF-1.6Effect of NE on MSCs proliferation, differentiationand migration in vitroObjective: To determine effect of NE on MSCs proliferation, differentiation andmigration in vitro. Methods: The rat jaw bone marrow MSCs were cultured with10-7mol/L NE or without. Population Doubling (PD) assay, BrdU incorporation assay andOsteogenic assay were performed. Migration was assayed using a24-well transwell inpolycarbonate porous membrane insert (pore diameter of8um). GFP-MSCsat a densityof5×10~5cells/ml each with NE10-7mol/L DMEM or without, were placed in the upperchamber. The lower chamber contained a concentration of25ng/ml SDF-1in600ul ofmedium. Following12h of incubation, the migrated MSCs were counted. Results: PD,BrdU+cells%and mineralized area%of NE10-7mol/L co-cultured MSCs were lowerthan non-NE co-cultured MSCs. The number of migrated GFP-MSCs in10-7mol/L NEtreatment group were markedly decreased. Conclusion: NE can negatively modulateMSCs proliferation and differentiation, and inhibit MSC migration in vitro.