Decellularized Tissue-Engineered Heart Valve Leaflets with Recellularization Potential
Decellularized Tissue-Engineered Heart Valve Leaflets with Recellularization Potential
复制标题
DOI：
10.1089/ten.tea.2012.0365
复制
发表时间：
2013-03-01
影响因子：
4.1
通讯作者：
Tranquillo, Robert T.
中科院分区：
文献类型：
--
作者：
Syedain, Zeeshan H.;Bradee, Allison R.;Tranquillo, Robert T.
组织工程心脏瓣膜(TEHV)已被认为是满足儿科患者临床需求的一种有前途的解决方案。体内研究表明，TEHV小叶在植入几个月后会收缩和反流。这归因于在TEHV培养过程中利用收缩细胞产生细胞外基质(ECM)。在这里，我们利用这样的细胞在纤维蛋白为基础的支架中培养出成熟的细胞外基质，在TEHV小叶中产生连合排列，然后使用洗涤剂去除这些细胞。此外，我们评估了潜在的非收缩细胞的再细胞化。建立了一种组织工程瓣叶模型，其力学各向异性和拉伸性能与绵羊肺瓣叶相似。用1%十二烷基硫酸钠和1%Triton洗涤剂处理后，纤维的拉伸性能没有变化。DNA定量和蛋白质印迹分析证实了细胞的去除。组织学和扫描电子显微镜成像显示ECM的组织和微结构没有明显变化。将人骨髓间充质干细胞(HMSC)接种于去细胞小叶表面，进一步检测脱细胞小叶的再细胞化能力，并在1周和3周两种培养条件下对其进行评价。选择一种培养液(M1)来维持MSC的表型，而第二种培养液(M2)用于潜在地将细胞分化为间质细胞的表型。细胞定量结果显示，M2次之，M1次之，工程小叶的细胞再分化浓度最高，脱细胞天然小叶的细胞侵袭最小。组织学检查显示M2小叶全层细胞侵袭，M1小叶部分侵袭。培养1周时，除CD90外，HMSC在两种培养液中均为MSC标记阳性，α-平滑肌肌动蛋白染色阳性，3周时未见MSC标记。这些结果表明，虽然与天然小叶相比，工程小叶具有相似的拉伸性能和胶原含量，但具有更好的再细胞化潜力。
Tissue-engineered heart valves (TEHV) have been proposed as a promising solution for the clinical needs of pediatric patients. In vivo studies have shown TEHV leaflet contraction and regurgitation after several months of implantation. This has been attributed to contractile cells utilized to produce the extracellular matrix (ECM) during TEHV culture. Here, we utilized such cells to develop a mature ECM in a fibrin-based scaffold that generates commissural alignment in TEHV leaflets and then removed these cells using detergents. Further, we evaluated recellularization with potentially noncontractile cells. A tissue-engineered leaflet model was developed with mechanical anisotropy and tensile properties comparable to an ovine pulmonary valve leaflet. No change in tensile properties occurred after decellularization using 1% sodium dodecyl sulfate and 1% Triton detergent treatment. Cell removal was verified by DNA quantitation and western blot analysis for cellular proteins. Histological and scanning electron microscope imaging showed no significant change in the ECM organization and microstructure. We further tested the recellularization potential of decellularized leaflets by seeding human mesenchymal stem cells (hMSC) on the surface of the leaflets and evaluated them at 1 and 3 weeks in two culture conditions. One medium (M1) was chosen to maintain the MSC phenotype while a second medium (M2) was used to potentially differentiate cells to an interstitial cell phenotype. Cellular quantitation showed that the engineered leaflets were recellularized to the highest concentration with M2 followed by M1, with minimum cell invasion of decellularized native leaflets. Histology showed cellular invasion throughout the thickness of the leaflets in M2 and partial invasion in M1. hMSC stained positive for MSC markers, but also for alpha-smooth muscle actin in both media at 1 week, with no presence of MSC markers at 3 weeks with the exception of CD90. These results show that engineered leaflets, while having similar tensile properties and collagen content compared to native leaflets, have better recellularization potential.