天津大学刘文广：3D打印仿生上皮/基质双层水凝胶支架用于角膜再生|Bioactive Materials

角膜是眼前部具有多层结构的透明组织，本文通过数字光固化3D打印技术（DLP）制备了聚乙二醇二丙烯酸酯(PEGDA)/甲基丙烯酰化明胶(GelMA)水凝胶，其具有足够力学性能、高透光率、高形状保真度、适宜的溶胀度和降解速率以及优异的细胞相容性，兔前板层角膜移植手术表明所构建的负载细胞的仿生双层水凝胶支架能够促进上皮愈合和基质再生。

01

研究内容简介

角膜主要由五层结构组成，从上到下分别为上皮层、前弹力层、基质层、后弹力层和内皮层，其对于折射、穿透光线具有重要作用，但由于烧伤、灼伤、外力损伤等原因，角膜疾病如今已经是致盲的主要原因之一。虽然现有的人工角膜（如 AlphaCor和Boston）具有高度的透明性，可以在短期内作为角膜替代物，但是由于其通常为不可降解以及生物惰性的材料，无法与自身组织相融合，因此舒适度差，美观性低，长期穿戴可能会对周围组织造成损伤。近些年来，组织工程以及3D打印技术迅猛发展，为人工角膜的个性化制备提供了一种强有力的方法，同时3D打印可以制备具有不同结构、不同尺寸参数、负载不同细胞的人工角膜，具有很大的发展前景。受此启发，本文以PEGDA/GelMA为墨水，使用数字光固化打印技术制备水凝胶，并对其理化性质、细胞相容性以及在体内对角膜愈合的影响进行表征（图1）。

Figure 1. (A) Components of ink and schematic illustration of network formation in PEGDA-GelMA hydrogel. (B) Description of the 3D bioprinting process and its application in a rabbit keratectomy model by using a bi-layer dome-shaped corneal scaffold consisting of rCECs-laden epithelia layer and rASCs-laden orthogonally aligned fibrous stroma layer.

本文通过改变PEGDA的固含量制备了三种比例的水凝胶。在拉伸和压缩测试中（图2），PEGDA-GelMA-20-5水凝胶具有82.2 kPa的拉伸强度，77.2 kPa的拉伸模量和100.7 kPa的压缩模量，能在保证手术操作的同时保留负载细胞的能力，压缩模量接近于天然角膜的压缩模量，有利于术后抵抗眼压。此外，PEGDA的加入还提高了GelMA水凝胶的韧性和打印性能。

Figure 2. Mechanical properties of GelMA and PEGDA-GelMA hydrogels. (A1) Representative tensile stress-strain curves and their corresponding (A2) tensile stress, (A3) elongation at break, and (A4) tensile modulus. (B1) Representative compressive stress-strain curves and their corresponding (B2) stress-strain curves before 50% strain, (B3) compressive stress, and (B4) compressive modulus. (C) Compressive process of GelMA-5 and PEGDA-GelMA-20-5 hydrogel by using a mechanical test machine. (D) Printed GelMA-5 and PEGDA-GelMA-20-5 hydrogel discs.

  如图3所示，其他理化性质测试表明此打印水凝胶能够在1天内达到溶胀平衡，一个月的时间内在胶原酶溶液中缓慢降解，并具有适宜的溶胀度和降解速率。透光率测试表明在600 nm处，水凝胶均能保持80%以上的透光率，并大于天然兔角膜的透光率。流变测试结果显示PEGDA-GelMA-20-5水凝胶具有3.1 kPa的储能模量，与天然角膜相近。

Figure 3. Characterizations of PEGDA-GelMA hydrogels. (A) The swelling degree of the hydrogels after 14 days incubation in PBS at 37 ℃. (B) Degradation curve of the hydrogels in the presence of collagenase (50 U/mL) in PBS at 37 ℃. (C) Optical transmittance of hydrogels and natural cornea between 400-800 nm. (D) Photographs showing transparency of native cornea, printed cornea and printed disc. (E) Frequency sweep curves of hydrogels at a frequency range from 0.1 to 10 Hz. (F) Averaged storage modulus (G') and loss modulus (G'') calculated from (E).

使用兔角膜上皮细胞测试水凝胶的细胞相容性（图4），结果显示水凝胶能够支持细胞的迁移、黏附和增殖。将凝胶前驱液与细胞进行混合并作为墨水进行打印，7天后测试细胞存活率，仍能达到90%，并且凝胶的透光率也能达到88.9%。

Figure 4. In vitro cytocompatibility of PEGDA-GelMA-20-5 hydrogel. (A) Representative live/dead images of rCECs in 2D scratch assay. (B) Quantification of relative cell density in the scratched area in the hydrogel and control samples. (C) Representative live/dead images of rCECs seeded on the surface of PEGDA-GelMA-20-5 hydrogel. (D) CCK-8 results of the cell proliferation on the hydrogel. (E) Representative live/dead image of rCECs encapsulated in the hydrogel at 7 days after bioprinting. (F) Transparency change of the rCECs-loaded hydrogels between 400-800 nm at 1, 3, 5, 7 days after bioprinting. Scale bars in live/dead images: 300 μm.

