背景:角膜碱烧伤可导致溃疡,穿孔,甚至由于上皮缺损和广泛的细胞坏死而导致的角膜失明,导致不良的愈合结果。前期研究发现,壳聚糖基原位水凝胶负载角膜缘上皮干细胞(LESCs)对角膜碱烧伤有一定的修复作用。然而,载体孔径不一致和细胞负载率低导致修复效果欠佳。在这项研究中,利用4D生物打印技术制备了一种孔径均匀、形状可调的壳聚糖基热敏凝胶载体(4D-CTH),以提高LESCs的转移能力。
方法:制备壳聚糖乙酸酯溶液,羧甲基壳聚糖,和特定浓度的β-甘油磷酸钠,并将它们按一定比例混合,以使用4D生物打印技术创建孔径均匀的支架。体外提取和培养大鼠LESCs(rLESCs),进行免疫荧光实验,观察deltaNp63细胞的阳性率,用于细胞鉴定。进行一系列实验以验证4D-CTH的细胞相容性,包括CCK-8测定以评估细胞毒性,划痕试验评估4D-CTH对rLESCs迁移的影响,和钙黄绿素-AM/PI细胞染色实验,检测4D-CTH对rLESCs增殖和形态的影响。建立大鼠角膜严重碱烧伤模型,使用4D-CTH将rLESC移植到受伤的角膜上,使用裂隙灯定期观察角膜混浊和新生血管形成,并通过荧光素钠染色评价上皮愈合。通过苏木精和伊红染色的角膜组织石蜡切片的组织学评估,评估4D-CTH负载的rLESCs对角膜碱烧伤的治疗效果。以及冷冻切片的免疫荧光染色。
结果:使用4D-CTH,将rLESCs转移到大鼠的碱烧伤伤口中。与传统治疗组(壳聚糖原位水凝胶包裹rLESCs)相比,4D-CTH-rLESC组对角膜损伤的修复效率明显增高,如较低的角膜混浊评分(1.2±0.4472vs0.4±0.5477,p<0.05)和新生血管评分(5.5±1.118vs2.6±0.9618,p<0.01),角膜上皮伤口愈合率明显较高(72.09±3.568%vs86.60±5.004%,p<0.01)。
结论:总之,4D-CTH-rLESC治疗组的角膜与正常角膜相似,角膜结构完整.这些发现表明,4D-CTH包裹的LESCs可显着加速碱烧伤后角膜伤口的愈合,可以被认为是治疗上皮缺损的快速有效方法。
BACKGROUND: Corneal alkali burns can lead to ulceration, perforation, and even corneal blindness due to epithelial defects and extensive cell necrosis, resulting in poor healing outcomes. Previous studies have found that chitosan-based in situ hydrogel loaded with limbal epithelium stem cells (LESCs) has a certain reparative effect on corneal alkali burns. However, the inconsistent pore sizes of the carriers and low cell loading rates have resulted in suboptimal repair outcomes. In this study, 4D bioprinting technology was used to prepare a chitosan-based thermosensitive gel carrier (4D-CTH) with uniform pore size and adjustable shape to improve the transfer capacity of LESCs.
METHODS: Prepare solutions of chitosan acetate, carboxymethyl chitosan, and β-glycerophosphate sodium at specific concentrations, and mix them in certain proportions to create a pore-size uniform scaffold using 4D bioprinting technology. Extract and culture rat LESCs (rLESCs) in vitro, perform immunofluorescence experiments to observe the positivity rate of deltaNp63 cells for cell identification. Conduct a series of experiments to validate the cell compatibility of 4D-CTH, including CCK-8 assay to assess cell toxicity, scratch assay to evaluate the effect of 4D-CTH on rLESCs migration, and Calcein-AM/PI cell staining experiment to examine the impact of 4D-CTH on rLESCs proliferation and morphology. Establish a severe alkali burn model in rat corneas, transplant rLESCs onto the injured cornea using 4D-CTH, periodically observe corneal opacity and neovascularization using a slit lamp, and evaluate epithelial healing by fluorescein sodium staining. Assess the therapeutic effect 4D-CTH-loaded rLESCs on corneal alkali burn through histological evaluation of corneal tissue paraffin sections stained with hematoxylin and eosin, as well as immunofluorescence staining of frozen sections.
RESULTS: Using the 4D-CTH, rLESCs were transferred to the alkali burn wounds of rats. Compared with the traditional treatment group (chitosan in situ hydrogel encapsulating rLESCs), the 4D-CTH-rLESC group had significantly higher repair efficiency of corneal injury, such as lower corneal opacity score (1.2 ± 0.4472 vs 0.4 ± 0.5477, p < 0.05) and neovascularization score (5.5 ± 1.118 vs 2.6 ± 0.9618, p < 0.01), and significantly higher corneal epithelial wound healing rate (72.09 ± 3.568% vs 86.60 ± 5.004%, p < 0.01).
CONCLUSIONS: In summary, the corneas of the 4D-CTH-rLESC treatment group were similar to the normal corneas and had a complete corneal structure. These findings suggested that LESCs encapsulated by 4D-CTH significantly accelerated corneal wound healing after alkali burn and can be considered as a rapid and effective method for treating epithelial defects.