| ObjectiveWith the development of life science,regenerative medicine in recent years has become a focus of research scientists.To construct tissue engineering bone repairing bone defect,vascularization rapidly is one of the most important problems.Endothelial Progenitor Cells(EPCs)are a branch of stem cells,which can promote vascularization in theory,and have the ability to provide nutritional to support the seed cells at the same time.The physiological characteristics of EPCs provide a new thinking way in repairing bone defect of tissue engineering bone.Our research team constructed biological tissue engineering bone with peripheral blood EPCs and autologous bone marrow mesenchymal stem cells(BMSCs)at the early stage.The PDPB showed better capillaries and osteogenesis ability in vivo.In this research we constructed indirect co-culture system with EPCs and BMSCs by transwell chambers in vitro.BMSCs growth,proliferation and multi-directional differentiation capacity and chemotactic movement ability of co-culture system were analyzed precisely.We speculated EPCs in co-culture system promoting BMSCs regulation mechanism by mRNA gene chip.It provided new theoretical basis and clinical application for BMSCs to construct tissue engineering bone to repair defects.Methods(1)Take 12 weeks rabbit bone marrow blood to receive the original rabbit BMSCs.According to the physiological characteristics of purified BMSCs by sticking wall growth,a microscope was applied to observe cell morphological changes.BMSCs were maintained its proliferation to the third-generation,surface antigen CD29,CD34,CD45 expression were detected to identify the BMSCs phenotype by flow cytometry.(2)Take 12 weeks of rabbit peripheral blood to receive the original rabbit EPCs by the stem cell separation and purification method.Double staining the third generation cells with DiI labeled acetyl Low Density Lipoprotein(DiI-acLDL)and Fluorescein labeled whin lectin-1(FITC-UEA-1).Cell morphological changes and the EPCs tube cavity structure were observed under microscope.(3)Different proportion of EPCs and BMSCs were put in Transwell Chambers respectively.The best proportion of cells was screened by cell counting method in vitro.(4)Corning Transwell Chambers(carbon polyester film,aperture 0.4 um)was applied to build BMSCs and EPCs indirect co-culture system.Collect EPCs and control the density of 1 × 105/mL on the bottom of the chamber on the Transwell membrane,the best proportion of BMSCs were put in the below room.The same amount BMSCs were put in 6-well culture plate plate as the control group.PDPBs were put in BMSCs cell suspension of two groups.Basal medium was added in plates for 7 days continuously and changed every two days.(5)Cell count plate was applied to count BMSCs growth of two groups for 10 days continuously.The growth curves of BMSCs of two groups were depicted(6)BMSCs of co-culture system and separately cultured BMSCs were induced osteogenesis separately.The activity of alkaline phosphatase was tested on days 1,3,7,14.Osteocalcin secretion was detected on days 1,3,7,14,21.BMSCs of co-culture system and separately cultured BMSCs were induced into fat separately.Oil red O was stained and analyzed.(7)Biological bones were put into bone defect model.Four groups eGFP positive staining rates were detected by immunohistochemical method at 2,4,8 weeks.(8)Endogenous stromal-derived factor 1(SDF-1),CXCR4,monocyte chemotactic protein 1(MCP-1)and its CCR2 chemokine receptor mRNA expression level were detected at 8 weeks with real-time quantitative PCR.SDF-1 and MCP-1 protein secretion were detected by ELISA at 8 weeks.CXCR4 and CCR2 protein expression were detected by western blot at 8 weeks.(9)Tissue engineering bone which were implanted into animals was made into slices.Masson staining was made to calculation collagen mass by pixels which on behalf of new bone formation at 2,4,8 weeks.(10)We collected two room cells of transwell co-culture system on days 7 to prepare mRNA chip samples.Total RNA were extracted with Trizol and expression difference were analyzed by Affymetrix company to filter pathway and speculate the regulatory mechanism.Results(1)Primitive BMSCs cultured to day 3 were spindle gathered growth shape.Adherent cells increased obviously and cells arranged loosely paving stones when EPCs were cultured to the third generation.By flow cytometry detection,BMSCs surface markers CD29 positive rate reached 97.1%,CD34 and CD45 were negative,which were consistent with the characteristics of BMSCs.DiI labeled acetyl low density lipoprotein cholesterol and fluorescein labeled lectin immunofluorescence double staining were positive,which were consistent with the EPCs characteristic.Unique tube cavity structure was formed on Matrigel by EPCs under microscope.(2)Different proportion of EPCs and BMSCs were put in Transwell Chambers respectively.The best proportion of cells was 10:1.(3)Partially deproteinized bones(PDPB)were smooth and porous structure under the electron microscope.No floater was seen after rinsing with distilled water.Seven days later,the cells grown on biological bone were observed under scanning electron microscopy.Cells attached to PDPB surface,generating a large number of filaments.