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Title: [Influence of porcine urinary bladder matrix and porcine acellular dermal matrix on wound healing of full-thickness skin defect in diabetic mice].
Author: Zhao P, Yang ML, Chu GP, Jia ZG, Zhou XJ, Lyu GZ.
Journal: Zhonghua Shao Shang Za Zhi; 2020 Dec 20; 36(12):1130-1138. PubMed ID: 33379849.
Abstract:
Objective: To compare the difference of pro-healing effect of porcine urinary bladder matrix (UBM) and porcine acellular dermal matrix (ADM) on full-thickness skin defect wounds in diabetic mice. Methods: Thirty-six type 2 diabetic BKS db/db mice aged 10 weeks were divided into UBM group and ADM group according to the random number table, with 18 mice in each group and preoperative molarity of non-fasting blood glucose higher than 16.6 mmol/L. A circular full-thickness skin defect wound with 6 mm in diameter was made on the back of each mouse, and porcine UBM and porcine ADM scaffolds were implanted into the wounds of both groups correspondingly. Immediately after operation and on post operation day (POD) 7, 14, and 28, wounds were observed generally. On POD 7, 14, and 28, 6 mice of each group were collected respectively to calculate the rate of wound epithelialization, and then the corresponding mice were sacrificed after calculation, and the wound tissue was harvested to make slices. Six slices of the mice in the 2 groups on POD 7 and 14 were respectively collected to stain with haematoxylin-eosin (HE), and 6 slices on POD 7 and 28 had Masson's staining, which were used to observe histopathological changes and scaffold degradation. On POD 7 and 14, 24 slices of each mouse in the 2 groups were collected respectively to detect alpha smooth muscle actin (α-SMA) and CD31 positive expression denoting the growth of myofibroblasts and neovessels respectively and observe the distribution and activation of macrophages with immunohistochemical staining. The wound tissue of mice in the 2 groups on POD 7 and 14 was harvested to detect mRNA expressions of fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor β(1) (TGF-β(1)) by real-time fluorescence quantitative reverse transcription polymerase chain reaction. The sample number of above-mentioned indexes in each group at each time point was 6. Data were statistically analyzed with analysis of variance for factorial design, t test, and Bonferroni correction. Results: (1) General observation showed that integration of UBM scaffold into the wounds of mice in UBM group on most time points was superior, and integration of ADM scaffold into the wounds of mice in ADM group on most time points was inferior. On POD 28, epidermis still did not form in some region of scaffold surface of wounds of mice in ADM group, while wounds of mice in UBM group were completely epithelialized. On POD 7, 14, and 28, wound epithelialization rates of mice in UBM group were respectively (22.4±6.4)%, (68.6±12.4)%, and 100.0%, all significantly higher than (4.5±2.2)%, (23.6±4.6)%, and (64.2±13.2)% in ADM group (t=7.427, 9.665, 7.655, P<0.01). (2) HE staining and Masson's staining showed that a large number of cells appeared in wound scaffold of mice in UBM group on POD 7; cells distributed in the whole region of UBM scaffold on POD 14; dermal tissue with structure similar to normal skin formed in the wounds and the fibrous morph of UBM scaffolds disappeared on POD 28. Only a small number of cells appeared in inside of wound scaffolds of mice in ADM group on POD 7; on POD 14, cells were