膀胱无细胞基质复合阴道平滑肌细胞修复兔阴道缺损

Bladder acellular matrix seeding with autologous cultured vaginal smooth cells repaired rabbit vaginal defect

目的探讨膀胱无细胞基质（BACM）复合阴道平滑肌细胞修复兔阴道缺损的可行性，寻求理想的阴道修复材料。方法将24只成年新西兰雌兔按随机数字表法分3组，每组8只，A组用于制备BACM和阴道无细胞基质（VACM），分别在B、c两组新西兰兔获取1cm^2阴道前壁组织，分离培养兔阴道平滑肌细胞，免疫组化染色鉴定。以1×10^7个细胞／cm。种植于BACM和VACM上，体外培养5d后形成VACM-平滑肌细胞复合物[对照组（C组）]和BACM-平滑肌细胞复合物[实验组（c组）]，用于修复兔中段阴道缺损，于移植后3、6、12周观察阴道修复情况。结果制备的BACM和VACM均为白色透明状，HE染色和扫描电镜观察为规则排列的网状胶原纤维，无细胞碎片；体外培养的阴道平滑肌细胞为梭形，呈典型的“峰和谷”样，免疫组化染色示仪-肌动蛋白阳性，种植到BACM和VACM上5h后，均可见部分细胞黏附于无细胞基质上，随时间推移，细胞逐渐增多并伸展成梭形，实验组和对照组移植后3周均可见基质表面已形成完整的上皮层，部分平滑肌细胞长入，移植后6周形成多层复合上皮，平滑肌束明显增多，移植后12周其组织结构与正常阴道组织结构相似，阴道造影证实修复部位阴道宽敞，未见明显狭窄和瘢痕形成，两组组织修复再生情况一致。结论BACM和VACM复合阴道平滑肌细胞修复阴道缺损的组织再生规律基本相同，但BACM来源广泛，具有良好的组织相容性，是一种较好的修复阴道缺损的支架材料。

Objective To evaluate the feasibility of bladder extracellular matrix （BACM） seeding with autologous cultured vaginal smooth muscle cells （VSMC） repaired rabbit vaginal defect. Methods This study included 24 female rabbits. BACM and vaginal acellular matrix （VACM） were obtained from 8 rabbits by decellularization process. The other 16 rabbits were randomly divided into experimental and con- trol groups. Vaginal tissue biopsies （ - 1 cm2） were harvested from female New Zealand rabbits. VSMC were cultured and stained with a-smooth muscle actin antibodies. Cultured VSMC ceils were seeded on BACM （ experimental group） or VACM （ control group） at a cell density of 1 x 107 cells/cm2. The cell-see- ded matrixes were cultured for 5days. In experimental group of 8 rabbits, a 2 cm segment of vagina was re- sected and replaced with BACM seeding with VSMC. Then the regenerative segment was studied with histo- logical technique by hematoxylin-eosin staining after 3, 6 and 12 weeks postoperative and vaginography was performed at 12 weeks postoperative. The 8 rabbits in control group underwent the exact same procedure as above but the vaginal defect was repaired with VACM seeding with VSMC. Results The prepared BACM and VACM were transparent,HE staining and scanning electron microscopy showed the two acellular matrix was both consisted of abundant network of fibers with the regular arrangement, without cellular debris. Pri-mary culture of VSMC was successfully established and passaged, and was uniformly spindle-shaped in the confluent state and showed a characteristic hill and valley＇formation. Immunohistochemical staining showed VSMC in culture stained positively with a-smooth muscle actin antibodies. VSMC began to adhere to the BACM 5 hour after implanting and the number of the adhered cells increased with time. Cells gradually ex- panded and showed the typical morphology of smooth muscle ceils. Three weeks after implantation in vivo, the luminal surface of matrix was completely covered by vaginal epithelial tissue, a layer of smooth muscle cells was formed in the outer surface of the matrix. Multilayered vaginal epithelial and improved develop- ment of organized muscle bundles was observed after 6 weeks. The regenerative tissue was equivalent to the normal vaginal tissue at 12 weeks postoperatively. Vaginography demonstrated the maintenance of full paten- cy and a wide vaginal caliber without fibrosis and graft rejection. There was no significant difference in all e- valuated items between experimental and control groups. Conclusions BACM has the same regenerative process as VACM in the replacement of vaginal defect. Moreover, BACM has wider source and appears to be an suitable material for vaginal replacement.