Differentiation Monitoring Of BMSCs And EPCs During Canine Urethral Defect Remediation Process

Urethral defects caused by congenital malformations,trauma,inflammation and cancer have been a subject in the field of urology research.There is currently no ideal method for repairing long tubular urethral defects.In the field of regenerative medicine,classical tissue engineering materials such as small intestine submucosa,bladder acellular matrix,and tissue engineering oral mucosa also have many problems such as limited donor tissue,immune reactions,fibrosis and calcification,and matrix contraction.More importantly,the efficacy and mechanism of action of cells transplanted into animals remains unclear.It is essential to track the fate of cells in the implanted host.Tracer and imaging methods can be used to solve some problems about the biodistribution of cells.Relevant animal models are essential to better understand the efficacy and safety of repairs.At present,there have been no reports on the monitoring of seed cell long-term migration and differentiation after repair of urethral defects.Therefore,we used a tissue-engineered amniotic membrane construct loaded with lentiviral-transfected GFP-BMSCs and RFP-EPCs to repair a 3 cm tubular urethral defect in the perineum of dogs in forms of surgical patch.20 healthy male mongrel dogs were selected and divided into 5 groups randomly,with 4 dogs in each group.After establishing the model of 3 cm tubular urethral defect at perineum,the urethral defect models were repaired with acellular amniotic matrix seeded with BMSCs,EPCs and BMSCs mixed with EPCs,respectively.Dogs treated with acellular amniotic membrane without implantation cells and defective canine as controls.The effect of urethral reconstruction was estimated by urination observation after surgery,retrograde urethrography,urethral anatomy check and histological analysis.Immunofluorescence was used to monitor the whereabouts and differentiation of both GFP-BMSCs and RFP-EPCs.The results of the study are as follows:1.Dogs in BMSCs+EPCs mixed group regained normal urination after operation,the new urethral canal was smooth and no urethral stricture was observed.The histological observation showed that the newborn epithelium was intact.In BMSCs group,a small-scale urethral stricture was found at perineum and scar was found in the newborn segment by dissection.In EPCs group,the urination time was prolonged and the urethral segment was narrow.The incomplete epithelialization was observed in the newborn segment by histological examination.Dogs in amniotic membrane repair group and autologous repair group had abnormal urination,urethra stricture,severe scarring and multiple shrinkage.Only single layer epithelium was found by histological examination.As for dogs in autologous repair group,there is almost no epithelial cell coverage in urethra.2.Monitoring of GFP-BMSCs and RFP-EPCs in tissue by immunofluorescence staining showed that the green fluorescence of BMSCs group was concentrated in the epithelial part of the newborn urethra,indicating that the transplanted BMSCs almost completely differentiated into urethral epithelial cells.The red fluorescence of EPCs group also distributed in the epithelial area,but the distribution was less,there were more blood vessels around the blood vessels,and the blood vessel contour was clearer.It was proved that most of the EPCs differentiated into vascular endothelial tissue and a few differentiated into urethral epithelial cells.In the BMSCs+EPCs mixed group,red and green fluorescence appeared in the urethral epithelium,and the green fluorescence is more.Red fluorescence appeared more around the blood vessels below the epithelium.It was proved that most of the transplanted BMSCs participate in the formation of new urethral epithelium with a small proportion of EPCs,and EPCs mainly participate in the angiogenesis and repair of blood vessels.In summary,tissue-engineered amniotic membranes loaded with BMSCs and EPCs can successfully repair 3 cm tubular urethral defects in the perineum of dogs.Differentiation monitoring demonstrated that BMSCs and EPCs are involved in the formation of new urinary tract epithelium,but based on BMSCs,EPCs mainly migrate to the vascular defect site and participate in the angiogenesis.The optimal effect of BMSCs mixed with EPCs in repairing urethral defects was demonstrated in both epithelialization and angiogenesis.