| Objectives:To evaluate porous silk fibroin scaffold combined with Adipose-derived mesenchymal stem cells (ADMSCs) repairing rabbit urethral defect.Methods:1. Experiment of ADMSCs isolation and culture and purification:The adipose tissue was obtained from epididymis region of rabbit and was digested with 0.15 % I-collagenase to culture ADMSCs. The growth curve of the 3rd passage cells was obtained by MTT and CD44 and Vimentin were observed by immunofluorescent staining.2. Experiment of porous silk fibroin scaffold combined with ADMSCs reparing rabbit urethral defect.39 New Zealand rabbits(3.0-4.5g, male) were randomly assigned into 3 groups:(1)control group(n=13): a segmental urethral defect was created by excising the posterior wall of urethra 2.5×1.0 cm~2 in size. (2) SF group( n=13): the urethral defect was repaired with the porous silk fibroin scaffold in inlay fashion. (3) SSF group(n=13): the porous silk fibroin scaffold combined with 1×106 ADMSCs was used to repair the urethral defect in inlay fashion. In all three groups, a urethral stenting catheter was placed for 3 weeks after operation. The animals in each group were sacrificed for pathological studies at 2w(n=2), 4w(n=9), 6w(n=2) after operation. Tissue sections were stained with hematoxylin-eosin for histological examination. The distriburion of the macrophages, BrdU positive ADMSCs, FⅧ-RAg positive cells ,α-smooth muscle actin positive cells, keratin positive cells in urethra were identified by immunohistochemistry. Perioperative urethrography was also performed to identify postoperative complications.Results:1. Experiment of ADMSCs isolation and culture and purification:After 2-hour of primary ADMSCs inoculation, cells adhered to wall. The cells changed shape into spindle alike cells after 3 days. After 5-7 days ADMSCs reached the aim of the passage. For the third passage of ADMSCs, MTT curve showed the lag phase in 1-2 days, the proliferation phase was on the 8th day, and the platform phase on 11th day. ADMSCs were positive for CD44 and Vimentin.2. Experiment of porous silk fibroin scaffold combined with ADMSCs reparing rabbit urethral defect:2.1 Urethrography examination: Preoperative urethrograms showed normal urethras in all animals. Before sacrifice, the postoperative complications of urethral stricture and fistula were demonstrated by urethrograms. The postoperative complication rate was 76.92% (10/13) in control group, significantly higher than the complication rate of 23.07% (3/13) in SF group (p<0.05) and 15.38% (2/13) in SSF group (p<0.05). There was no significant difference in the complication rate between SSF group and SF group (p>0.05).2.2 morphology examination:Macroscopically, mucous membrane of urethra was absent in control group and hyperemia and edema was observed in the urethra defect area at 2w after operation. At 4w and 6w the urethra consisted of scar formation and segmental urethral stricture. In SF group and SSF group porous silk fibroin scaffold was exposed in urethra lumens at 2w after operation. At 4w and 6w silk fibroin scaffold was covered by mucous membrane of urethra, but part of silk material fell off.Histological examination:No urothelium was formed in control group and a large amount of lymphocyte infiltration was observed in urethra defect area at 2w after operation. A large amount of fibrous tissue was formed underneath the segmental defect of urethral epithelium at 4w. Lymphocyte infiltration was a constant finding up to the 6th postoperative week. The lining of the urethra epithelium was irregular. There were a trifle of blood vessels and scattered smooth muscle fiber underneath the urethral epithelium.In SF group silk fibroin was not degraded and less lymphocytes were infiltrated from 2w to 6w. At 2w after operation, no urethral epithelium was formed in scaffold and less blood vessels and collagen grew under the scaffold. Until 4w urethral epithelium formed multilayer. And blood vessels and smooth muscle fiber and fibrous tissue grew along the large part of the porous silk fibroin scaffold. 3-4 layers of epithelial cells were formed at 6w and blood vessels and smooth muscle fiber and fibrous tissue grew along scaffold.In SSF group no urethral epithelium was formed in scaffold at 2w after operation, but much more blood vessels and collagen grow under scaffold than in SF group. The scaffold was not degraded and less lymphocytes infiltrated at 2w after operation. At 4w urethral epithelium formed 3-4 layers in irregular distribution. Larger blood vessels and smooth muscle fiber and fibrous tissue grew along porous silk fibroin scaffold. And scaffold was not degraded and less lymphocytes infiltrated. 6-7 layers of epithelial cells were formed that arranged regularly and larger amount of urethral tissue grew along scaffold at 6w. The scaffold was resolving into much more small pieces and less lymphocytes infiltration was found.2.3 Distribution of macrophages and the BrdU and the FⅧ-RAg and theα-SMA and the keratin positive cells in urethra:2w, 4w, 6w after operation, macrophages in SSF group and SF group were positive in the gap and edge of the scaffold. Macrophages in the SSF group was 11.66±1.58/HP, much less than SF group (13.88±2.08/HP) at 4w after operation (P < 0.05).2w, 4w, 6w after operation, BrdU positive cell were located diffusely in the silk fibroin and more in the the urethral epithelium conjoin.The FⅧ-RAg positive cells were seen in urethral defect area in SSF group, SF group and control group as well at 2w, 4w, 6w after operation. At the 4th postoperative week, the FⅧ-RAg positive cells were 23.44±2.40/HP,20.77±2.38/HP in SSF group and SF group respectively, significantly higher than in control group (15.11±1.61) (P < 0.01). There were also significant difference in the FⅧ-RAg positive cells between SSF group and SF group at 4w after operation (P < 0.05).2w, 4w, 6w after operation,α-SMA positive cells were observed in the urethral defect area in SSF, SF group and control group. At 4 weeks of operation theα-SMA positive cells were 33.00±3.27/HP,29.00±3.20/HP in SSF group and SF group respectively, significantly higher than in control group(16.11±1.53/HP )(P < 0.01). There were also significant difference in theα-SMA positive cells between SSF group and SF group at 4w after operation (P < 0.05).In the SSF group and SF group keratin positive cells was observed and displayed in stratified columnar epithelial structure similar to normal urethra. In contrast keratin positive cells were poor in the control group and lack of stratified columnar epithelial structure.Conclusion:ADMSCs was easy to harvest,isolate and multiply in vitro, and the porous silk fibroin scaffold combined with ADMSCs is feasible for urethral tissue engineering.
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