Effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats
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摘要:
目的 探讨载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响。 方法 采用实验研究方法。通过油包水乳化法制备聚乙烯醇/海藻酸钠微球（单纯微球）、P311微球及异硫氰酸荧光素标记的牛血清白蛋白（FITC-BSA）微球，于光学显微镜/倒置荧光显微镜下观察形貌。制备壳聚糖溶液，将壳聚糖溶液和β-磷酸甘油二钠水合物混合制备单纯温敏水凝胶，在单纯温敏水凝胶中加入相应物质制备载单纯微球和载P311微球的温敏水凝胶，观察4种液体在37 ℃时倾斜状态下的形态变化，冷冻干燥后于扫描电子显微镜下观察微观形貌。取18只3~4周龄雄性SD大鼠，分为不进行任何处理的正常组及于背部脊柱两侧分别制作1个全层皮肤缺损创面并进行相应处理的敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组、载P311微球水凝胶组，每组3只。取5组全层皮肤缺损大鼠，于伤后0（即刻）、5、10、15 d 观察创面愈合情况，计算伤后5、10、15 d创面愈合率；取5组全层皮肤缺损大鼠伤后15 d创面和创缘组织及正常组大鼠相同部位正常皮肤组织，行苏木精-伊红染色观察组织学变化，行免疫组织化学染色观测CD31及血管内皮生长因子（VEGF）的表达，采用蛋白质印迹法检测CD31及VEGF的蛋白表达。样本数均为3。对数据行单因素方差分析、重复测量方差分析及Bonferroni校正。 结果 单纯微球呈球形，表面疏松多孔；P311微球及FITC-BSA微球表面光滑无孔隙，且FITC-BSA微球散发出均匀的绿色荧光；3种微球直径基本一致，为33.1~37.7 μm。在37 ℃时倾斜状态下，与壳聚糖溶液及单纯温敏水凝胶相比，载微球的2种水凝胶结构更稳定。载微球的2种水凝胶网状结构较壳聚糖溶液、单纯温敏水凝胶更致密，且其横断面可见直径约30 μm的微球。伤后15 d内，5组大鼠创面均不同程度愈合，其中载P311微球水凝胶组大鼠创面愈合情况最好。敷贴组、壳聚糖组大鼠伤后5、10、15 d创面愈合率分别为（26.6±2.4）%、（38.5±3.1）%、（50.9±1.5）%，（47.6±2.0）%、（58.5±3.6）%、（66.7±4.1）%，均明显低于载P311微球水凝胶组的（59.3±4.8）%、（87.6±3.2）%、（97.2±1.0）%，P<0.05或P<0.01；单纯水凝胶组大鼠伤后10、15 d及载单纯微球水凝胶组大鼠伤后15 d创面愈合率分别为（76.0±3.3）%、（84.5±3.6）%、（88.0±2.6）%，均明显低于载P311微球水凝胶组（P<0.05）。正常组大鼠正常皮肤中可见表皮、毛囊及皮脂腺，未见CD31和VEGF阳性表达；载P311微球水凝胶组大鼠伤后15 d创面已几乎完全上皮化，创面血管、毛囊、皮脂腺生成及CD31和VEGF阳性表达较其他4组全层皮肤缺损大鼠明显增加，CD31和VEGF蛋白表达均较其余5组大鼠明显增加（P<0.01）。 结论 载P311微球的温敏壳聚糖水凝胶，可以缓释包载的药物，延长药物作用时间，并通过促进创面血管生成及再上皮化，促进全层皮肤缺损大鼠创面愈合。 Abstract:Objective To explore the effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats. Methods The method of experimental study was adopted. The polyvinyl alcohol/sodium alginate microspheres (simple microspheres), P311 microspheres, and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) microspheres were prepared by water-in-oil emulsification, and then their morphology was observed under a light microscope/inverted fluorescence microscope. Chitosan solution was prepared, chitosan solution and β-glycerol phosphate disodium hydrate were mixed to prepare simple thermosensitive hydrogels, and thermosensitive hydrogels loaded with simple microspheres or P311 microspheres were prepared by adding corresponding substances in simple thermosensitive hydrogels. The morphological changes of the prepared four liquids in the state of tilt was observed at 37 ℃. After being freeze-dried, the micromorphology of the prepared four liquids was observed under a scanning electron microscope. Eighteen 3-4-week-old male Sprague-Dawley rats were divided into normal group without any treatment, dressing group, chitosan group, hydrogel alone group, simple microspheres-loaded hydrogel group, and P311 microspheres-loaded hydrogel group, which were inflicted with one full-thickness skin defect wound on both sides of the back spine and were dealt correspondingly, with 3 rats in each group. Rats with full-thickness skin defects in the five groups were