Skip to main content
NCBI home page
Chinese Journal of Burns logoLink to Chinese Journal of Burns
. 2021 May 20;37(5):460–468. [Article in Chinese] doi: 10.3760/cma.j.cn501120-20210104-00005

负载大鼠表皮干细胞的聚己内酯-乙酸纤维素纳米纤维支架对大鼠全层皮肤缺损创面愈合的影响及其机制

Effects and mechanisms of polycaprolactone-cellulose acetate nanofiber scaffold loaded with rat epidermal stem cells on wound healing of full-thickness skin defects in rats

Zhixiao Lin 1, Yuheng Zhang 1, Rong Huang 1, Xueyong Li 1,*
PMCID: PMC11917331  PMID: 33894697

Abstract

Objective

To explore the effects and mechanisms of polycaprolactone-cellulose acetate (PCL-CA) nanofiber scaffold loaded with rat epidermal stem cells (ESCs) on wound healing of full-thickness skin defects in rats.

Methods

The experiment research method was applied. The primary ESCs were isolated from 1-3 d old Sprague-Dawley (SD) rats (undefined gender) by rapid adherent method and cultured by rapid adherent method. ESCs of the first passage were used for the subsequent experiments after the positive expressions of integrin β1 and cytokeratin 19 (CK19) in primary cells were identified respectively by flow cytometey and immunofluorescence method. PCL-CA nanofiber scaffolds with polycaprolactone and cellulose acetate as components were prepared by electrospinning technique. The topological structure of the nanofiber scaffolds was determined and the diameter of 25 fibers was measured by scanning electron microscope. The constructed PCL-CA nanofiber scaffolds were used as the culture substrate for ESCs, which were cultured in keratinocytes (KCs) medium to construct ESCs-nanofiber scaffold complex (hereinafter referred to as ESCs scaffold). After 3 days of culture, the morphology of ESCs in the scaffold and their relationship was observed by scanning electron microscopy. The ESCs in ESCs scaffold were set as PCL-CA nanofiber scaffold group, and the ESCs cultured with KCs medium in culture dishes coated with type Ⅳ collagen were set as type Ⅳ collagen group. Western blotting was used to detect the protein expression level of CK19 in ESCs in the two groups after 3 days of culture (n=3). The protein expressions of CK19 and proliferating nuclear antigen (PCNA) in ESCs in the two groups were detected by immunofluorescence method after 7 days of culture. A circular full-thickness skin wound of about 2 cm in diameter was prepared on both left and right sides of the back of 15 male SD rats aged 6-8 weeks. The rats were then equally divided into blank control group without implantation, scaffold alone group implanted with PCL-CA nanofiber scaffold, and ESCs scaffold group implanted with ESCs scaffold which were constructed after 3 days of culture according to the random number table. The percentage of wound areas on post injury day (PID) 3, 7, 14, and 21 was calculated (n=5). The new skin tissue at the wound edge was collected on PID 21, the wound healing quality was evaluated by Masson staining, and the protein expression levels of Notch1, Jagged1, and Hes1, which are key proteins of Notch signaling pathway, were detected by Western blotting (n=3). Data were statistically analyzed with one-way analysis of variance, one-way analysis of variance, analysis of variance for repeated measurement, independent sample t test, and Bonferroni correction.

Results

The constructed PCL-CA nanofiber scaffolds had a porous, mesh-like, and multilayered three-dimensional structure, in which the surface of the fibers was smooth and non-porous, and the fiber diameter was (383±24) nm. The ESCs in ESCs scaffold showed intact cellular structures and were tightly attached to the scaffold after 3 days of culture. The cells were interconnected and fully extended on the surface of the scaffold to form a membrane. After 3 days of culture, the protein expression level of CK19 of ESCs in PCL-CA nanofiber scaffold group was significantly higher than that in type Ⅳ collagen group (t=24.56, P < 0.01). After 7 days of culture, compared with those in type Ⅳ collagen group, there was no significant change in the proportion of PCNA positive cells of ESCs in PCL-CA nanofiber scaffold group, while the proportion of CK19 positive cells was higher. On PID 3, 7, 14, and 21, the percentages of wound areas of rats in ESCs scaffold group were (78.0±1.8)%, (40.9±2.0)%, (17.9±1.1)%, and (5.0±1.0)%, respectively, which were significantly lower than (84.2±1.9)%, (45.4±2.6)%, (21.8±1.7)%, and (10.1±1.1)% in blank control group (t=5.42, 3.09, 4.33, 7.58, P < 0.05 or P < 0.01) and (82.7±1.2)%, (44.8±2.0)%, (22.4±2.4)%, and (10.3±2.4)% in scaffold alone group (t=4.98, 3.11, 3.84, 4.57, P < 0.05 or P < 0.01), while the percentages of wound areas of rats between blank control group and scaffold alone group were similar (t=1.47, 0.39, 0.47, 0.22, P > 0.05). On PID 21, the layer of new skin at the wound edge of rats in each group was intact; compared with that in blank control group or scaffold alone group, the new skin tissue at the wound edge of rats in ESCs scaffold group had more orderly collagen arrangement; the scaffolds in the new skin at the wound edge of rats were completely degraded in ESCs scaffold group and scaffold alone group. On PID 21, the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in scaffold alone group were similar to those in blank control group (t=1.70, 1.94, 0.18, P > 0.05), while the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in ESCs scaffold group were significantly higher than those in scaffold alone group (t=13.31, 22.07, 20.71, P < 0.01).