sparsely distributed in ADM scaffolds; on POD 28, the morph of originally robust collagen fiber of ADM scaffolds was still clear and visible. (3) On POD 7, a large number of accumulated myofibroblasts and neovessels appeared in the lower layers of scaffolds of wounds of mice in UBM group; on POD 14, evenly distributed myofibroblasts and neovessels appeared in the upper layers of UBM scaffolds, and most vessels were perfused. On POD 7 and 14, myofibroblasts were sparsely distributed in scaffolds of wounds of mice in ADM group with no or a few neovascular structures perfused unobviously. On POD 7 and 14, α-SMA positive expressions in scaffolds of wounds of mice in UBM group were significantly higher than those in ADM group (t=25.340, 6.651, P<0.01); CD31 positive expressions were also significantly higher than those in ADM group (t=34.225, 10.581, P<0.01). (4) On POD 7, a large number of macrophages appeared in the lower layers of scaffolds of wounds of mice in UBM group; on POD 14, macrophages infiltrated into the internal region of UBM scaffolds, and M2 polarization occured without M1 polarization. On POD 7, a small number of macrophages appeared in the bottom of scaffolds of wounds of mice in ADM group; on POD 14, macrophages were few in internal region of ADM scaffold, and neither M2 polarization nor M1 polarization occurred. (5) On POD 7 and 14, mRNA expressions of FGF-2, VEGF, PDGF, and TGF-β(1) in the wound tissue of mice in UBM group were all significantly higher than those in ADM group (t=7.007, 14.770, 10.670, 8.939; 7.174, 7.770, 4.374, 4.501, P<0.01). Conclusions: Porcine UBM scaffold is better than porcine ADM in facilitating wound repair and dermis reconstruction of full-thickness skin defects in diabetic mice through the induction of myofibroblasts and macrophages immigration, the promotion of neovascularization and expression of pro-healing growth factors. 目的： 对比猪膀胱脱细胞基质(UBM)和猪脱细胞真皮基质(ADM)对糖尿病小鼠全层皮肤缺损创面促愈合效果的差异。 方法： 将36只10周龄雄性2型糖尿病BKS db/db小鼠按照随机数字表法分为UBM组和ADM组，每组18只，术前非空腹血糖物质的量浓度大于16.6 mmol/L，于每只小鼠背部制作直径6 mm圆形全层皮肤缺损创面，相应植入猪UBM和猪ADM支架。术后即刻及7、14、28 d，行创面大体观察。术后7、14和28 d，每组各取小鼠6只计算创面上皮化率，计算后处死相应小鼠，取创面组织制作切片。收集2组小鼠术后7与14 d各6张切片行苏木精-伊红(HE)染色、术后7与28 d各6张切片行Masson染色，观察组织病理学变化及支架降解情况。收集2组小鼠术后7、14 d各24张切片，采用免疫组织化学法检测α平滑肌肌动蛋白(α-SMA)和CD31阳性表达量，以分别反映肌成纤维细胞(Fb)、新生血管生长情况；观察巨噬细胞的分布和活化。取2组小鼠术后7、14 d创面组织，采用实时荧光定量反转录PCR法检测成纤维细胞生长因子2(FGF-2)、血管内皮生长因子(VEGF)、血小板衍生生长因子(PDGF)、转化生长因子β(1)(TGF-β(1))mRNA含量。上述指标每组各时间点样本数为6。对数据行析因设计方差分析、t检验及Bonferroni校正。 结果： (1)大体观察显示，UBM组小鼠创面术后大部分时间点与UBM支架融合佳，ADM组小鼠创面术后大部分时间点与ADM支架融合差；术后28 d，ADM组小鼠创面支架表面部分区域仍未形成表皮，UBM组小鼠创面完全上皮化。术后7、14和28 d，UBM组小鼠创面上皮化率分别为(22.4±6.4)%、(68.6±12.4)%、100.0%，均明显高于ADM组[(4.5±2.2)%、(23.6±4.6)%、(64.2±13.2)%，t＝7.427、9.665、7.655，P<0.01]。(2)HE染色和Masson染色显示，术后7 d，UBM组小鼠创面支架出现大量细胞；术后14 d，细胞占据UBM支架的全部区域；术后28 d，创面形成与正常皮肤结构类似的真皮组织，并且UBM支架原有的纤维形态消失。术后7 d，ADM组小鼠创面支架内部仅出现少量细胞；术后14 d，细胞散布于ADM支架之中；术后28 d，ADM支架原有粗大的胶原纤维形态仍清晰可见。(3)术后7 d，UBM组小鼠创面支架底层出现大量聚集的肌Fb，出现新生血管；术后14 d，UBM支架上层出现均匀分布的肌Fb、新生血管，并且大部分血管实现灌注。术后7、14 d，ADM组小鼠创面支架中仅散在分布肌Fb，无或少量未明显灌注的新生管腔结构。术后7、14 d，UBM组小鼠创面支架中α-SMA阳性表达量明显高于ADM组(t＝25.340、6.651, P<0.01)，CD31阳性表达量也明显高于ADM组(t＝34.225、10.581, P<0.01)。(4)术后7 d，UBM组小鼠创面支架底层出现大量巨噬细胞；术后14 d，巨噬细胞迁入UBM支架内部，发生M2型极化、无M1型极化。术后7 d，ADM组小鼠创面支架底部出现少量巨噬细胞；术后14 d，ADM支架内部巨噬细胞稀少，未发生M2型或M1型极化。(5)术后7、14 d，UBM组小鼠创面组织中FGF-2、VEGF、PDGF和TGF-β(1)的mRNA表达量均明显高于ADM组(t＝7.007、14.770、10.670、8.939，7.174、7.770、4.374、4.501, P<0.01)。 结论： 猪UBM支架通过诱导肌Fb和巨噬细胞迁入、促血管新生与促愈合生长因子的表达，促进糖尿病小鼠全层皮肤缺损创面的修复和真皮重建，效果优于猪ADM。.

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