collected, the wound healing was observed on post injury day (PID) 0 (immediately), 5, 10, and 15, and the wound healing rates on PID 5, 10, and 15 were calculated. The wound and wound margin tissue of rats with full-thickness skin defects in the five groups on PID 15 and normal skin tissue in the same site of rats in normal group were collected, hematoxylin and eosin staining was conducted to observe the histological changes, immunohistochemical staining was performed to observe the expressions of CD31 and vascular endothelial growth factor (VEGF), and Western blotting was conducted to detect the protein expressions of CD31 and VEGF. The number of samples was all three. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, and Bonferroni correction. Results Simple microspheres were spherical, with loose and porous surface. The surfaces of P311 microspheres and FITC-BSA microspheres were smooth without pores, and the FITC-BSA microspheres emitted uniform green fluorescence. The diameters of the three microspheres were basically consistent, being 33.1 to 37.7 μm. Compared with chitosan solution and simple thermosensitive hydrogel, the structures of the two microspheres-loaded hydrogels were more stable in the state of tilt at 37 ℃. The two microspheres-loaded hydrogels had denser network structures than those of chitosan solution and simple thermosensitive hydrogel, and in the cross section of which microspheres with a diameter of about 30 μm could be seen. Within PID 15, the wounds of rats in the five groups were healed to different degrees, and the wound healing of rats in P311 microspheres-loaded hydrogel group was the best. On PID 5, 10, and 15, the wound healing rates of rats in dressing group and chitosan group were (26.6±2.4)%, (38.5±3.1)%, (50.9±1.5)%, (47.6±2.0)%, (58.5±3.6)%, and (66.7±4.1)%, respectively, which were significantly lower than (59.3±4.8)%, (87.6±3.2)%, (97.2±1.0)% in P311 microspheres-loaded hydrogel group (P<0.05 or P<0.01). The wound healing rates of rats in hydrogel alone group on PID 10 and 15, and in simple microspheres-loaded hydrogel group on PID 15 were (76.0±3.3)%, (84.5±3.6)%, and (88.0±2.6)%, respectively, which were significantly lower than those in P311 microspheres-loaded hydrogel group (P<0.05). The epidermis, hair follicles, and sebaceous glands could be seen in the normal skin of rats in normal group, without positive expressions of CD31 or VEGF. The wounds of rats in P311 microspheres-loaded hydrogel group on PID 15 were almost completely epithelialized, with more blood vessels, hair follicles, sebaceous glands, and positive expressions of CD31 and VEGF in the wounds than those of rats with full-thickness skin defects in the other four groups, and more protein expressions of CD31 and VEGF than those of rats in the other five groups. Conclusions The P311 microspheres-loaded thermosensitive chitosan hydrogel can release the encapsulated drug slowly, prolong the drug action time, and promote wound healing in rats with full-thickness skin defects by promoting wound angiogenesis and re-epithelialization. -