Conclusions

PCL-CA nanofiber scaffolds can inhibit the differentiation of ESCs of rats without affecting their proliferation in vitro. ESCs scaffolds constructed through using PCL-CA nanofiber scaffolds as the carrier to culture ESCs of rats can significantly promote the wound healing of full-thickness skin defects in rats, and the mechanism may be related to the activation of Notch signaling pathway.

Keywords: Nanofibers, Wound healing, Cell differentiation, Epidermal stem cells, Notch signaling pathway

创面的迁延不愈极易引发感染、瘢痕增生等并发症, 也会使其转化成慢性创面。慢性创面修复是当前创面修复领域的一大难题。每年都有大量的医疗资源被用于治疗慢性创面[1], 如何加速创面修复, 避免创面因难以愈合转化成慢性创面是创面修复研究中的重要一环。成体干细胞在创面修复中应用广泛[2-4]。表皮干细胞（ESC）作为表皮层基底部的细胞参与了创面修复的过程, 利用移植ESC进行创面修复具有良好的临床应用前景[5-6]。单纯的细胞膜片由于机械强度低、难以保存等原因, 临床应用受限。目前, 已有研究者将人羊膜组织提取物、脱细胞真皮等生物材料作为载体应用于ESC的移植[7-8]；但是由于这些生物材料获取难度较高, 需要寻找更加廉价且易制备的生物材料。

采用静电纺丝技术制备纳米纤维支架价格低廉, 制备的支架因具有高比表面积及孔隙率、与天然ECM结构相似的三维网络结构, 在组织工程修复中被广泛应用, 具备体外模拟在体ESC微环境的潜力[9-12]。作为静电纺丝技术可选用的原材料, 聚己内酯与乙酸纤维素具有无毒害、生物相容性好等特性[13-15]。因此, 本研究制备以聚己内酯、乙酸纤维素为组分的聚己内酯-乙酸纤维素（PCL-CA）纳米纤维支架, 研究支架对ESC干细胞特性的影响, 并进一步研究利用制备的支架构建的ESC-纳米纤维支架复合物（下称ESC支架）在大鼠全层皮肤缺损创面修复中的作用及相关分子机制。

1. 材料与方法

本实验研究遵循空军军医大学动物实验伦理委员会和国家有关实验动物管理和使用的有关规定。

1.1. 动物及主要试剂与仪器来源

15只健康无特殊病原体级6~8周龄雄性SD大鼠（体重约200 g）、30只1~3 d龄健康无特殊病原体级SD大鼠（雌雄不明）均购自空军军医大学实验动物中心, 许可证号：SCXK（陕）2019-001。兔抗大鼠细胞角蛋白19（CK19）多克隆抗体、兔抗大鼠Notch1单克隆抗体、兔抗大鼠GAPDH单克隆抗体、辣根过氧化物酶（HRP）标记的山羊抗兔IgG抗体购自美国Proteintech公司, 兔抗大鼠Jagged1单克隆抗体、兔抗大鼠Hes1单克隆抗体、Alexa Fluor 488标记的山羊抗兔IgG抗体、花青素3标记的山羊抗小鼠IgG抗体购自美国CST公司, 小鼠抗大鼠增殖细胞核抗原（PCNA）单克隆抗体购自美国Abcam公司, KC培养基购自美国ScienCell公司, 聚己内酯、乙酸纤维素、中性蛋白酶、人胎盘Ⅳ型胶原购自美国Sigma-Aldrich公司。静电纺丝用LSP02-1B型可编程注射泵购自保定兰格恒流泵有限公司, JSM-6700F型扫描电子显微镜购自日本电子株式会社, Olympus FV1000型激光扫描共聚焦显微镜、BX53型光学显微镜购自日本奥林巴斯公司。

1.2. 大鼠原代ESC的分离培养及鉴定

取30只1~3 d龄SD大鼠的背部皮肤, 用剪刀分割为2.0 cm×0.5 cm左右条状皮片。加入配制好的1 g/L中性蛋白酶溶液, 浸没皮片, 置于4 ℃冰箱内过夜。次日取出皮片, 于37 ℃下复温30 min。分离皮片的表皮层、真皮层, 用PBS冲洗表皮层3次。剪碎表皮层, 加入2.5 g/L胰蛋白酶（含0.2 g/L乙二胺四乙酸）3~4 mL, 37 ℃下消化表皮层5 min, 用含体积分数10%胎牛血清的KC培养基中和胰蛋白酶, 充分振荡。消化液经200目滤网过滤2次, 收集滤液, 800×g离心5 min, 弃去上清液。用不含胎牛血清的KC培养基重新悬浮收集的细胞, 接种于包被有Ⅳ型胶原的培养皿中, 37 ℃下于体积分数5%二氧化碳培养箱（培养箱条件下同）中培养15 min后弃去悬浮细胞, 理论上剩余贴壁细胞即为ESC[16-17]。取原代细胞, 用流式细胞仪检测ESC特异性标志物整合素β1表达, 用免疫荧光法检测ESC特异性标志物CK19的表达, 结果二者均为阳性。使用KC培养基培养原代细胞, 待细胞生长至70%~80%融合时, 以1∶2比例进行传代, 常规培养至第1代细胞, 用于后续实验。

