In situ repair of full-thickness skin defects by handheld electrospun biodegradable nanofibers

doi:10.3969/j.issn.2095-4344.1513

Abstract

Abstract:

BACKGROUND: The traditional electrospinning nanofiber manufacturing process is relatively complicated, which requires high manufacturing conditions and cannot meet the needs of rapid tissue repair in emergency events such as trauma and burn/scald.

OBJECTIVE: To observe the effect of handheld electrospun polylactic acid/gelatin degradable nanofiber membrane on the in situ repair of mouse skin defects.

METHODS: Handheld electrospun polylactic acid/gelatin degradable nanofiber membranes were prepared by self-made 3D printing handheld electrospinning device, and the contact angle and water vapor transmission rate were then measured. Fetal rat fibroblasts were cultured with 100%, 50%, 20% polylactic acid/gelatin degradable nanofiber membrane extracts, and the residual solvent toxicity of the materials was evaluated by cell counting kit-8 cytotoxicity assay. Fetal rat fibroblasts were co-cultured with polylactic acid/gelatin degradable nanofiber membrane (experimental group), and cells cultured alone were set as control. Cell proliferation was detected by Alamar blue method, cell viability was observed by live/dead staining, and cell morphology was observed by scanning electron microscope. A full-thickness skin defect of 2 cm in diameter was made on the back of 18 Balb/c mice. The experimental group was inlaid with handheld electrospun polylactic acid/gelatin degradable nanofiber membrane for in situ repair followed by gauze dressing. The control group was treated with gauze dressing at the defect site. Eight weeks after operation, hematoxylin-eosin and Masson staining were used to observe the repair of skin defects.

RESULTS AND CONCLUSION: (1) The contact angle of polylactic acid/gelatin degradable nanofiber membrane was (32.68±5.68)°, indicating a hydrophilic material suitable for cell adhesion. The 24-hour water vapor transmission rate was (4.21±0.11)×10³ g/m², which met the requirements of external skin dressing. Different concentrations of polylactic acid/gelatin degradable nanofiber membrane extract had no obvious cytotoxicity. (2) In the experimental group, fetal rat fibroblasts had the cell viability equivalent to control cells, but exhibited faster proliferation rate and longer proliferation time. (3) Results from the hematoxylin-eosin and Masson staining showed that the full-thickness skin defect healed in the experimental group, with the material being completely degraded and the hair follicles being regenerated. In the control group, the defect healed incompletely. To conclude, handheld electrospun polylactic acid/gelatin degradable nanofibers can implement the in situ repair of mouse full-thickness skin defects.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Nanofibers, Gelatin, Tissue Engineering

CLC Number:

R459.9

R318

Chen Hongrang, Zhang Haitao, Deng Kunxue, Li Yongsheng, Shen Yun, Xu Tao, Zhang Xinqiong. In situ repair of full-thickness skin defects by handheld electrospun biodegradable nanofibers[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(2): 257-264.

Figures/Tables 3

2.1 手持静电纺丝纳米纤维膜材料的特性 2.1.1 电镜观察手持式静电纺丝装置原位电纺样品的电镜图，见图2A，可见经原位电纺后，聚乳酸/明胶纺丝液被拉伸、固化为相对均匀的纳米纤维，纤维平均直径为(584.44±188.43) nm。直径分布统计发现，其直径分布符合高斯正态分布，见图2B所示。 2.1.2 傅里叶变换红外光谱聚乳酸/明胶纳米纤维膜、聚乳酸纳米纤维膜及明胶纳米纤维膜的傅里叶变换红外光谱图，见图2C所示，可见聚乳酸纳米纤维在1 758，1 452，1 183，1 130，1 087 cm-1出现的吸收峰分，别对应C=O的伸缩振动吸收峰、-CH3的伸缩振动吸收峰、C-O反对称伸缩振动吸收峰、C-O-C伸缩振动吸收峰、C-O对称伸缩振动吸收峰；明胶纳米纤维在1 593，1 462，1 246 cm-1出现的吸收峰，对应酰胺Ⅰ带的N-H变形振动吸收峰、酰胺Ⅱ带的N-H伸缩振动吸收峰、酰胺Ⅲ 带的N-H伸缩振动吸收峰。当二者复合后，聚乳酸/明胶纳米纤维基本遵从聚乳酸纳米纤维的红外特征，归属于明胶的特征吸收峰基本消失，证明少量明胶的加入，对聚乳酸的特征基团不产生大的影响；同时，还发现聚乳酸/明胶纳米纤维在1 650 cm-1出现了吸收峰，对应酰胺Ⅰ带C=O伸缩振动吸收峰，说明明胶和聚乳酸之间产生了微弱的化学作用，这有助于明胶在聚乳酸中的均匀分布。 2.1.3 X射线衍射聚乳酸/明胶纳米纤维膜、聚乳酸纳米纤维膜及明胶纳米纤维膜的X射线衍射结果，见图2D所示，可见聚乳酸纳米纤维在16.6°位置出现结晶峰，对应聚乳酸的(200)晶面。明胶纳米纤维在20°左右出现馒头峰。当二者复合后，聚乳酸/明胶纳米纤维也仅在16.6°位置出现结晶峰，并且X射线衍射曲线与聚乳酸纳米纤维没有明显差别，说明明胶的加入对复合材料结晶性能无影响。 2.1.4 水接触角聚乳酸/明胶纳米纤维膜、聚乳酸纳米纤维膜及明胶纳米纤维膜的水接触角，见图2E所示。聚乳酸纳米纤维的水接触角为(134.48±3.43)°，属于疏水材料范围。研究采用的明胶纳米纤维未经过交联处理，因此当水滴接触明胶纳米纤维膜后，会迅速弥散开来，且明胶纳米纤维易溶于水，所以无法得到正常的水接触角，增大明胶纳米纤维膜的厚度并缩短拍照时间，可得到明胶纳米纤维的水接触角为(11.02±10.34)°，属于亲水材料范围。当二者复合后，测得聚乳酸/明胶纳米纤维膜的水接触角为(32.68± 5.68)°，属于亲水材料范围，而且这个水接触角在最适宜细胞黏附的30°-50°的范围内[24]。因此，聚乳酸/明胶纳米纤维膜的水接触角满足细胞黏附需求。 2.1.5 水蒸气透过率聚乳酸/明胶纳米纤维膜、聚乳酸纳米纤维膜及明胶纳米纤维膜的水蒸气透过率，见图2F所示。作为皮肤上的外敷料，除需保护创面不受外界环境影响，原位电纺的纳米纤维膜还需要有一定的透气透湿性能。经测试发现，因聚乳酸纳米纤维为疏水性，24 h透湿率仅为(0.272±0.09)×103 g/m2。因明胶纳米纤维溶于水，当进行透湿测试时，膜片很快被水浸湿后破裂，无法得到透湿率，因此不具备原位电纺的实际应用价值。当二者复合后，聚乳酸/明胶纳米纤维膜的24 h透湿率可达到(4.21±0.11)× 103 g/m2，可满足皮肤外敷料的要求[25]。综上所述，聚乳酸/明胶纳米纤维膜材料特性可满足皮肤修复材料以及原位电纺电纺的要求，因此选取聚乳酸/明胶纳米纤维膜进行后续生物学测试。"

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