3D-Printed membrane as an alternative to amniotic membrane for ocular surface/conjunctival defect reconstruction: An in vitro & in vivo study
3D-Printed membrane as an alternative to amniotic membrane for ocular surface/conjunctival defect reconstruction: An in vitro & in vivo study
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DOI：
10.1016/j.biomaterials.2018.05.013
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发表时间：
2018-08-01
期刊：
影响因子：
14
通讯作者：
Tayebi, Lobat
中科院分区：
文献类型：
--
作者：
Dehghani, Shima;Rasoulianboroujeni, Morteza;Tayebi, Lobat
背景资料：本研究的目的是评估一种特定的3D打印膜设计的手术处理和临床适用性，该膜设计使用明胶，弹性蛋白和透明质酸钠混合物制造用于结膜重建，并将其与羊膜（AM）进行比较，羊膜通常用于此类surgery.Methods：采用3D打印技术制造基于梯度设计的膜。在印刷之前，采用流变仪来优化油墨组合物。然后在物理和机械性能方面对印刷的膜进行充分表征。分别用mu测定法和扫描电镜（SEM）观察人角膜缘上皮细胞的活力、增殖和粘附。在体内实验之前，由三名外科医生评价每种膜的手术处理。通过将明胶基膜和AM植入诱导的兔结膜缺损（n = 8）进行体内评价。从第1天至第28天记录临床观察结果，包括上皮形成、炎症严重程度、瘢痕组织形成和肉芽组织的存在。在第28天对所有摘除的眼睛进行组织学检查。除了H&E染色之外，还进一步使用包括过碘酸希夫染色、Masson三色染色和α-SMA的免疫组织化学染色的特异性染色来分别评估杯状细胞增殖、愈合的上皮下基质和瘢痕组织形成以及肌成纤维细胞的存在。在所有检查的组合物中，发现8%w/v明胶、2%w/v弹性蛋白和0.5%w/v透明质酸钠的共混物适合于印刷。印刷膜具有良好的光学特性（无色透明），与AM相比，手术操作明显更容易。在膜上培养的上皮细胞显示适当的活力和增殖，并且SEM图像显示膜表面上适当的细胞粘附。临床观察表明，膜和AM移植眼的上皮形成时间相似（约3周），但膜组从第1天到第28天的临床炎症水平显著较低（p = 0.01），这是使用打印膜优于AM的关键优势。组织学检查显示愈合的上皮细胞在细胞形态和细胞层方面具有相似的质量。然而，在明胶基膜移植组中观察到每100个细胞中杯状细胞的密度是对照组的两倍。仅在3只膜中观察到降解的植入物残留，但在7只AM移植眼中观察到降解的植入物残留。与膜相比，含有AM的切片中的炎症和肉芽肿反应显著更高（分别为p < 0.01和p = 0.01）。AM组的α-SMA染色更明显，但与明胶基膜无显著差异（p = 0.25）。所设计的明胶基膜提供了成功构建眼表/结膜缺损所需的必要物理和机械特性，并且由于更可预测的降解模式，可被认为是AM的有前景的替代品，愈合上皮上杯状细胞密度较高，炎症较少，瘢痕组织形成减少。(C)2018爱思唯尔有限公司版权所有
Background: The aim of this study was to evaluate the surgical handling and clinical applicability of a specific 3D-printed membrane design fabricated using a gelatin, elastin and sodium hyaluronate blend for conjunctival reconstruction and compare it with amniotic membrane (AM), which is normally used in such surgeries.Methods: 3D printing technique was employed to fabricate the membrane based on gradient design. Prior to printing, rheometry was employed to optimize the ink composition. The printed membranes were then fully characterized in terms of physical and mechanical properties. In vitro viability, proliferation and adhesion of human limbal epithelial cells were assessed using mu assay and scanning electron microscopy (SEM), respectively. Prior to in vivo experiment, surgical handling of each membrane was evaluated by three surgeons. In vivo evaluation was conducted through implanting the gelatin-based membranes and AM on induced conjunctival defects in rabbits (n = 8). Clinical observations, including epithelialization, inflammation severity, scar tissue formation and presence of granulation tissue, were recorded from day 1 through day 28. Histological examination was performed on all enucleated eyes on day 28. In addition to H&E staining, specific stains including Periodic Acid Schiff staining, Masson's Trichrome staining and immuno-histochemical staining for alpha-SMA were further used to assess goblet cell proliferation, healed sub-epithelial stroma and scar tissue formation and the presence of myofibroblasts, respectively.Results: Among all the examined compositions, a blend of 8% w/v gelatin, 2% w/v elastin and 0.5% w/v sodium hyaluronate was found to be appropriate for printing. The printed membranes had favorable optical characteristics (colorless and transparent), and the surgical handling was significantly easier compared to AM. Epithelial cells cultivated on the membranes indicated suitable viability and proliferation, and SEM images presented appropriate cell adhesion on the surface of the membranes. Clinical observations suggested similar epithelialization time (approximately 3 weeks) for both the membrane and AM grafted eyes but significantly lower levels of clinical inflammation in the membrane group from day 1 through day 28 (p = 0.01), which is a key advantage of using the printed membranes over the AM. Histological examination showed similar qualities in the healed epithelium in terms of cell morphology and cell layers. However, twice the density of goblet cells per 100 cells was observed in the gelatin-based membrane grafted group. Remnant of the degraded implant was seen in only 3 of the membranes, but in 7 of the AM grafted eyes. Inflammation and granulomatous reaction was significantly higher in sections containing the AM compared to membrane (p < 0.01 and p = 0.01, respectively). alpha-SMA staining was more evident, but not significantly different from the gelatin-based membrane, for the AM group (p = 0.25).Conclusion: The designed gelatin-based membrane offers the necessary physical and mechanical characteristics needed for successful ocular surface/conjunctival defect construction and may be considered a promising alternative to AM due to a more predictable degradation pattern, higher goblet cell density on the healed epithelium, less inflammation and reduced scar tissue formation. (C) 2018 Elsevier Ltd. All rights reserved.