1.3. PCL-CA纳米纤维支架的制备及表征观测

将8 g聚己内酯溶解于100 g以2∶1质量比配制的二氯甲烷和N, N-二甲基甲酰胺混合溶液中, 制备质量分数为8%的聚己内酯溶液。将14 g乙酸纤维素溶解于100 g以2∶1质量比配制的丙酮和N, N-二甲基乙酰胺混合溶液中, 制备质量分数为14%的乙酸纤维素溶液。以2∶1质量比混合质量分数为8%的聚己内酯溶液和质量分数为14%的乙酸纤维素溶液, 获得PCL-CA混合溶液。利用可编程注射泵在电压15 kV、距离接受平面10 cm、流速0.25 mL/h、电纺时间1 h的条件下制备PCL-CA纳米纤维支架。将制备的支架喷金后, 于2 000、15 000倍扫描电子显微镜下观察支架拓扑结构, 用ImageJ软件（美国国立卫生研究院）处理图片并计算支架中纤维直径大小, 测算样本量为25。制备的PCL-CA纳米纤维支架经空军军医大学第二附属医院供应科环氧乙烷消毒灭菌后用于后续实验。

1.4. ESC支架的构建及表征观察

将PCL-CA纳米纤维支架剪裁为稍大于100 mm培养皿形状后, 按1×105个/cm2接种ESC, 使用KC培养基, 于培养箱中培养构建ESC支架, 培养3 d, 于500、1 500倍扫描电子显微镜下观察支架中细胞形态及其与支架的关系。

1.5. PCL-CA纳米纤维支架对ESC分化和增殖的影响

1.5.1. ESC中CK19蛋白表达

采用蛋白质印迹法检测。同1.4构建ESC支架（样本数为3）, 以其中的ESC作为PCL-CA纳米纤维支架组；另取ESC, 按1×105个/cm2接种于用Ⅳ型胶原包被的3个培养皿中用KC培养基培养, 设为Ⅳ型胶原组。培养3 d, 提取2组细胞总蛋白, 使用蛋白质定量试剂盒进行定量。取25 μg总蛋白样品, 行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳, 湿法转膜, 用50 g/L脱脂奶粉溶液封闭30 min。分别加入兔抗大鼠CK19多克隆一抗、兔抗大鼠GAPDH单克隆一抗（稀释比均为1∶1 000）, 4 ℃孵育过夜。加入HRP标记的山羊抗兔IgG二抗（稀释比为1∶2 000）, 室温孵育60 min。增强化学发光法显色, 采用ImageJ软件处理图像。以GAPDH为内参照, 以目的条带灰度值与GAPDH灰度值的比值为目的蛋白的相对表达水平。本实验重复3次, 结果取均值。

1.5.2. ESC中PCNA及CK19蛋白表达

采用免疫荧光法检测。将PCL-CA纳米纤维支架剪裁为稍大于24孔板孔格形状后, 同1.4接种ESC、培养构建ESC支架, 以其中的ESC作为PCL-CA纳米纤维支架组；另取ESC, 以1×105个/cm2接种于用Ⅳ型胶原包被的24孔板的3孔中, 用KC培养基培养, 设为Ⅳ型胶原组。培养7 d, 取2组细胞固定后, 采用山羊血清封闭15 min。加入小鼠抗大鼠PCNA单克隆一抗、兔抗大鼠CK19多克隆一抗（稀释比均为1∶500）, 4 ℃孵育过夜。加入花青素3标记的山羊抗小鼠IgG二抗、Alexa Fluor 488标记的山羊抗兔IgG二抗（稀释比均为1∶1 000）, 室温孵育1 h。4', 6-二脒基-2-苯基吲哚共染后, 使用抗荧光淬灭剂封片。在200倍激光扫描共聚焦显微镜下观察2组ESC中PCNA和CK19蛋白阳性表达情况, PCNA阳性染色为绿色, CK19阳性染色为红色。

1.6. ESC支架对大鼠全层皮肤缺损创面愈合的影响

1.6.1. ESC支架构建及大鼠分组处理

将PCL-CA纳米纤维支架剪裁为直径30 mm的圆形, 按1×107个/cm2接种ESC, 使用KC培养基, 于培养箱中培养3 d构建ESC支架, 用于后续移植。将15只6~8周龄SD大鼠按随机数字表法分为空白对照组、单纯支架组和ESC支架组, 每组5只。大鼠均以10 g/L戊巴比妥钠按50 mg/kg腹腔注射麻醉, 于每只大鼠背部左右两侧各制备1个直径约为2 cm的圆形切口, 进行深至肌筋膜的全层皮肤切除, 获得全层皮肤缺损创面模型。伤后即刻, 空白对照组大鼠创面不行植入处理, 加压包扎；单纯支架组大鼠创面植入PCL-CA纳米纤维支架后加压包扎；ESC支架组大鼠创面植入ESC支架后加压包扎。

