The Research On EPC-BMSC Co-culture Combined With PDPBB Tissue Engineering Bone System Transplantation

[Objective]In this study,EPCs and BMSCs derived from bone marrow were used to construct a stem cell co-culture system,to explore the mechanism of cell mutual regulation in the EPC-BMSC co-culture system.At the same time,the partially deproteinized biologic bone(PDPBB)prepared by our research group was incorporated into the tissue engineering bone system to study the co-transplantation of PDPBB.In addition,the regulatory mechanism of FoxO1 gene on EPCs promoting BMSCs homing was also studied in this study.[Methods](1)After the SD rats were killed by cervical dislocation,EPCs and BMSCs from bone marrow were isolated and cultured by the adherence method of bilateral femur and tibia.EPCs were identified by tubule formation test,immunodouble staining and CD31 immunofluorescence staining.BMSCs were identified by inducing osteogenic,adipogenic and chondrogenic differentiation of BMSCs and using different differentiation staining kits.(2)The cultured EPCs and BMSCs were co-cultured,and the EPC/BMSCs were pure EPCs,2:1,1:1,1:2 and pure BMSCs were co-cultured,and the proportion of cells in the co-culture was determined by observing the cell morphology and cell proliferation experiments.(3)Transwell chamber was used to construct EPC-BMSC co-culture model.Total RNA of cells was extracted,and the differential genes between the co-culture system and the single culture group were analyzed by transcriptome sequencing technology.(4)The effect of BMSCs on the tubule-forming ability of EPCs in EPC-BMSC co-culture system was studied by tubule-forming experiment.IL15RA gene expression of BMSCs in the EPC-BMSC co-culture system was verified by qPCR,and the expression of Osteopontin(OPN)and Osteopontin(OC)in BMSCs under the co-culture system was detected simultaneously to study the effect of EPCs on osteogenic ability of BMSCs.(5)PDPB and PDPBB were prepared from the commercial cancellous bone of porcine spine by surface deproteinization.The maximum stress and elastic modulus of PDPB,PDPBB and the decalcified bone matrix scaffolds were compared under compression test.The surface microstructure of the materials was observed by scanning electron microscopy.(6)The EPC-BMSC co-culture system was inoculated on PDPB,PDPBB and decalcified bone matrix,respectively.The biocompatibility of the three scaffolds was evaluated by cell proliferation assay and scanning electron microscopy.The EPCs,BMSCs and EPC-BMSC co-culture systems were inoculated on PDPBB scaffolds,respectively.Cell growth and cell adhesion on the scaffold surface were evaluated by cell proliferation assay and scanning electron microscopy.(7)FoxO1 gene expression in EPCs derived from bone marrow was knocked down by RNAi technique,and FoxO1 gene expression was detected by qPCR and Western blot techniques from mRNA and protein levels,respectively.After stable knockdown FoxO1 gene EPCs were cultured,the expression of SDF-1 gene in EPCs after knockdown of FoxO1 gene was detected by qPCR to determine the relationship between the two.(8)The homing effect of EPCs with stable knockdown of FoxO1 gene on BMSCs was studied in Transwell chamber.The expression of SDF-1 gene in lower compartment EPCs in the chemotaxis model was detected by qPCR compared with that in the control group.[Results](1)EPCs and BMSCs derived from bone marrow were successfully isolated and cultured by adherence method.The EPCs and BMSCs derived from bone marrow were positive for double immunostaining and CD31 immunofluorescence staining,and had the ability of tube formation.BMSCs were successfully induced to differentiate into osteoblasts,adipocytes and chondroblasts.(2)When EPCs and BMSCs were co-cultured in different ratios,cell proliferation experiments showed that when the ratio of EPC/BMSCs was 2:1,the cells could maintain a high proliferation rate.The cell proliferation activity was the highest on the third day,and the optimal ratio of EPC/BMSC was 2:1.(3)The results of transcriptome sequencing indicated that BMSCs in the EPC-BMSC co-culture system had 3 different genes at the transcriptional level compared with BMSCs in the control group,of which 2 were up-regulated and 1 was down-regulated.Compared with the control group,there were 11 differential genes in the transcription level of EPCs in the co-culture group,including 8 up-regulated genes and 3 down-regulated genes.Among them,the up-regulated differential gene IL15Ra in the co-culture group might be related to the osteogenesis and bone mineralization of BMSCs.(4)There was no significant difference in the length of tubules and the number of branch nodes of EPCs in the co-culture system at the early stage of tubulation(2h)compared with the control group,indicating that there was no significant change in the tubulation ability of EPCs in the co-culture system.BMSCs in the co-culture system up-regulated the expression of IL15RA and OPN genes,while the expression of OC gene was not significantly different,indicating that EPCs can promote the osteogenic ability of BMSCs.(5)The results of compression experiments showed that the maximum stress of PDPBB and PDPB was similar,while the maximum stress of decalcified bone matrix was less than that of PDPB and PDPBB,the difference was statistically significant.The surface of PDPBB was observed by scanning electron microscopy and filamentous fibronectin was found.(6)The cell proliferation activity of the EPC-BMSC co-culture system on the decalcified bone matrix was the highest,followed by the PDPBB scaffold,while the cell proliferation activity of the EPC-BMSC co-culture system on the PDPB scaffold was the lowest,the difference was statistically significant.By scanning electron microscopy,it was found that the EPC-BMSC co-culture system had the best growth on the decalcified bone matrix and PDPBB surface,while the cell morphology of the EPC-BMSC co-culture system on PDPB surface was chaotic.The EPC-BMSC co-culture system had better cell adhesion to PDPBB surface in the decalcified bone matrix,but fewer pseudopods were found on PDPB surface.The proliferation activity of EPC-BMSC co-culture system on PDPBB surface was higher than that of EPCs or BMSCs alone,and the difference was statistically significant.It was observed under scanning electron microscope that BMSCs were beneficial to the cell adhesion of EPCs on the scaffold surface.(7)EPCs treated by RNAi technology were detected by qPCR,and the expression of FoxO1 gene was significantly decreased,the difference was statistically significant,while the expression of SDF-1 gene was not significantly different from the control group,indicating that FoxO1 and SDF-1 had no direct relationship.(8)In the Transwell chamber model,there was no significant difference in the number of BMSCs migration between the EPCs with stable knockdown of FoxO1 gene expression and the control group,and there was no significant difference in the expression of SDF-1 gene between the control group and the EPCs under the Transwell chamber model by qPCR detection.[Conclusions](1)In this study,EPCs and BMSCs were successfully isolated from whole bone marrow cells by adherence method,and the optimal ratio of EPC/BMSC in EPC-BMSC co-culture system was 2:1.In the EPC-BMSC co-culture system,the osteogenic ability of BMSCs promoted by EPCs may be related to the IL15RA gene,while BMSCs had no significant effect on the tubule formation ability of EPCs.Through compression experiment,cell proliferation experiment and scanning electron microscope observation of cell adhesion,PDPBB was proved to be an ideal scaffold material in tissue engineering bone,and EPC-BMSC co-culture system could enhance the adhesion of EPCs on the surface of scaffold.(2)FoxO1,as a transcription factor,has no significant effect on the expression of SDF-1 gene in EPCs,and FoxO1 has no direct regulation on the homing of BMSCs promoted by EPCs.