| Objective:To evaluate silk fibroin scafflod combined with adipose-derived mesenchymal stem cells(ADMSCs) reparing rabbit tunica albuginea defect,and observe biocompatibility and degradation of SF in vivo.Methods:1. ADMSCs isolation and culture and purification. The adipose tissue was obtained near epididymis region of rabbit and was digested with 0.15% I-collagenase to culture ADMSCs.2. Silk fibroin scaffold combined with ADMSCs reparing rabbit tunica albuginea defect.62 New Zealand rabbits(2.5-3.0kg, male) were randomly divided into five groups : (1) DC group (defect control, n=14):a segmental tunica albuginea defect was created by excising the dorsal tunica albuginea 8×4mm in size. (2) SF group (silk fibroin treatment, n=14):the tunica albuginea defect was repaired with the silk fibroin scaffold 6×10mm in size in running suture. (3) SSF group (cells seeded silk fibroin treatment, n=14):the silk fibroin scaffold combined with 1×106 ADMSCs was used to repair the tunica albuginea defect 6×10mm in size in running suture. (4) TV group (tunica vaginalis treatment, n=14): a 6×10mm tunica vaginalis was harvested from the same rabbit ,then the autologous free graft of tunica vaginalis was used to repair the tunica albuginea defect in running suture. (5) NC group (normal control, n=6): unoperated healthy rabbits were used as normal controls. Penile straightness were confirmed by an intracavernosal injection with saline before and after operation. In every treatment groups, the animals were sacrificed for pathological studies at 2nd week (n=2),6th week (n=6),12th week (n=6) after operation. For histological examination.tissue sections were stained with hematoxylin-eosin, Sirius red for observing collagen fibers, and Hart's technique for observing elastic fibers. The macrophage infiltration was evaluated with immunofluorescence method.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 in 3 days.After 3 passages, the cells purity reached 95% or more.2. Experiment of silk fibroin scafflod combined with ADMSCs reparing rabbit tunica albuginea defect:2.1 The results of artificial erection examination: On preoperative artificial erection examination pinile curvature was not found in all rabbits. There were increased rates of penile curvature in DC group, while none was found in TV group at the 6th and 12th week after operation. At the 12th week, one case of penile curvature was found in SF group and SSF group respectively..2.2 Histological examination:2.2.1 At the 2nd week time point, large amounts of blood vessels and moderate amounts of erythrocytes were observed in tunica albuginea wound area, while there were large amounts of lymphocytes and fibroblasts infiltrating the wound area in DC group. At the 6th week, the amount of blood vessel , erythrocyte and fibroblasts decreased, while the amount of new collagen fiber formation increased remarkably in a chaotic pattern. At the 12th week, blood vessels and erythrocyte could hardly be seen, new collagen fibers increased remarkably but distributed in a chaotic pattern.2.2.2 In SF group at the 2nd week time point silk fibroin degraded into several large pieces, and small amounts of blood vessels were obserbed in tunica albuginea wound area. Large amounts of fibroblasts were found infiltrating the wound area,and moderate amounts of blood vessels were distributed in the area. At the 6th week time point, silk fibroin degraded into many long strips, fibroblasts diminished notably, and collagen fibers grew along the silk scaffold. At the 12th w, most of silk fibers degraded completely, and only a small amount of residue silk frbroin remain degraded into small short strips. A growing number of new collagen fibers was observed repairing the wound area and remolding in an ordered pattern.2.2.3 In SSF group the degradation of silk fibroin was analogous with SF group from the 2nd week to the 12th week. At the 2nd week silk fibroin degraded into several large pieces, moderate amounts of blood vessels were found in the wound area and large amounts of fibroblasts infiltrated in the wound area. At the 6th week, collagen fibers grew along the silk scaffold , and small amounts of blood vessels were found. a few of fibroblasts infiltrated in the wound area. At the 12th week, a growing number of new collagen fibers was observed repairing the wound area mostly and distributing in an ordered pattern. small amounts of bood vessels ditributed scatteredly.2.2.4 In TV group at the 2nd week time point, the profile of tunica vaginalis still existed, small amounts of blood vessels were observed in the wound area and large amounts of fibroblasts infiltrated in the wound area. At the 6th week, the profile of tunica vaginalis were hardly to be seen, collagen fibers grew into the tunica vaginalis. small amounts of blood vessels were found and small amounts of fibroblasts infiltrated in the area. At the 12th week, a growing number of new collagen fibers was found reparing the tunica vaginalis and distributing in an ordered pattern. a small amount of fibroblasts were found.2.3 Evaluation of macrophages in operated area:The values for the DC, SF, SSF, and TV group at the 6th week time point were respectively 6.8±1.63/HP, 4.1±0.87/HP,3.8±0.78/HP, and 3.7±0.94/HP. SF group, SSF group and TV group were significantly fewer than the DC group(p<0.01). The SF group ,SSF group and TV group were not significantly difference(p>0.05).2.4 Evaluation of collagen fibers I and III by Serius red staining and polarizing microscope: At the 12th week time point, collagen fibers I took up the main part of the collagen fibers at SF ,SSF ,and TV group. There were no significant differences in the number of collagen fibers of either type among the SF,SSF and TV group.While the number of collagen fibers I in DC group was significantly fewer than the other three groups at the 12th week time point (p<0.01) and the collagen fibers III at DC group was significantly more than the other three groups(p<0.01).2.5 Evaluation of elastic fibers by Hart,s technique There were significant differences in the number of elastic fibers in the same group between the 6th week and 12th week time point(p<0.05). At the 12th week time point , compared with the SSF and TV group ,there were significantly fewer elastic fibers in the SF group(p<0.05). The number of elastic fibers in the DC group were significantly fewer than the other three groups(p<0.01).Conclusion:Silk fibroin is an ideal scaffold for tissue engineering ,and silk fibroin combined with ADMSCs can achieve similar effect with autologous graft at repairing tunica albuginea defect. |
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