1.6.2. 创面面积百分率

于伤后0（即刻）、3、7、14、21 d, 对各组大鼠选定的1个创面进行拍照, 伤后3 d打开包扎后去除, 计算伤后3、7、14、21 d创面面积百分率, 创面面积百分率=当前时间点创面面积÷伤后0 d时创面面积×100%。

1.6.3. 创面愈合质量

伤后21 d, 取各组大鼠拍照后创面部分创缘新生皮肤组织行Masson染色, 在40倍光学显微镜下观察皮肤层次、胶原纤维排列和纳米纤维支架降解情况, 以评估创面愈合质量。胶原纤维、黏液、软骨呈蓝色, 细胞质、纤维素、神经胶质呈红色, 细胞核呈黑蓝色。

1.6.4. 新生皮肤组织中Notch1、Jagged1、Hes1蛋白表达

伤后21 d, 每组取3只大鼠, 取大鼠拍照后创面部分创缘新生皮肤组织, 每只大鼠1个样本。提取组织蛋白, 同1.5.1采用蛋白质印迹法检测Notch1、Jagged1、Hes1蛋白表达。其中一抗为兔抗大鼠Notch1单克隆抗体、兔抗大鼠Jagged1单克隆抗体、兔抗大鼠Hes1单克隆抗体、兔抗大鼠GAPDH单克隆抗体（稀释比均为1∶1 000）, 二抗为HRP标记的山羊抗兔IgG抗体（稀释比为1∶2 000）, 结果处理同1.5.1。

1.7. 统计学处理

采用SPSS 18.0、Prism GraphPad 8.0统计软件进行数据分析。计量资料数据均符合正态分布, 以x ± s表示, 2组间比较行独立样本t检验, 单一时间点多组间总体比较行单因素方差分析, 多个时间点多组间总体比较行重复测量方差分析, 组间多重比较行Bonferroni校正。P < 0.05为差异有统计学意义。

2. 结果

2.1. PCL-CA纳米纤维支架的表征

制备的PCL-CA纳米纤维支架具有疏松多孔的网格状多层立体结构, 其中的纤维表面光滑无孔隙, 见图 1。制备的PCL-CA纳米纤维支架中纤维直径为（383±24）nm。

图 1.

图 1

Open in a new tab

聚己内酯-乙酸纤维素纳米纤维支架结构观察。1A.整体形貌观察可见支架具有疏松多孔的网格状多层立体结构  扫描电子显微镜×2 000, 图中标尺为10 μm；1B.表面形貌观察可见支架各纤维直径相仿, 纤维表面光滑无孔隙扫描电子显微镜×15 000, 图中标尺为1 μm

2.2. ESC支架的表征

培养3 d, ESC支架中的ESC具有完整的细胞结构且与支架紧密贴合, 细胞间相互连接, 细胞充分伸展在纳米纤维支架表面形成膜片, 见图 2。

图 2.

图 2

Open in a new tab

表皮干细胞（ESC）-纳米纤维支架复合物中大鼠ESC形态及其与支架的关系观察。2A.细胞充分伸展在聚己内酯-乙酸纤维素纳米纤维支架表面形成膜片  扫描电子显微镜×500, 图中标尺为50 μm；2B.细胞间相互连接, 细胞具有完整的结构且与支架紧密贴合  扫描电子显微镜×1 500, 图中标尺为10 μm

2.3. ESC中CK19与PCNA蛋白表达

蛋白质印迹法检测结果显示, 培养3 d, Ⅳ型胶原组ESC中CK19的蛋白表达水平为1.02±0.03, 明显低于PCL-CA纳米纤维支架组的1.71±0.04（t=24.56, P < 0.001）, 见图 3。免疫荧光法检测结果显示, 培养7 d, 与Ⅳ型胶原组相比, PCL-CA纳米纤维支架组ESC中PCNA表达阳性细胞比例无明显变化, CK19表达阳性细胞比例更高, 见图 4。

图 3.

蛋白质印迹法检测2组大鼠表皮干细胞培养3 d CK19蛋白表达

注：CK19为细胞角蛋白19, GAPDH为3-磷酸甘油醛脱氢酶；1.Ⅳ型胶原组, 2.聚己内酯-乙酸纤维素纳米纤维支架组

图 3

Open in a new tab

图 4.

2组大鼠表皮干细胞（ESC）培养7 d后CK19与PCNA的蛋白表达  花青素3-Alexa Fluor 488-4', 6-二脒基-2-苯基吲哚×200, 图中标尺为50 μm。4A、4B、4C、4D.分别为Ⅳ型胶原组细胞核染色、CK19染色、PCNA染色、细胞核及CK19与PCNA染色重叠图片, 细胞核完整, CK19表达少, PCNA表达明显；4E、4F、4G、4H.分别为聚己内酯-乙酸纤维素纳米纤维支架组细胞核染色、CK19染色、PCNA染色、细胞核及CK19与PCNA染色重叠图片, 细胞核完整, CK19表达明显多于图 4B, PCNA表达与图 4C相似

注：细胞核阳性染色为蓝色, 细胞角蛋白19（CK19）阳性染色为红色, 增殖细胞核抗原（PCNA）阳性染色为绿色

图 4

Open in a new tab

2.4. 大鼠创面面积百分率及愈合质量

伤后3、7、14、21 d, 单纯支架组与空白对照组大鼠创面面积百分率相近（P > 0.05）, ESC支架组大鼠创面面积百分率明显小于空白对照组和单纯支架组（P < 0.05或P < 0.01）, 见图 5、表 1。

图 5.

