S1 The improvement of chirurgical incision healing using scaffold prepared by the fiber drawing method in a rabbit model

Dataset for the publication: Figure 1. Preparation of the scaffolds for in vitro and in vivo testing. The principle of the drawing method lies in pulling a single fibre from a droplet of polymer solution (A). For in vitro testing, fibers were fixed on inserts fitting in wells of 24-well plate in three directions (B). The scaffold for in vivo testing consisted of a nanofibrous frame on which drawn fibers were fixed in three dimensions (C). Figure 2. PCL fibers drawn in three dimensions visualised by scanning electron microscopy. Magnification 100✕, scale bar 500 μm (A), magnification 500✕, scale bar 100 μm (B) Figure 3. In vitro test of drawn fibres biocompatibility. Cell metabolic activity of 3T3 fibroblasts cultured on the PCL microfibers increased during the experiment (A) (p<0.05, p<0.001 is indicated by *). 3T3 fibroblasts adhered on fibres were visualised using SEM on day 21 (B; magnification 500✕, scale bar 100 μm). The cell distribution was detected using DAPI staining and visualisation using fluorescent microscopy (C, D, E; magnification 10✕, scale bar 100 µm). It is visible that cell number increased on day 7 (D) and 21 (E) compared to day 1 (C). Figure 4. In vivo test composite scaffold. The incision in linea alba was sutured (A) and left without any further treatment in the control group. Alternatively, the scaffold was applied on the incision (B) and fixed to the abdominal wall (C). The abdominal wall with implanted scaffold (D) or without (E) was explanted after 6 weeks and examined from the biomechanical and histological point of view. Figure 5. Average values of the elasticity in tension and maximal stress of the non-treated incision and the incision treated with scaffold 6 weeks after surgery. The maximal stress of the tore sample was on the same level but the elasticity in traction of the incision treated with the scaffold reached a significantly higher value. The level of statistical significance for the assays is designated above the mean values (p<0.05). Figure 6. The α-smooth muscle actin immunohistochemical evaluation of the samples and visualisation of the type I collagen stained with the picrosirius red in the polarised light. (A, E) Incision in the tissue samples uncovered with the scaffold. (B, F) The surrounding area uncovered with the scaffold. (C, G) Incision in the tissue samples covered with the scaffold. (D, H) The surrounding area covered with the scaffold. The incision covered with the scaffold (C) contained comparable amount of myofibroblasts as the samples with no covering (A). The surrounding area without scaffold covering contained a very few myofibroblasts (B). Wounds covered with the scaffold showed a more regular distribution of type I collagen (G, H) compared to uncovered defect (E, F). (A, B, C, D) scale bar 100 µm; (E, F, G, H) scale bar 200 µm. Figure 7. Evaluation of microvessels within the samples using CD31 immunohistochemistry and overall trichrome staining of the samples. (A, E) Incision in the tissue samples uncovered with the scaffold. (B, F) The surrounding area uncovered with the scaffold. (C, G) Incision in the tissue samples covered with the scaffold. (D, H) The surrounding area covered with the scaffold. The density of microvessels in the incision was comparable in the samples with (C) and without scaffold (A). The surrounding area covered with scaffold (D) contained more microvessels than the samples without covering (B). The tissue of the incision was less organized and contained adipose tissue (E, F). Wounds supported