新型细胞外基质源性神经修复材料的制备与评价

韩锋; 魏帅; 张健; 张宇轩; 李超超; 孟繁琪; 管延军; 刘修志; 许文静; 彭江

doi:10.3969/j.issn.2095-5227.2022.04.017

摘要:

背景周围神经损伤及缺损是临床常见病、多发病，长段缺损的治疗效果并不理想。

目的探索新型细胞外基质源性神经修复材料的制备方法并评价其物理化学性质。

方法无菌条件下提取新鲜猪坐骨神经，剔除神经外脂肪、血管等结缔组织备用。新鲜组：将剥除好的猪坐骨神经冷冻干燥，作为对照组；0.5% SDS 48 h组：将剥除好的猪坐骨神经置于浓度为0.5%的十二烷基硫酸钠(sodium dodecyl sulfate，SDS)溶液中，去细胞处理48 h，后冷冻干燥；0.5% SDS 48 h + scCO₂组：重复0.5% SDS 48 h组制备后，将获取的神经置于超临界二氧化碳(supercritical carbon dioxide，scCO₂)中行去除脂肪处理。将制备好的三组神经细胞外基质移植物分别行物理性能测定(外观和规格尺寸)、组织学观察(HE染色、Laminin/DAPI免疫荧光染色和油红O脂肪染色)、DNA含量测定和扫描电镜观察等。

结果大体观察，处理后的神经长度稍缩短，直径稍增大，呈乳白色半透明状，大体轮廓仍完整，质地较处理前疏松；HE染色显示，0.5% SDS 48 h组和0.5% SDS 48 h + scCO₂组神经均未见细胞核；Laminin(层粘连蛋白)/DAPI染色显示，0.5% SDS 48 h组和0.5% SDS 48 h + scCO₂组神经内膜管状结构存在，未见细胞核；油红O脂肪染色显示，0.5% SDS 48h组脂肪未见明显减少；0.5% SDS 48 h + scCO₂组脂肪明显减少；DNA含量测定显示，0.5% SDS 48h组DNA含量下降(706.7 ng/mg降至59.6 ng/mg，P ＜ 0.01)，0.5% SDS 48 h + scCO₂组DNA含量下降(59.6 ng/mg降至49.2 ng/mg，P ＜ 0.05)；扫描电镜观察，0.5% SDS 48 h组和0.5% SDS 48 h + scCO₂组内部可见明显的神经内膜管状结构，未见髓鞘和轴突。

结论 1)本实验制备的新型细胞外基质源性神经修复材料，清除了内部的髓鞘、轴突和脂肪等抑制神经再生的成分；同时保留了层粘连蛋白和神经内膜管状结构等促进神经再生的成分和结构。2)综合评价两组神经修复材料的物理结构和生物化学成分，0.5% SDS 48 h + scCO₂组优于0.5% SDS 48 h组。

Abstract:

Background Peripheral nerve injury and defect is commonly seen, but the treatment to long segment defect is not satisfying.

Objective To explore the preparation method of a new extracellular matrix-derived nerve repair material and evaluate its physicochemical properties.

Methods Fresh pig sciatic nerve was extracted under aseptic condition,and connective tissue such as fat and blood vessels were removed. Fresh group: the stripped porcine sciatic nerve was freeze-dried as the control group; 0.5% SDS 48 h group: the exfoliated porcine sciatic nerve was placed in 0.5% sodium dodecyl sulfate (SDS) solution for 48 hours, then freeze-dried; 0.5% SDS 48 h + scCO₂ group: after repeated 0.5% SDS 48 h preparation, the obtained nerve was placed in supercritical carbon dioxide (scCO₂) for fat removal. The prepared nerve extracellular matrix grafts were evaluated by physical properties (appearance and size), histological observation (HE staining, Laminin/DAPI immunofluorescence staining and oil red O fat staining), DNA content determination and scanning electron microscope observation.

Results In general observation, the length of the treated nerve was slightly shortened, the diameter was slightly increased, the nerve was milky white and translucent, the general outline was still intact and the texture was looser than that before treatment; HE staining showed that there was no nucleus in 0.5% SDS 48 h group and 0.5% SDS 48 h + scCO₂ group; Laminin / DAPI staining showed that there was intimal tubular structure in 0.5% SDS 48 h group and 0.5% SDS 48 h + scCO₂ group, but no nucleus was found in 0.5% SDS 48 h group and 0.5% SDS 48 h + scCO₂ group. Oil red O fat staining showed that there was no significant decrease in fat in the 0.5% SDS 48 h group; Fat decreased significantly in the 0.5% SDS 48 h + scCO₂ group; DNA content determination, 0.5% SDS 48 h group of DNA content decreased (706.7 ng/mg decline into 59.6 ng/mg, P ＜ 0.01), 0.5% SDS 48 h + CO₂ group of DNA content decreased (59.6 ng/mg decline into 49.2 ng/mg, P ＜ 0.05); Scanning electron microscope showed that obvious intimal tubular structure could be seen in the 0.5% SDS 48 h and 0.5% SDS 48 h + scCO₂ groups, but no myelin sheath and axon were found in the 0.5% SDS 48 h and 0.5% SDS 48 h + scCO₂ groups.

Conclusion 1) The new extracellular matrix-derived nerve repair material prepared in this experiment can remove the internal substances that inhibit nerve regeneration, such as myelin sheath, axons and fat. At the same time, the components and structures that promote nerve regeneration such as laminin and intimal tubular structure were retained. 2) The comprehensive evaluation of physical structure and biochemical composition of the 0.5% SDS 48 h + scCO₂ group was superior to that of the 0.5% SDS 48 h group.

新型细胞外基质源性神经修复材料的制备与评价

Preparation and evaluation of novel extracellular matrix-derived nerve repair materials