3组全层皮肤缺损创面大鼠伤后各时间点创面情况。5A、5B、5C.分别为空白对照组、单纯支架组、ESC支架组伤后0 d创面情况, 大小均为直径约2 cm圆形；5D、5E、5F.分别为空白对照组、单纯支架组、ESC支架组伤后7 d创面情况, 图 5E与图 5D相近, 图 5F创面面积较图 5D、5E明显缩小；5G、5H、5I.分别为空白对照组、单纯支架组、ESC支架组伤后21 d创面情况, 图 5H与图 5G相近, 图 5I创面面积较图 5G、5H显著缩小

注：单纯支架组移植聚己内酯-乙酸纤维素纳米纤维支架, 表皮干细胞（ESC）支架组移植以前述支架为载体培养ESC 3 d构建的ESC-纳米纤维支架复合物

图 5

Open in a new tab

表 1.

3组全层皮肤缺损创面大鼠伤后各时间点创面面积百分率比较（%, x ± s）

组别 创面数（个） 3 d 7 d 14 d 21 d
注：单纯支架组移植聚己内酯-乙酸纤维素纳米纤维支架, 表皮干细胞（ESC）支架组移植以前述支架为载体培养ESC构建的ESC-纳米纤维支架复合物；处理因素主效应, F=52.76, P < 0.001；时间因素主效应, F=8 190.00, P < 0.001；两者交互作用, F=3.22, P=0.005；t1值、P1 值, t2值、P2 值分别为单纯支架组、ESC支架组与空白对照组各时间点比较所得；t3值、P3值为ESC支架组与单纯支架组各时间点比较所得
空白对照组 5 84.2±1.9 45.4±2.6 21.8±1.7 10.1±1.1
单纯支架组 5 82.7±1.2 44.8±2.0 22.4±2.4 10.3±2.4
ESC支架组 5 78.0±1.8 40.9±2.0 17.9±1.1 5.0±1.0
t1值 1.47 0.39 0.47 0.22
P1值 0.554 0.999 0.999 0.999
t2值 5.42 3.09 4.33 7.58
P2值 0.002 0.049 0.012 < 0.001
t3值 4.98 3.11 3.84 4.57
P3值 0.005 0.043 0.030 0.015
Open in a new tab

伤后21 d, 各组大鼠创缘新生皮肤层次完整；与空白对照组、单纯支架组相比, ESC支架组大鼠创缘新生皮肤组织表现出更加整齐的胶原纤维排列；ESC支架组与单纯支架组大鼠创缘新生皮肤组织中均未见支架残留, 支架完全降解。见图 6。

图 6.

3组全层皮肤缺损创面大鼠伤后21 d创缘新生皮肤完整性、胶原排列情况及支架移植组支架降解情况  Masson×40, 图中标尺为100 μm。6A.空白对照组皮肤层次完整, 胶原纤维排列混乱；6B.单纯支架组皮肤层次完整, 胶原纤维排列混乱, 未见支架残留；6C.ESC支架组皮肤层次完整, 胶原纤维排列较图 6A、6B整齐有序, 未见支架残留

注：单纯支架组移植聚己内酯-乙酸纤维素纳米纤维支架, 表皮干细胞（ESC）支架组移植以前述支架为载体培养ESC 3 d构建的ESC-纳米纤维支架复合物

图 6

Open in a new tab

2.5. 大鼠创缘新生皮肤组织中Notch1、Jagged1、Hes1蛋白表达

伤后21 d, 3组大鼠创缘新生皮肤组织中Notch1、Jagged1、Hes1蛋白表达水平总体比较, 差异均有统计学意义（F=65.01、47.22、33.28, P < 0.001）。单纯支架组大鼠创缘新生皮肤组织中的Notch1、Jagged1、Hes1蛋白表达水平与空白对照组相比无明显差异（t=1.70、1.94、0.18, P=0.762, 0.933, 0.813）, ESC支架组大鼠创缘新生皮肤组织中的Notch1、Jagged1、Hes1蛋白表达水平显著高于单纯支架组（t=13.31、22.07、20.71, P < 0.001）。见图 7。

图 7.

