A Study Into Effects Of Intermittent Axial Compressive Stress On Adhesion,Proliferation And Osteogenic Differentiation Ability Of Tissue Engineered Bone Seed Cells

Objectives:Large segmental bone defects are an important problem in clinical treatment,and tissue engineered bone(TEB)transplantation in combination with mechanical stress stimulation provides a new solution to this issue.Our previous studies found that intermittent axial compressive stress stimulation combined with deproteinized and defatted cancellous bone scaffolds could effectively promote bone defect healing in rabbits.However,the mechanism of how this stress stimulation combined with TEB can promote the healing of bone defect is still unclear.This study further explored the effects of this stress on TEB seed cells so as to determine its mechanism for promoting bone defect repairing.Methods:To investigate this,bone derived mesenclhymal stem cells(B-MSCs),bone marrow derived mesenchymal stem cells(BM-MSCs)and bone-bone marroxw derived mesenchymal stem cells(B-BM-MSCs)were extracted from New Zealand rabbits(n=5).Comparison in terms of the expression of stem cell surface marker molecules,proliferation ability and osteogenic differentiation ability among the 3 types of MSCs w ere made,and the optimal seed cells to construct TEB were selected.The selected optimal seed cells were labeled with green fluorescent protein and then co-cultured v.with the deproteinized and defatted cancellous bone scaffolds to obtain TEB.TEB was divided into two groups(n=5),one group was subjected to intermittent axial compressive stress stimulation,and the other group was commonly cultured.After 2 weeks,comparison in terms of survival rate,adhesion ability,proliferation ability,osteogenic differentiation ability,as well as expression and paracrine level of TGF-(31,BMP-2 and IGF-1 were made through living fluorescence limaging system and scanning electronic microscope,cell adhesion test.CCK-8 cell proliferation experiment,osteogenic differentiation dyeing assay.western blot and enzyme-linked immunosorbent assay.Results:Mature BM-MSCs,B-MSCs and B-BM-MSCs shared the same cell morphology and stem cell surface marker molecules.The proliferation ability of B-BM-MSCs and BM-MSCs was better than that of B-MSCs(p<0.0 1).The osteogenic differentiation ability of B-BNM-MSCs and B-MSCs was better than that of BM-MSCs(p<0.05).hence B-BM-MSCs were selected as the optimal seed cells for constructing TEB.The fluorescence imaging system and scanning electronic microscopy showed that the growth of seed cells in stress-stimulation group was better than that in non-stress stimulation group.The fluorescence intensity of the two groups was(2.15±0.21)×108,(1.56±0.28)×108,P<0.05.The number of cells in the two groups/the mirror field per 200 times was 62.33±7.99,43.83±7.10.P<0.05.Cell adhesion experiments showed that the adhesion ability of seed cells in the stress-stimulated group was stronger than that in the non-stress-stimulated group(p<0.001).CCK-8 assay showed that the proliferation ability of seed cells in stress-stimulated group was better than that in non-stress-stimulated group at the 48th to 96th hours.and the difference was statistically significant(p<0.05).Osteogenic induction staining showed that the osteogenic differentiation ability of seed cells in stress-stimulation group was stronger than that in non-stress stimulation group.The positive rate of dyeing of ALP staining in the two groups was(25.06±2.25)%.(14.32±4.55)%,p<0.05.The positive rate of dyeing of Ca nodule staining was(38.78±3.36)%,(30.49±2.33)%.p<0.05.The cytokine assay showed that the expression of TGF?1 and BMP-2 in the seed cells and the amount of TGF-?1.BMP-2 and IGF-1 secreted by the seed cells to the surrounding medium in the stress-stimulated group was higher than those in the non-stress-stimulated group(p<0.05).Conclusion:B-BM-MSCs have good proliferation and osteogenic differentiation ability,and they can be used as seed cells that are better than B-MSCs and BM-MSCs for TEB construction.Intermittent axial compressive stress stimulation of 10N,1Hz and 4h/day can effectively enhance TEB seed cells in terms of adhesion,proliferation and osteogenic differentiation ability.In this process,the expression of TGF-?1 and BMP-2 in TEB seed cells as well as the amount of the secretion of TGF-?1,BMP-2 and IGF-1 by seed cells to the surrounding medium have significantly improved.This provides a new theoretical basis for the clinical combination of intermittent axial compression stress stimulation and TEB transplantation in the treatment of large segmental bone defects.