为了考察墨水的可打印性，使用DLP打印机，对PEGDA-GelMA-20-5进行不同模型的打印。如图5所示，经过逐层光固化，此墨水可以成功打印出兔子、皇冠、带孔圆片，并具有足够的强度和透明性。为了模拟角膜基质层中正交的胶原纤维结构，对水凝胶进行正交结构的打印，结果显示水凝胶的打印精度较高，显微镜下的正交部分清晰可见，细胞分布均匀且具有较高活性。通过改变光吸收剂的浓度和曝光时间，本文还打印出了负载兔角膜上皮细胞的角膜结构，其具有良好的光滑度和适宜的厚度、曲率，与天然角膜相似。

Figure 5. Printability evaluation of PEGDA-GelMA-20-5 hydrogel. (A1) 3D model and (A2) digital photo of printed rabbit. (B1) Digital photo and (B2) micrograph of printed perforated disc. (C1) 3D model and (C2) digital photo of printed crown. (C3) Top view and (C4) side view of crown after immersion in PBS. (D1) 3D model, (D2) digital photo and (D3) micrograph of printed orthogonally aligned fibrous structure. (D4) Evaluation of live/dead rCECs viability in orthogonally aligned fibrous object after culturing for 2 days. (E) Printed hydrogel after immersion in cell culture medium for 2 days. Printed hydrogel disc (F1) with and (F2) without photoabsorber. White circle denoted the predetermined printing area. (G) Printed corneal scaffold. Scale bars: 300 μm.

为了考察PEGDA-GelMA水凝胶在体内对角膜愈合的影响，本文打印出负载兔角膜上皮细胞的上皮层以及负载兔脂肪间充质干细胞的正交纤维基质层，并进行兔前板层角膜移植术将水凝胶移植入角膜损伤处。1个月的裂隙灯检测（图6）以及组织学染色（图7）表明，水凝胶能够加速上皮愈合和基质再生，正交纤维结构的存在和干细胞的双重作用为角膜基质细胞提供了最有效的再生环境。

Figure 6. In vivo evaluation of the 3D printed bi-layer corneal scaffold in a rabbit model of ALK. (A) Surgical procedures of keratoplasty, including removing a certain part of corneal epithelium-stroma by a trephine and spatula, implantation of the hydrogel, and overlying suture. (B) Representative slit lamp and cobalt blue photographs after in vivo application of hydrogel to rabbit cornea at different time points. Progressive reduction in the size of corneal epithelial defect (green area in the central cornea) implicated epithelial migration over hydrogel and defect.

Figure 7. Histological and gene expression analysis after application of the 3D printed bi-layer corneal scaffold in a rabbit model of ALK. (A1) H&E staining and immunostaining with (A2) Col I and (A3) CK3 antibodies (green) of the corneas at 1 day post-surgery. (B1) H&E staining and immunostaining with (B2) Col I, (B3) CK3, (B4) LUM, and (B5) SMA antibodies (green) at 28 days post-surgery. The red arrows and rectangles denoted the uncompleted epithelium and stroma region, respectively. In all images, nuclei were co-stained with DAPI (blue). The scale bars were 200 μm. Thickness of (C1) total, (C2) epithelium, and (C3) stroma layer of cornea at 28 days post-surgery obtained from H&E images. The relative gene expressions of (D1) KERA, (D2) ALDH, (D3) AQP1 in corneas, which were quantified by PCR at 28 days post-surgery. Values reflected fold change in mRNA expression over normal cornea.

综上所述，此3D打印仿生上皮/基质双层水凝胶具有与角膜相近的理化性质，优异的细胞相容性和体内生物相容性，能够加速角膜愈合，减少瘢痕的产生，具备应用于角膜再生与修复中的潜力。  

02

论文第一/通讯作者简介

第一作者：何彬彬

硕士，天津大学材料学院，研究方向为生物医用水凝胶。

通讯作者：韩泉洪

博士，天津市眼科医院，主任医师，主要从事玻璃体视网膜治疗。

通讯作者：刘文广

博士，天津大学材料学院，讲席教授。主要从事生物医用高分子水凝胶研究，创建了基于侧链氨基酸多重氢键交联的超分子聚合物水凝胶，提出了自增稠/自增强3D打印超分子水凝胶墨水的设计理念，发展了水下和湿态环境应用黏合剂、止血剂及心肌补片。发表学术论文200余篇，2018-2020 Elsevier中国高被引学者，授权发明专利40余项。

03

资助信息

上述研究工作得到了国家自然科学基金（51733006）和国家重点研发计划基金（2018YFA0703100）支持。

04

原文信息

Binbin He, Jie Wang, Mengtian Xie, Miaoyi Xu, Yahan Zhang, Huijie Hao, Xiaoli Xing, William Lu, Quanhong Han*, Wenguang Liu*. 

3D Printed Biomimetic Epithelium/Stroma Bilayer Hydrogel Implant for Corneal Regeneration. 

Bioactive Materials, 6, (2022) 3085–3096.

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Bioactive Materials是一本高质量英文期刊，目前已经被SCIE、PubMed Central、Scopus、Embase收录。同时本刊还入选了2019年中国科技期刊卓越行动计划--“高起点新刊”项目。

2022年Bioactive Materials 获得影响因子16.874 ，在Materials Science，Biomaterials领域排名第一。

位于《2021年中国科学院文献情报中心期刊分区表》1区，TOP期刊。

CiteScore 2021: 14.3。