蛋白质印迹法检测3组全层皮肤缺损创面大鼠伤后21 d创缘新生皮肤组织中Notch1、Jagged1、Hes1蛋白表达。7A.条带图；7B.条形图（x ± s, 样本数为3）

注：GAPDH为3-磷酸甘油醛脱氢酶；条带图中1、2、3分别为空白对照组、单纯支架组、表皮干细胞（ESC）支架组；单纯支架组移植聚己内酯-乙酸纤维素纳米纤维支架, ESC支架组移植以前述支架为载体培养ESC 3 d构建的ESC-纳米纤维支架复合物；与单纯支架组比较, aP < 0.01

图 7

Open in a new tab

3. 讨论

ESC的干细胞特性与其移植后对创面修复的效果息息相关, 因此作为ESC移植载体的支架能否维持ESC干细胞特性将影响ESC支架移植于创面后对创面的修复质量[18-20]。细胞外微环境的变化影响着ESC干细胞特性的维持[21-24]。虽然采用静电纺丝技术制备的纳米纤维支架具有体外模拟在体ESC干细胞龛微环境的潜力, 但该类支架能否作为ESC移植载体支架、用作载体支架后能否对ESC干细胞特性起维持作用尚未被阐明。本研究团队通过对构建的PCL-CA纳米纤维支架进行表征观察了解到, 支架具有疏松多孔的复层网状结构, 纤维直径为（383±24）nm, 与ECM中网状结构纤维直径相仿, 高度模拟了细胞外微环境, 具有成为ESC移植载体支架的潜力。随后本研究团队使用制备的PCL-CA纳米纤维支架作为载体在体外培养ESC一定时间后构建了ESC支架, 扫描电子显微镜拍摄的图片显示细胞与支架之间贴合紧密, 细胞间相互连接, 说明可以利用PCL-CA纳米纤维支架作为移植载体, 构建ESC支架。

采用静电纺丝技术制备的纳米纤维支架因自身制备组分、支架表面拓扑结构的不同, 对不同种类干细胞的分化调控作用不尽相同[25-33]。本研究的体外细胞实验中, 通过蛋白质印迹法和免疫荧光法实验观察到, PCL-CA纳米纤维支架组ESC中干细胞特异性标志物CK19表达水平相比于Ⅳ型胶原组培养皿环境培养更高, PCNA的表达水平相比于Ⅳ型胶原组无明显变化。该结果说明制备的PCL-CA纳米纤维支架抑制了ESC的分化, 能够维持其干细胞特性, 且不会影响ESC的增殖潜力, 从而不会影响构建的ESC支架的在体应用。

既往研究表明, 利用纳米纤维支架负载细胞修复创面效果显著[34-35]。本研究团队通过在体实验研究利用PCL-CA纳米纤维支架作为载体培养ESC构建的ESC支架对大鼠全层皮肤缺损创面愈合的影响。大体观察及Masson染色显示, 在伤后相同时间点, 与空白对照组和单纯支架组相比, ESC支架组大鼠创面面积更小；伤后21 d时, ESC支架组大鼠创缘新生皮肤组织中胶原排列较其余2组更整齐且支架被完全降解。以上结果显示, 负载ESC的PCL-CA纳米纤维支架能够促进大鼠全层皮肤缺损创面的愈合。

移植异体ESC在伤口愈合过程中促进愈合的机制涉及多种信号通路[36]。Notch信号通路的激活在皮肤组织中能促进基底层ESC分化成为上层表皮细胞继而参与创面修复和皮肤自更新[37]。Notch和Wnt信号通路已被证实参与调控伤口愈合过程, 激活Notch通路可以显著促进创面愈合[38-39]。本研究团队利用蛋白质印迹法对伤后21 d各组大鼠创缘新生皮肤组织中Notch信号通路关键蛋白的表达水平进行检测, 结果显示, 单纯支架组中受体蛋白Notch1、配体蛋白Jagged1和效应靶分子蛋白Hes1这3个Notch信号通路关键蛋白的相对表达水平与空白对照组相近, 而ESC支架组中这3个Notch信号通路关键蛋白的相对表达水平显著高于单纯支架组。以上结果说明ESC支架可以通过激活创面组织中的Notch通路促进创面的愈合, 但本研究未阐明ESC支架是如何激活组织中的Notch通路的, 此外, 本研究未对异体ESC植入体内后是否发生分化、分化是否在促进创面修复中发挥作用进行深入研究, 这些将是本研究团队后续的研究目标。

综上所述, 本研究利用静电纺丝技术, 以对人体安全无害的、具有良好生物相容性与生物降解性的PCL-CA为原材料构建了纳米纤维支架；通过体外构建ESC支架实验了解到PCL-CA纳米纤维支架作为ESC支架构建的载体, 具有抑制ESC体外分化作用且对ESC的增殖潜力无影响；通过在体实验了解到利用ESC支架能够显著促进大鼠全层皮肤缺损创面的愈合, 其机制可能与Notch信号通路的激活有关。

Funding Statement

国家自然科学基金面上项目（81671929）

General Program of National Natural Science Foundation of China (81671929)

