Bone marrow mononuclear cells-differentiated vascular endothelial progenitor cells for urethral defect repair in rabbits / 中国组织工程研究

ABSTRACT

BACKGROUND: How to solve the source of material substitute for repair of urethral dcfoct and improve the blood supply of new urethra has become a critical problem in the urethral repair and reconstruction.OBJECTIVE: To investigate the effects of endothelial progenitor cells (EPCs) on improving blood circulation in the new urethra following urethral defect repair.DESIGN,TIME AND SETTING: In vivo tissue engineering experiment,performed at the Laboratory of Deparanent of Surgery,First Affiliated Hospital of Zhengzhou University between January 2006 and February 2008.MATERIALS Thirteen 3-5-month-old male Japanese rabbits were included for this study.Of them,one was used for preparation of bone marrow mononuclear cells,and the remaining twelve rabbits were divided into EPC repair group (n=8) and model group (n=4).METHODS: Under the aseptic condition,bone marrow was taken from the rabbit bilateral anterior superior lilac spine.Mononuclear cells isolated by Percoll method were induced in vitro using medium supplemented with vascular endothelial growth factors (VEGFs) and bovine basic fihroblast growth factors.When covering the whole bottom of culture flask,the mononuclear cells were digested with trypsin for passage.Animal models of urethral defect were developed in the two groups.One piece of aseptic fresh acellular human amnion (1 cm2) was sutured to each defected urethra using 0/6 DG suture for forming urethra.In the EPC repair group,1010/L passage 3 rabbit bone marrow mononuclear cell suspension was injected to two anastomotic stomas of the new urethra,0.1 mL for each stoma.The subcutaneous tissue was interruptedly sutured to the formed urethra using 0/6 DG suture.In addition,0.5 mL bone marrow mononuclear cell suspension was added to the region between each anastomotic stroma and newly repaired urethra.The same procedure was performed in the model group except that bone marrow mononuclear cell suspension was replaced by cell-free medium.At weeks 4 and 12 after surgery,paraffin sections of urethral tissue were made.MAIN OUTCOME MEASURES: Identification of cellular morphology; vascular regeneration of urethral tissue after urethral defect repair.RESULTS: After surgery,rabbit bone marrow mononuclear cells adhesively grew in vitro.Four days later,these cells exhibited rapid clone-like growth.Ten days later,they had typical slabstone-like change,presenting with strip-shaped and bundle-shaped growth.The phenotype of cultured cells gradually turned from CD34+/CD 133+/CD31+ to CD34+/CD 1337CD31+.At weeks 4 and 12 after surgery,the number of regenerate,d blood capillaries in the urethral tissue was significantly greater in the EPC repair group than in the model group (t=-9.034 to 5.985,P ＜ 0.01).CONCLUSION: Rabbit bone marrow mononuclear cells-differentiated EPCs can apparently improve local blood circulation in the urethral defect repair.