应用缓释TGF-β3的胶原/聚乳酸己内酯纳米电纺膜构建组织工程气管补片的实验研究

The congenital tracheal stenosis has a higher incidence in infants with congenital heart diseases. It is difficult to restore the long segment tracheal stenosis because of deficiency of suitable cartilage graft by traditional surgery. Recently, tissue-engineered tracheal patch (TETP) may be a promising approach to restore long segment tracheal stenosis because of its histocompatibility and growth potential. We have previously demonstrated that the electrospun collagen/poly(L-lactic acid-co-ε-caprolactone) (collagen/PLCL) nanofibrous membranes seeded with chondrocytes by a sandwich model could facilitate high quality of well-distributed neocartilage formation without central necrosis. However, the source of chondrocytes is limited. The selection of mesenchymal stem cells (MSCs) for engineering cartilage needs inducing factors in the culture environment, such as transforming growth factor-β3 (TGF-β3). However, there were some popular problems, such as long time for culture in vitro, inducing factors easily inactivated, low induction efficiency, poor quality for the formation of newcartilage with central necrosis. Thus, we conceived that the inducing factor TGF-β3 could be loaded into the collagen/PLCL membranes for TETP by coaxial electrospinning, which would make TGF-β3 has the ability of controlled release. This new scaffold may increase the induction rate of TGF-β3, resulting the formation of a more ideal cartilage tissue. Therefore, we will choose human umbilical cord Wharton's jelly-derived mesenchymal stem cells as cell source, combining with collagen/PLCL membranes loaded with TGF-β3 for engineering tracheal patch. Eventually, we will restore the trachea defect by the TETP in the rabbit tracheal defect model. We will provide a new method of therapy for most of patients with congenital tracheal stenosis, especially for the patients simultaneously complicated with congenital heart diseases.

婴幼儿先天性气管狭窄在先心病人群中发生率较高，对于长段气管狭窄，目前治疗手段有限，预后欠佳，组织工程气管软骨补片有望成为修复长段气管狭窄的有效手段。我们前期利用胶原/聚乳酸己内酯纳米电纺膜复合软骨细胞成功构建出良好软骨组织，但软骨细胞来源有限，应用间充质干细胞构建软骨需要体外添加诱导分化因子，如转化生长因子（TGF）-β3，存在的普遍问题是体外培养时间过长，生长因子易失活，使诱导效率低，形成的软骨不均质。由此，我们提出利用同轴静电纺技术将TGF-β3载入该材料中，使其具备控释功能的设想，有望缩短体外培养时间，提高诱导分化效率，得到更为均质稳定的组织。故本课题拟采用脐带间充质干细胞为种子细胞，复合缓释TGF-β3的胶原/聚乳酸己内酯电纺膜，探讨体内外构建软骨的可行性，并最终构建组织工程气管补片修复兔气管缺损，以期为广大气管狭窄患者提供新的治疗思路和手段。

婴幼儿气管狭窄的治疗仍然是临床难题，患儿病死率高，其主要原因是缺乏理想的补片材料，难以实施狭窄矫治术，基于干细胞的组织工程技术为解决这一临床难题提供了新的希望，气管补片包括两大关键因素：支架材料和种子细胞。间充质干细胞来源相对广泛，体外扩增能力强，且具有多向分化潜能，因此常被用作组织工程的种子细胞。静电纺制备的纳米纤维在形态和尺寸上能仿生天然细胞外基质，利于细胞的粘附、迁移、生长及分化。现有的组织工程气管补片构建方案通常先对种子细胞进行体外成软骨诱导培养，之后将其种植到支架材料上，进而体内成熟后应用。但这种构建方式有明显的缺点：体外诱导时间过长；生长因子易失活，使诱导效率低；生长因子难以渗透到组织深层，形成的软骨不均质。因此，本项目为缩短体外构建时间的同时提高软骨分化诱导效率，利用同轴静电纺丝法将TGF-β3载入电纺膜中以制备具有控释功能的补片材料，保证生长因子缓慢释放，以提供稳定而高效诱导BMSCs成软骨的内环境，从而达到稳定且高效的诱导效果，缩短体外培养时间。.本项目主要研究内容分为：制备负载TGF-β3的“壳-芯”结构collagen/PLCL电纺膜并评价相关特性以确定最佳成分比例，探究该电纺膜体外、体内成软骨效果，利用该材料构建组织工程气管补片并应用于修复气管缺损。经系列探究，我们确定collagen/PLCL的最适构成比为25:75，该比例的电纺膜能够稳定缓释TGF-β3，且在体内、体外均能达到良好的软骨诱导分化效果，通过“三明治”法构建C型气管补片后，能够成功用于兔气管缺损修复试验。.综上，本项目成功利用同轴电纺技术将TGF-β3载入collagen/PLCL电纺膜中，复合BMSCs成功构建组织工程气管补片并用于气管部分缺损的移植修复，缩短了补片的体外构建时间。本项目为气管狭窄患而提供新的治疗思路和手段，尤其使合并气管狭窄的先心病患儿从中受益，在一期治疗先心病的同时获得矫治气管狭窄的机会，以减少围手术期并发症及改善患儿预后，具有广阔的临床应用前景。