Footnotes

利益冲突  所有作者均声明不存在利益冲突

References

  • 1.Izadi K, Ganchi P. Chronic wounds. Clin Plast Surg. 2005;32(2):209–222. doi: 10.1016/j.cps.2004.11.011. [DOI] [PubMed] [Google Scholar]
  • 2.Xie WG, Zhou XQ, Hu WG, et al. Pterostilbene accelerates wound healing by modulating diabetes-induced estrogen receptor β suppression in hematopoietic stem cells[J/OL]. Burns Trauma, 2021, 9: tkaa045[2021-01-04]. https://pubmed.ncbi.nlm.nih.gov/33654697/. DOI: 10.1093/burnst/tkaa045.
  • 3.Fujiwara O, Prasai A, Perez-Bello D, et al. Adipose-derived stem cells improve grafted burn wound healing by promoting wound bed blood flow[J/OL]. Burns Trauma, 2020, 8: tkaa009[2021-01-04]. https://pubmed.ncbi.nlm.nih.gov/32346539/. DOI: 10.1093/burnst/tkaa009.
  • 4.Hu P, Yang QX, Wang Q, et al. Mesenchymal stromal cells-exosomes: a promising cell-free therapeutic tool for wound healing and cutaneous regeneration[J/OL]. Burns Trauma, 2019, 7: 38[2021-01-04]. https://pubmed.ncbi.nlm.nih.gov/31890717/. DOI: 10.1186/s41038-019-0178-8.
  • 5.Jackson CJ, Tønseth KA, Utheim TP. Cultured epidermal stem cells in regenerative medicine. Stem Cell Res Ther. 2017;8(1):155. doi: 10.1186/s13287-017-0587-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Huang SB, Hu ZC, Wang P, et al. Rat epidermal stem cells promote the angiogenesis of full-thickness wounds. Stem Cell Res Ther. 2020;11(1):344. doi: 10.1186/s13287-020-01844-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ji SZ, Xiao SC, Luo PF, et al. An epidermal stem cells niche microenvironment created by engineered human amniotic membrane. Biomaterials. 2011;32(31):7801–7811. doi: 10.1016/j.biomaterials.2011.06.076. [DOI] [PubMed] [Google Scholar]
  • 8.赵 跃华, 张 永红, 林 凯. 人表皮干细胞复合裸鼠脱细胞真皮支架制备的人工皮肤. 中国组织工程研究. 2016;20(25):3771–3777. doi: 10.3969/j.issn.2095-4344.2016.25.019. [DOI] [Google Scholar]
  • 9.Xue JJ, Wu T, Dai YQ, et al. Electrospinning and electrospun nanofibers: methods, materials, and applications. Chem Rev. 2019;119(8):5298–5415. doi: 10.1021/acs.chemrev.8b00593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Madl CM, Heilshorn SC. Engineering hydrogel microenvironments to recapitulate the stem cell niche. Annu Rev Biomed Eng. 2018;20:21–47. doi: 10.1146/annurev-bioeng-062117-120954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Jin GR, Prabhakaran MP, Ramakrishna S. Stem cell differentiation to epidermal lineages on electrospun nanofibrous substrates for skin tissue engineering. Acta Biomater. 2011;7(8):3113–3122. doi: 10.1016/j.actbio.2011.04.017. [DOI] [PubMed] [Google Scholar]
  • 12.Rho KS, Jeong L, Lee G, et al. Electrospinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing. Biomaterials. 2006;27(8):1452–1461. doi: 10.1016/j.biomaterials.2005.08.004. [DOI] [PubMed] [Google Scholar]
  • 13.Naseri-Nosar M, Salehi M, Hojjati-Emami S. Cellulose acetate/poly lactic acid coaxial wet-electrospun scaffold containing citalopram-loaded gelatin nanocarriers for neural tissue engineering applications. Int J Biol Macromol. 2017;103:701–708. doi: 10.1016/j.ijbiomac.2017.05.054. [DOI] [PubMed] [Google Scholar]
  • 14.El Khatib M, Mauro A, Di Mattia M, et al. Electrospun PLGA fiber diameter and alignment of tendon biomimetic fleece potentiate tenogenic differentiation and immunomodulatory function of amniotic epithelial stem cells. Cells. 2020;9(5):1207. doi: 10.3390/cells9051207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Heidari M, Bahrami SH, Ranjbar-Mohammadi M, et al. Smart electrospun nanofibers containing PCL/gelatin/graphene oxide for application in nerve tissue engineering. Mater Sci Eng C Mater Biol Appl. 2019;103:109768. doi: 10.1016/j.msec.2019.109768. [DOI] [PubMed] [Google Scholar]
  • 16.Wang P, Shu B, Xu YB, et al. Basic fibroblast growth factor reduces scar by inhibiting the differentiation of epidermal stem cells to myofibroblasts via the Notch1/Jagged1 pathway. Stem Cell Res Ther. 2017;8(1):114. doi: 10.1186/s13287-017-0549-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Guo AH, Jahoda CAB. An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor cells. Exp Dermatol. 2009;18(8):720–726. doi: 10.1111/j.1600-0625.2009.00900.x. [DOI] [PubMed] [Google Scholar]
  • 18.Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med. 2014;6(265):265sr6. doi: 10.1126/scitranslmed.3009337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Pellegrini G, Ranno R, Stracuzzi G, et al. The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation. 1999;68(6):868–879. doi: 10.1097/00007890-199909270-00021. [DOI] [PubMed] [Google Scholar]