with scaffolds showed good integration of the scaffold both with granular connective tissue and dense collagenous connective tissue (G, H). Scale bar 100 µm. Figure 8. Paired comparison of the samples covered and uncovered with scaffold. Left column - differences between the healing incision treated with scaffold or without the treatment. The incision treated with scaffold contained greater fraction of type I collagen (A). (A) Fraction of type I collagen in the healing incision. (B) Fraction of actine-positive myofibroblasts (actin) in the healing incision. (C) Density of microvessels in the healing incision. Right column - differences between the healing surrounding areas covered with scaffold or without covering. The tissue of the surrounding area covered with scaffold had a greater fraction of myofibroblasts (B). (A) Fraction of type I collagen in the healing surrounding area. (B) Fraction of myofibroblasts (actin) in the healing surrounding area. (C) Fraction of microvessels in the healing surrounding area. Figure 9. Paired comparison of the healing incision and the surrounding. Left Column - all the samples were covered with the scaffold. The incisions contained greater fraction of type I collagen (A) and higher density of microvessels (C). (A) The quantitative histological evaluation of the content of type I collagen. (B) The quantitative histological evaluation of the content of myofibroblasts (actin). (C) The quantitative histological evaluation of the microvessel density. Right column - all the samples were uncovered with the scaffold. The incisions contained greater fraction of type I collagen (A), greater fraction of myofibroblasts (B) and higher density of microvessels (C). (A) The quantitative histological evaluation of the content of type I collagen. (B) The quantitative histological evaluation of the fraction of myofibroblasts. (C) The quantitative histological evaluation of the microvessel density.本数据集配套发表文章： 图1 体外与体内测试用支架的制备流程。拉伸法的核心原理为从聚合物溶液液滴中拉制单根纤维（图A）。针对体外测试，将纤维以三维方向固定于适配24孔板孔位的插入式支架上（图B）。体内测试用支架由纳米纤维框架构成，拉制纤维以三维形式固定于该框架之上（图C）。 图2 经三维拉制的聚己内酯（PCL）纤维，通过扫描电子显微镜（SEM）成像。放大倍数100×，标尺为500 μm（图A）；放大倍数500×，标尺为100 μm（图B）。 图3 拉制纤维的体外生物相容性测试。实验期间，培养于PCL微纤维上的3T3成纤维细胞的代谢活性逐步升高（图A），*标注对应p<0.05、p<0.001的统计学显著性水平。于第21天通过扫描电子显微镜观察到黏附于纤维表面的3T3成纤维细胞（图B；放大倍数500×，标尺为100 μm）。通过4',6-二脒基-2-苯基吲哚（DAPI）染色结合荧光显微镜成像检测细胞分布（图C、D、E；放大倍数10×，标尺为100 μm）。可见相较于第1天（图C），第7天（图D）与第21天（图E）的细胞数量均显著增加。 图4 复合支架的体内测试。对腹白线切口进行缝合（图A），对照组不施加任何额外干预措施。实验组则将支架覆盖于切口处（图B）并固定于腹壁（图C）。术后6周，将植入支架（图D）与未植入支架（图E）的腹壁组织取出，分别从生物力学与组织学维度进行检测分析。 图5 未处理切口与支架处理切口术后6周的拉伸弹性及最大应力平均值。撕裂样本的最大应力处于相近水平，但支架处理切口的拉伸弹性显著更高。检测的统计学显著性水平标注于均值上方（p<0.05）。 图6 样本的α-平滑肌肌动蛋白（α-SMA）免疫组织化学评估，以及偏振光下天狼星红染色的I型胶原成像结果。（A、E）未覆盖支架的组织样本切口区域。（B、F）未覆盖支架的组织样本周围区域。（C、G）覆盖支架的组织样本切口区域。（D、H）覆盖支架的组织样本周围区域。覆盖支架的切口区域（图C）的肌成纤维细胞数量与未覆盖支架的样本（图A）相当。未覆盖支架的周围区域仅含极少量肌成纤维细胞（图B）。相较于未覆盖缺损区域（图E、F），覆盖支架的伤口的I型胶原分布更为规整（图G、H）。（A、B、C、D）标尺为100 μm；（E、F、G、H）标尺为200 μm。 图7 采用CD31免疫组织化学染色评估样本内微血管密度，以及对样本进行整体三色染色分析。（A、E）未覆盖支架的组织样本切口区域。（B、F）未覆盖支架的组织样本周围区域。（C、G）覆盖支架的组织样本切口区域。（D、H）覆盖支架的组织样本周围区域。切口区域的微血管密度在覆盖支架（图C）与未覆盖支架的样本中无显著差异。覆盖支架的周围区域（图D）的微血管数量多于未覆盖支架的样本（图B）。未覆盖支架的切口组织排列较为紊乱，且包含脂肪组织（图E、F）。经支架支持的伤口可实现支架与肉芽结缔组织及致密胶原结缔组织的良好整合（图G、H）。所有图像标尺均为100 μm。 图8 覆盖与未覆盖支架的样本的配对比较。左列：支架处理与未处理的愈合切口之间的差异。支架处理的切口含有更高比例的I型胶原（图A）。（A）愈合切口内的I型胶原占比。（B）愈合切口内的肌动蛋白阳性肌成纤维细胞占比。（C）愈合切口内的微血管密度。右列：覆盖与未覆盖支架的愈合周围组织之间的差异。覆盖支架的周围组织含有更高比例的肌成纤维细胞（图B）。（A）愈合周围组织内的I型胶原占比。（B）愈合周围组织内的肌成纤维细胞占比。（C）愈合周围组织内的微血管密度。 图9 愈合切口与周围组织的配对比较。左列：所有样本均覆盖支架。切口区域含有更高比例的I型胶原（图A）与更高密度的微血管（图C）。（A）I型胶原含量的定量组织学评估。（B）肌成纤维细胞（肌动蛋白）含量的定量组织学评估。（C）微血管密度的定量组织学评估。右列：所有样本均未覆盖支架。切口区域含有更高比例的I型胶原（图A）、更高比例的肌成纤维细胞（图B）与更高密度的微血管（图C）。（A）I型胶原含量的定量组织学评估。（B）肌成纤维细胞占比的定量组织学评估。（C）微血管密度的定量组织学评估。