Objective: To observe the changes in the number and function of bone marrow-derived endothelial progenitor cells (EPCs) after bone-marrow stimulation, and to investigate the possible mechanism of improving ischemic limb disease after bone-marrow stimulation through autologoue bone-marrow stem cell implantation.
Methods: Twelve male Lewis rats, weighing 200-250 g, were classified into the bone marrow stimulation group (n = 6) and the control group (n = 6). In the stimulation group, the bone marrow of each rat was stimulated by injection of recombinant human granulocyte-macrophage colony-stimulatory factor. Mononuclear cells were harvested from bone marrow and cultured in EBM-2 medium. After 7-day culture, EPCs were stained by 1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein/fluorescein isothiocyanate-ulex europaeus agglutinin 1, and the double positive cells were counted by the fluorescent microscope. The adhesive ability of EPCs was determined by counting the number of re-cultured EPCs. The unilateral ischemia hindlimb model was made with 12 Lewis rats. Three days later, EPCs were transplanted into the ischemic tissues. According to different sources of EPCs, the 12 rats were divided into 2 groups: the stimulation group (n=6) and the control group (n = 6). At 3 weeks after EPCs transplantation, the quantity of the collateral vascular was observed by digital subtraction angiography (DSA).
Results: After 7-day culture, the number of EPCs in the stimulation and control groups was (145.2 +/- 37.0)/HP and (95.2 +/- 39.4)/HP, respectively, and there was significant difference between the two groups (P < 0.05). Meanwhile, the number of adhesive EPCs in the stimulation and control groups was (21.8 +/- 4.3)/HP and (15.0 +/- 5.2)/HP, respectively, and the difference between the two groups was significant (P < 0.05). At 3 weeks after the EPCs implantation, the number of the collateral vascular was significantly larger in the stimulation group (4.2 +/- 1.2) compared with the control group (2.7 +/- 0.8), (P < 0.05).
Conclusion: Bone marrow stimulation increases the number of EPCs and improves the function concurrently, which may be the reason why autologoue bone-marrow stem cell implantation improves the curative effect of ischemic limb diseases after bone-marrow stimulation.