  • 20.Wang YW, Beekman J, Hew J, et al. Burn injury: challenges and advances in burn wound healing, infection, pain and scarring. Adv Drug Deliv Rev. 2018;123:3–17. doi: 10.1016/j.addr.2017.09.018. [DOI] [PubMed] [Google Scholar]
  • 21.Ghadially R. 25 years of epidermal stem cell research. J Invest Dermatol. 2012;132(3 Pt 2):797–810. doi: 10.1038/jid.2011.434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Chermnykh E, Kalabusheva E, Vorotelyak E. Extracellular matrix as a regulator of epidermal stem cell fate. Int J Mol Sci. 2018;19(4):1003. doi: 10.3390/ijms19041003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Nel AE, Mädler L, Velegol D, et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009;8(7):543–557. doi: 10.1038/nmat2442. [DOI] [PubMed] [Google Scholar]
  • 24.Kucharzewski M, Rojczyk E, Wilemska-Kucharzewska K, et al. Novel trends in application of stem cells in skin wound healing. Eur J Pharmacol. 2019;843:307–315. doi: 10.1016/j.ejphar.2018.12.012. [DOI] [PubMed] [Google Scholar]
  • 25.Zhang YS, Yao JJ. Imaging biomaterial-tissue interactions. Trends Biotechnol. 2018;36(4):403–414. doi: 10.1016/j.tibtech.2017.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Zhang C, Xie B, Zou YJ, et al. Zero-dimensional, one-dimensional, two-dimensional and three-dimensional biomaterials for cell fate regulation. Adv Drug Deliv Rev. 2018;132:33–56. doi: 10.1016/j.addr.2018.06.020. [DOI] [PubMed] [Google Scholar]
  • 27.Othman Z, Cillero Pastor B, van Rijt S, et al. Understanding interactions between biomaterials and biological systems using proteomics. Biomaterials. 2018;167:191–204. doi: 10.1016/j.biomaterials.2018.03.020. [DOI] [PubMed] [Google Scholar]
  • 28.Huang R, Li WZ, Lv XX, et al. Biomimetic LBL structured nanofibrous matrices assembled by chitosan/collagen for promoting wound healing. Biomaterials. 2015;53:58–75. doi: 10.1016/j.biomaterials.2015.02.076. [DOI] [PubMed] [Google Scholar]
  • 29.Bhowmick S, Scharnweber D, Koul V. Co-cultivation of keratinocyte-human mesenchymal stem cell (hMSC) on sericin loaded electrospun nanofibrous composite scaffold (cationic gelatin/hyaluronan/chondroitin sulfate) stimulates epithelial differentiation in hMSCs: in vitro study. Biomaterials. 2016;88:83–96. doi: 10.1016/j.biomaterials.2016.02.034. [DOI] [PubMed] [Google Scholar]
  • 30.Zhang C, Yuan HH, Liu HH, et al. Well-aligned chitosan-based ultrafine fibers committed teno-lineage differentiation of human induced pluripotent stem cells for Achilles tendon regeneration. Biomaterials. 2015;53:716–730. doi: 10.1016/j.biomaterials.2015.02.051. [DOI] [PubMed] [Google Scholar]
  • 31.Chen HL, Huang XB, Zhang MM, et al. Tailoring surface nanoroughness of electrospun scaffolds for skeletal tissue engineering. Acta Biomater. 2017;59:82–93. doi: 10.1016/j.actbio.2017.07.003. [DOI] [PubMed] [Google Scholar]
  • 32.Tarassoli SP, Jessop ZM, Al-Sabah A, et al. Skin tissue engineering using 3D bioprinting: an evolving research field. J Plast Reconstr Aesthet Surg. 2018;71(5):615–623. doi: 10.1016/j.bjps.2017.12.006. [DOI] [PubMed] [Google Scholar]
  • 33.Stejskalová A, Almquist BD. Using biomaterials to rewire the process of wound repair. Biomater Sci. 2017;5(8):1421–1434. doi: 10.1039/c7bm00295e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Chogan F, Mirmajidi T, Rezayan AH, et al. Design, fabrication, and optimization of a dual function three-layer scaffold for controlled release of metformin hydrochloride to alleviate fibrosis and accelerate wound healing. Acta Biomater. 2020;113:144–163. doi: 10.1016/j.actbio.2020.06.031. [DOI] [PubMed] [Google Scholar]
  • 35.Ding S, Xu Y, Yan X, et al. Effect of collagen scaffold with Bcl-2-modified adipose-derived stem cells on diabetic mice wound healing. Int J Low Extrem Wounds. 2020;19(2):139–147. doi: 10.1177/1534734619880055. [DOI] [PubMed] [Google Scholar]
  • 36.Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 2003;83(3):835–870. doi: 10.1152/physrev.2003.83.3.835. [DOI] [PubMed] [Google Scholar]
  • 37.Rangarajan A, Talora C, Okuyama R, et al. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. EMBO J. 2001;20(13):3427–3436. doi: 10.1093/emboj/20.13.3427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Yang RH, Qi SH, Shu B, et al. Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway. Biosci Rep. 2016;36(4):e00364. doi: 10.1042/BSR20160034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Shi Y, Shu B, Yang RH, et al. Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately. Stem Cell Res Ther. 2015;6(1):120. doi: 10.1186/s13287-015-0103-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Chinese Journal of Burns are provided here courtesy of Chinese Medical Association

RESOURCES