口腔黏膜复合血管化平滑肌构建轴型膀胱壁瓣的研究

The key challenge in tissue engineering is the establishment of an efficient vascularization for tissue constructs guaranteeing long-term survival and function. The lack of vascularization may lead to the shrinkage of the tissue after implantation. To fabricate vascularized composite tissue that containing smooth muscle and epithelial layers with their own axial vessels may provide an effective method for bladder reconstruction and may pave the way for the vascularization in tissue engineering. In our previous studies, we have found that the tissue expander capsule can be used as a vascular bed for smooth muscle tissue cultivation. The vascularized capsule can also be used for autologous oral mucosa cultivation, and the size of oral mucosa can be increased with the expansion of the expander. Based on these preliminary experiments, we plan to implant silicone tissue expanders near the superficial circumflex iliac (SCI) artery and vein to induce axial-pattern vascularized capsule formation. Then the cell sheets composed of SMCs, EPCs and their extracellular matrix were transplanted on the capsule. After vascularized smooth muscle tissue formation, the expanded oral mucosa cultivated on the other side of the vascularized capsule were seperated and transplanted onto the surface of the vascularized smooth muscle tissue. With the interaction of the smooth muscle and the oral mucosa, an axial-pattern vascularized composite tissue containing smooth muscle and epithelial layers will be generated. After that, with the SCI vessels as the vascular pedicle, this axial-pattern composite tissue containing smooth muscle and epithelial layers will be raised and transplanted to bladder wall defects. By this method, we will find out the feasibility of using the tissue expander capsule as a vascular bed to cultivate vascularized composite tissue with its own axial vessels.

体外构建组织植入体内后无法及时获得血供而萎缩是目前组织工程构建面临的主要问题。构建有轴心血管支持并含血管化良好的上皮和平滑肌的复合组织瓣进行组织工程膀胱重建是解决该问题的重要途径。前期研究发现扩张器包膜可作为血管床培育含轴心血管的平滑肌组织瓣，且可作为在体血管床培育口腔黏膜组织片并使其面积随扩张器刺激而增大。在此基础上，本研究拟在兔双侧旋髂浅动静脉旁埋置扩张器形成含轴心血管的包膜，将平滑肌-内皮祖细胞膜片置于一侧该包膜血管床上充分血管化，再将另一侧培育的随扩张器扩张而增大面积的口腔黏膜组织片移植至该血管化肌层上方，在体构建轴型的含上皮和平滑肌的复合膀胱组织瓣，最后以旋髂浅动静脉为血管蒂将构建的该岛状复合组织瓣转移至膀胱壁缺损区进行膀胱重建，探讨以扩张器包膜为在体血管床培育轴型含上皮和平滑肌的复合膀胱组织瓣的可行性，以解决组织工程血管系统构建的难题。

在结构上和功能上类似正常膀胱组织的组织工程化膀胱移植物是临床膀胱重建的一种治疗选择。构建血管化复合组织是当前膀胱组织工程研究领域的主要挑战。本研究拟探讨采用口腔黏膜结合血管化平滑肌构建带血管蒂的血管化膀胱移植物的可行性。我们将6层平滑肌细胞膜片分两次（每次6层，间隔2天）移植到包膜血管床上培育7天形成了血管化平滑肌组织。将口腔黏膜移植到血管化平滑肌上培育7天，结果显示口腔黏膜存活良好并成功构建了带血管蒂的血管化膀胱移植物，该膀胱移植物具有类似正常膀胱组织的全层结构、适当的组织厚度、丰富的血管化水平以及有效的黏膜屏障功能。随后，构建膀胱全层缺损模型，并分别以带血管蒂的包膜组织（带蒂包膜组）、游离的血管化膀胱移植物（游离移植物组）以及带血管蒂的血管化膀胱移植物（带蒂移植物组）修复膀胱缺损。天狼星红染色和免疫组化染色结果显示带蒂移植物组重建的膀胱组织纤维含量最低、平滑肌含量最高，血管化水平也较高。同时，也只有在带蒂移植物组中才能在重建部位观察到连续的上皮层。此外，膀胱形态和功能学检查表明带蒂移植物组重建的膀胱组织具有更好的顺应性及良好的黏膜延展性。综上所述，研究结果表明口腔黏膜可以很好地同血管化平滑肌相结合并构建出带血管蒂的、充分血管化的且具有适当结构的组织工程化膀胱移植物。该血管化膀胱移植物可以促进膀胱结构重塑以及功能恢复，有望成为临床膀胱重建的替代选择。