Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold.
Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold.
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DOI：
10.1007/s10792-022-02418-y
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发表时间：
2023-01
影响因子：
1.6
通讯作者：
Perez, Victor L.
中科院分区：
文献类型：
--
作者：
Xie, Jiajun;Gao, Qi;del Prado, Zelmira Nunez;Venkateswaran, Nandini;Mousa, Hazem M.;Salero, Enrique;Ye, Juan;De Juan-Pardo, Elena M.;Sabater, Alfonso L.;Perez, Victor L.
利用使用聚-（ε-己内酯）（PCL）的熔融电写入（MEW）技术结合2步共培养策略开发结膜双层合成构建体。 使用内部制造的MEW打印机制造使用PCL的熔融电写入支架。从供体组织中分离人结膜基质细胞（CjSC）和上皮细胞（CjEC）。通过首先接种CjSC并培养4周以建立基质层，然后接种CjEC并再共培养2周来进行2步共培养方法。通过形态学和免疫荧光和qPCR上的标志物表达对培养的细胞进行表征。使用活力测定评估所产生的构建体的细胞增殖。使用扫描电子显微镜（SEM）、共聚焦显微镜和免疫荧光成像评估双层形态。使用qPCR评估细胞外基质组分和TGF-β的表达。CjSC呈梭形，波形蛋白阳性，而CjECs呈多边形，CK 13阳性。CjSC在4周培养标记时显示出一致的增殖和与支架的最佳粘附。由CjSC组成的基质层和CjEC组成的上皮层组成的2层结构在共聚焦显微镜、SEM和免疫荧光上得到赞赏。CjSC分泌胶原蛋白（I、V、VI型），但与天然组织的量不同，而TGF-β的产生是相当的。3D打印的熔融电写入PCL支架与支架的2步共培养条件配对允许结膜的双层基质和上皮重建的第一近似，这可以潜在地改善眼表面重建中的治疗武器库。在线版本包含补充材料，可通过10.1007/s10792-022-02418-y获得。
To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction. The online version contains supplementary material available at 10.1007/s10792-022-02418-y.
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