Key words:
- Biocompatibile materials /
- Hydrogel /
- Microspheres /
- P311 /
- Angiogenesis /
- Wound repair
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参考文献
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1 3种聚乙烯醇/海藻酸钠微球的形貌观察。1A.单纯微球呈球形 光学显微镜×200；1B、1C.分别为单纯微球和P311微球，表面结构疏松多孔 光学显微镜×200；1D. 异硫氰酸荧光素标记的牛血清白蛋白微球表面光滑无空隙，且发出均匀绿色荧光 倒置荧光显微镜×200
2 壳聚糖溶液及3种温敏水凝胶在37 ℃水浴3 min后的形貌。2A.壳聚糖溶液仍为液态；2B、2C、2D.分别为单纯温敏水凝胶、载单纯微球的温敏水凝胶、载P311微球的温敏水凝胶，均变成凝胶态，且图2C、2D温敏水凝胶结构较图2B更加稳定
注：微球为聚乙烯醇/海藻酸钠微球，温敏水凝胶由β-磷酸甘油二钠水合物与壳聚糖制成
3 壳聚糖溶液及3种温敏水凝胶冻干后的横断面结构 扫描电子显微镜×200。3A.壳聚糖呈多孔网状结构；3B.温敏水凝胶网状结构较图3A更加致密；3C、3D.分别为载单纯微球的温敏水凝胶及载P311微球的温敏水凝胶，微球被包裹在凝胶中，且凝胶结构较图3A、3B更加致密
注：微球为聚乙烯醇/海藻酸钠微球，温敏水凝胶由β-磷酸甘油二钠水合物与壳聚糖制成；图中箭头指示微球
4 5组全层皮肤缺损大鼠伤后各时间点创面情况。4A、4B、4C、4D.分别为敷贴组伤后0（即刻）、5、10、15 d创面情况，伤后15 d仍有部分创面未愈合；4E、4F、4G、4H.分别为壳聚糖组伤后0、5、10、15 d创面情况，图4F创面面积较图4E明显缩小，图4G、4H创面面积相近；4I、4J、4K、4L及4M、4N、4O、4P.分别为单纯水凝胶组及载单纯微球水凝胶组伤后0、5、10、15 d创面情况，创面面积均较组内前一时间点明显缩小，但伤后15 d均有小部分创面未愈合；4Q、4R、4S、4T.分别为载P311微球水凝胶组伤后0、5、10、15 d创面情况，创面逐渐缩小，且伤后15 d创面已基本愈合
注：右侧圆形物为参照物；创面均贴手术敷贴，微球为聚乙烯醇/海藻酸钠微球，水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶
5 正常组大鼠正常皮肤组织及5组全层皮肤缺损大鼠伤后15 d创面组织学观察 苏木精-伊红×40。5A.正常组大鼠正常皮肤组织毛囊、皮脂腺排列整齐；5B.敷贴组创面仅有少许血管生成；5C、5D.分别为壳聚糖组和单纯水凝胶组，创面部分上皮化，有少许血管生成；5E.载单纯微球水凝胶组创面大部分上皮化，有部分皮脂腺、毛囊及血管生成；5F.载P311微球水凝胶组创面已几乎完全上皮化，有大量皮脂腺、毛囊及血管生成
注：黑色箭头指示皮脂腺，黄色箭头指示毛囊，红色箭头指示新生血管；正常组大鼠取与其他5组大鼠相同部位正常皮肤组织；创面均贴手术敷贴，微球为聚乙烯醇/海藻酸钠微球，水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶
6 正常组大鼠正常皮肤组织及5组全层皮肤缺损大鼠伤后15 d创面组织中CD31及VEGF阳性表达（均为棕色） 二氨基联苯胺-苏木精×400。6A.正常组未见CD31阳性表达；6B、6C、6D、6E、6F.分别为敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组、载P311微球水凝胶组，CD31阳性表达逐渐增多，图6F中CD31阳性表达明显多于图6B、6C、6D、6E；6G.正常组未见VEGF阳性表达；6H、6I、6J、6K、6L.分别为敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组及载P311微球水凝胶组，VEGF阳性表达逐渐增多，图6L中VEGF阳性表达明显多于图6H、6I、6J、6K
注：正常组大鼠取与其他5组大鼠相同部位正常皮肤组织；创面均贴手术敷贴，微球为聚乙烯醇/海藻酸钠微球，水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶；VEGF为血管内皮生长因子
7 蛋白质印迹法检测正常组大鼠正常皮肤组织及5组全层皮肤缺损大鼠伤后15 d创面组织中CD31、VEGF蛋白表达水平
注：创面均贴手术敷贴，微球为聚乙烯醇/海藻酸钠微球，水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶；VEGF为血管内皮生长因子，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4、5、6分别指示正常组、敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组、载P311微球水凝胶组
表1 5组全层皮肤缺损大鼠伤后各时间点创面愈合率比较（%，
组别 鼠数（只） 5 d 10 d 15 d 敷贴组 3 26.6±2.4a 38.5±3.1a 50.9±1.5a 壳聚糖组 3 47.6±2.0b 58.5±3.6a 66.7±4.1a 单纯水凝胶组 3 50.4±3.0 76.0±3.3b 84.5±3.6b 载单纯微球水凝胶组 3 57.6±2.7 83.2±3.7 88.0±2.6b 载P311微球水凝胶组 3 59.3±4.8 87.6±3.2 97.2±1.0 F值 52.34 106.55 129.84 P值 <0.001 <0.001 <0.001 注：创面均贴手术敷贴，微球为聚乙烯醇/海藻酸钠微球，水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶；处理因素主效应，F=323.44，P<0.001；时间因素主效应，F=245.30，P<0.001；两者交互作用，F=4.24，P=0.005；与载P311微球水凝胶组相比，aP<0.01，bP<0.05 
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