(4)Proliferation activity of co-culture system BMSCs growth trend curve was higher than BMSCs cultured alone group.After BMSCs of co-culture system and BMSCs cultured alone group were induced osteogenesis separately,calcified nodules were observed.Alkaline phosphatase kit showed alkaline phosphatase activity of BMSCs cultured alone group reduced at day 11,while co-culture system BMSCs at day 14.ELISA results showed osteocalcin secretion of two groups BMSCs were with significant difference after day 3.(5)After BMSCs of co-culture system group and BMSCs cultured alone group were induced into fat separately,cells morphology changed,the cells shape from spindle became into round,oval,etc.A certain amount of lipid droplets emerged in co-culture system group and BMSCs cultured alone group after induced 48h.The quantitative analysis of lipid deposition by oil red O staining showed BMSCs of co-culture system group forming fat ability was better than that of BMSCs cultured alone group at day 7.(6)Immunohistochemical staining of eGFP positive staining rate indicated that co-culture cell group and BMSCs cultured alone group were with no significant difference at 2 weeks.4weeks later,eGFP positive staining rate of co-culture group was higher than the other groups.(7)Stromal cell derived factor-1(SDF-1)mRNA level in co-culture system group was significantly higher than bone marrow mesenchymal stem cells group,endothelial progenitor cells group and unseeded cell group.SDF-1 receptor CXCR4 mRNA level in co-culture system group was also higher than the other groups.Monocyte chemotactic protein-1(MCP-1)mRNA level in co-culture system group was higher than the other groups.However,there was no significant difference of MCP-1 receptor CCR2 in co-culture system group compared with other groups.(8)At 8 weeks,SDF-1 expression in co-culture system group was significantly higher than the other groups.And MCP-1 expression of co-culture system group was only higher than unseeded group.CXCR4 expression in co-culture system group was higher than the other groups.There was no significant difference of CCR2 protein expression in co-culture system group and other groups.(9)Masson staining result showed de-protein biological bone built with co-culture cell can effectively promote bone defect repairing.The amount of new collagen composite of co-culture group was higher than EPCs culture alone group and unsedded group at 2 weeks.4 weeks later the amount of new collagen composite of co-culture group was higher than all the other groups.(10)Set co-culture system group/cell cultured alone group>2 as increased genes and<0.5 as decreased genes as standard.Gene chip result of co-culture system group BMSCs and BMSCs cultured alone group showed a total of 1772 differentially expressed genes,and 664 of them were increased,1108 of them were decreased.Enrichment of significant genes were chromosomes,centromere region of cell membrane and organelles,DNA wrappers,etc.Significant enrichment of channel name:cell cycle,p53 signaling pathway,ketone metabolism,transcription information system of cancer,etc.(11)Gene chip result of co-culture system group EPCs and EPCs cultured alone group showed a total of 1473 differentially expressed genes,and 595 of them were increased,878 of them were decreased.Enrichment of significant genes:intracellular organelles,mitotic cell processes,chromosome,etc.Significant enrichment of channel name:cell cycle,DNA replication,p53 signaling pathway,purine metabolism,etc.Conclusion(1)The methods we received cells were reliable and effective for separation and purification rabbit BMSCs and EPCs.Surface markers were consistent with the characteristics of BMSCs by flow cytometry.Double staining result were consistent with the EPCs characteristic.(2)Corning Transwell Chambers(carbon polyester film,aperture 0.4 um)was suitable to build BMSCs and EPCs indirect co-culture system in vitro.(3)Primary culture of rabbit BMSCs could differentiate into osteoblast,fat cells and show their features after osteogenesis and fat inducing.(4)We can effectively compare biological differences of co-culture system group BMSCs and BMSCs cultured alone group.Co-culture system group BMSCs showed stronger cell proliferation activity,more potential of osteogenesis and fat differentiation ability.(5)Immunohistochemical staining results of eGFP positive staining rate indicated that co-culture cell group had better promotion of exogenous BMSCs to defect homing.(6)EPCs promoting BMSCs chemotactic movement was closely related to SDF-1/CXCR4 and MCP-1/CCR2 chemotactic axis.The SDF-1/CXCR4 axis was more evidenced in this chemotactic movement.(7)Deprotein biological bone built with BMSCs and EPCs could be more effectively in promoting bone defect repair.(8)Through BMSCs and EPCs indirect co-culture system with corning transwell Chambers compared to BMSCs cultured alone and EPCs cultured alone,we can effectively filter out different expressed genes,which will be foundation of rabbit BMSCs and peripheral blood EPCs interaction mechanism and clarify possible mechanism. |
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