Wu Xiaoyu, Li Haibo, Yin Jianjian, Liu Chun, Wu Siyu, Liu Jun, Ma Jiayi, Dai Ting, Zhao Hongbin
Medical Research Centre, the Affiliated Changzhou No.2 People's Hospital, Nanjing Medical University, Changzhou Jiangsu, 213164, P. R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2022 Mar 15;36(3):352-361. doi: 10.7507/1002-1892.202108142.
To construction the telmisartan/collagen/polycaprolactone (PCL) nerve conduit and assess its effect on repairing sciatic nerve defect in rats.
The 60% collagen/hexafluoroisopropanol (HFIP) solution and 40% PCL/HFIP solution were prepared and mixed (collagen/PCL solution). Then the 0, 5, 10, and 20 mg of telmisartan were mixed with the 10 mL collagen/PCL solution, respectively. Telmisartan/collagen/PCL nerve conduits were fabricated via high voltage electrospinning technology. The structure of nerve conduit before and after crosslinking was observed by using scanning electron microscope (SEM). The drug release efficiency was detected by sustained release method. RAW264.7 cells were cultured with lipopolysaccharide to induce inflammation, and then co-cultured with nerve conduits loaded with different concentrations of telmisartan for 24 hours. The mRNA expressions of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) were detected by using real-time fluorescence quantitative PCR. Forty adult Wistar rats were randomly divided into 4 groups ( =10). After preparing 15-mm-long sciatic nerve defect, the defect was repaired by cross-linked nerve conduits loaded with 0, 5, 10, and 20 mg telmisartan in groups A, B, C, and D, respectively. After operation, the general condition of rats was observed after operation; the sciatic function index (SFI) was tested; the bridging between the nerve conduit and sciatic nerve, and the integrity of nerve conduit were observed; the tissue growth in nerve conduit and material degradation were observed by HE staining; the expressions of CD86 (M1 macrophage marker), CD206 (M2 macrophage marker), myelin basic protein (MBP), and myelin protein 0 (P0) in new tissues were also observed by immunohistochemical staining; the expressions of neurofilament 200 (NF-200) and S-100β in new tissues were assessed by immunofluorescence staining.
The general observation showed that the inner diameter of the nerve conduit was 1.8 mm and the outer diameter was 2.0 mm. After cross-linking by genipin, the nanofiber became thicker and denser. The drug release test showed that the telmisartan loaded nerve conduit could be released gradually. With the increase of telmisartan content in nerve conduit, the iNOS mRNA expression decreased and the Arg-1 mRNA expression increased; and the differences between 20 mg group and other groups were significant ( <0.05). experiment showed that all animals in each group survived until the completion of the experiment. The SFI was significantly higher in groups C and D than in groups A and B at different time points ( <0.05) and in group D than in group C at 6 months after operation ( <0.05). HE staining showed that there were significantly more new tissues in the middle of the nerve conduit in group D after operation than in other groups. Immunohistochemical staining showed that CD86 and CD206 stainings were positive in each group at 1 month after operation, among which group D had the lowest positive rate of CD86 and the highest positive rate of CD206, and there were significant differences in the positive rate of CD206 between group D and groups A, B, and C ( <0.05); the MBP and P0 stainings were positive in groups C and D at 6 months, and the positive rate in group D was significantly higher than that in group C ( <0.05). Immunofluorescence staining showed that the NF-200 and S-100β expressions in group D were significantly higher than those in other groups.
Telmisartan/collagen/PLC nerve conduit can promote the sciatic nerve defect repair in rats through promoting the polarization of M1 macrophages to M2 macrophages, and the nerve conduit loaded with20 mg telmisartan has the most significant effect.
构建替米沙坦/胶原蛋白/聚己内酯（PCL）神经导管，并评估其对大鼠坐骨神经缺损的修复效果。
制备60%胶原蛋白/六氟异丙醇（HFIP）溶液和40% PCL/HFIP溶液并混合（胶原蛋白/PCL溶液）。然后将0、5、10和20 mg替米沙坦分别与10 mL胶原蛋白/PCL溶液混合。通过高压静电纺丝技术制备替米沙坦/胶原蛋白/PCL神经导管。使用扫描电子显微镜（SEM）观察交联前后神经导管的结构。采用缓释法检测药物释放效率。用脂多糖培养RAW264.7细胞诱导炎症，然后与加载不同浓度替米沙坦的神经导管共培养24小时。采用实时荧光定量PCR检测诱导型一氧化氮合酶（iNOS）和精氨酸酶1（Arg-1）的mRNA表达。40只成年Wistar大鼠随机分为4组（每组n =10）。制备15 mm长的坐骨神经缺损后，A、B、C、D组分别用加载0、5、10和20 mg替米沙坦的交联神经导管修复缺损。术后观察大鼠的一般情况；检测坐骨神经功能指数（SFI）；观察神经导管与坐骨神经的桥接情况以及神经导管的完整性；通过HE染色观察神经导管内的组织生长和材料降解情况；通过免疫组织化学染色观察新组织中CD86（M1巨噬细胞标志物）、CD206（M2巨噬细胞标志物）、髓鞘碱性蛋白（MBP）和髓鞘蛋白0（P0）的表达；通过免疫荧光染色评估新组织中神经丝200（NF-200）和S-100β的表达。
大体观察显示神经导管内径为1.8 mm，外径为2.0 mm。经京尼平交联后，纳米纤维变粗且致密。药物释放试验表明，加载替米沙坦的神经导管可逐渐释放药物。随着神经导管中替米沙坦含量的增加，iNOS mRNA表达降低，Arg-1 mRNA表达增加；20 mg组与其他组之间差异有统计学意义（P<0.05）。实验表明每组所有动物均存活至实验结束。不同时间点C组和D组的SFI显著高于A组和B组（P<0.05），术后6个月D组高于C组（P<0.05）。HE染色显示术后D组神经导管中部的新组织明显多于其他组。免疫组织化学染色显示术后1个月每组CD86和CD206染色均为阳性，其中D组CD86阳性率最低，CD206阳性率最高，D组与A、B、C组CD206阳性率差异有统计学意义（P<0.05）；术后6个月C组和D组MBP和P0染色为阳性，D组阳性率显著高于C组（P<0.05）。免疫荧光染色显示D组NF-和S-100β表达显著高于其他组。
替米沙坦/胶原蛋白/聚己内酯神经导管可通过促进M1巨噬细胞向M2巨噬细胞极化促进大鼠坐骨神经缺损修复，加载20 mg替米沙坦的神经导管效果最显著。
