Department of Dermatology, Huadong Hospital, Fudan University, Shanghai 200040, PR China.
Shanghai Skin Disease Hospital, Institute of Photomedicine, Tongji University School of Medicine, Shanghai, PR China.
Photodiagnosis Photodyn Ther. 2021 Dec;36:102480. doi: 10.1016/j.pdpdt.2021.102480. Epub 2021 Aug 8.
A distressing issue of diabetic ulcer (DU) is its poor healing feature with limited clinical solutions. We have previously shown that 5-aminolevulinic acid photodynamic therapy (ALA-PDT) is a promising alternative to the currently limited measures for DU. Mesenchymal stem cells (MSCs) transplantation has been believed to impose certain therapeutic effect on restoration of injury. Thus, this study aims to explore whether the combination of MSCs and ALA-PDT will exert a more advanced curative effect on DU.
Diabetic mice were induced by intraperitoneal injection of streptozotocin (STZ, 60 mg/kg/d) for consecutive 5 days. A full-thickness skin injury (diameter 6 mm) was created in the center of the back of each mouse, and then 10 μl of methicillin-resistant Staphylococcus aureus (MRSA) suspension was added to establish an infected DU model. All DU models were randomly divided into four groups: Untreated group, MSCs group, ALA-PDT group, and ALA-PDT combined with human umbilical cord mesenchymal stem cells (hUC-MSCs) (ALA-PDT + MSCs) group. The wound sizes were recorded by a digital camera, and the healing rates were calculated using Image J software. Bacterial loads on wounds were measured using CFU (Colony forming units) analysis. The epithelialization, inflammatory cells infiltration and granulation tissue formation were monitored by Haematoxylin and eosin (H&E) staining, and the corresponding semi-quantitative score was matched. Growth and pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA).
Either ALA-PDT or injection of hUC-MSCs resulted in a rapid wound closure compared with the untreated, while their combination brought about the most prominent healing. On day 12, healing rates of the untreated, MSCs, ALA-PDT and ALA-PDT + MSCs were 40.56% ± 7.06%, 74.23 ± 4.83%, 84.03 ± 3.53%, 99.67 ± 0.49%, respectively. The bacterial burden reductions were approximately 1.58 logs (97.36%, P < 0.05), 2.34 logs (99.54%, P < 0.01), 4.50 logs (nearly 100%, P < 0.001) for MSCs, ALA-PDT and ALA-PDT + MSCs, respectively. Histology revealed reduced inflammatory cells and improved collagen precipitation and angiogenesis after hUC-MSCs and ALA-PDT treatment compared to the untreated. The combined therapy leaded to a more intact epithelium, similar to the healthy. Finally, ELISA revealed that the property of ALA-PDT to stimulate transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) and inhibit IL (interleukin) -1β and IL-6 outweighed that of hUC-MSCs, and this function of the combination overwhelmed that of any single therapy.
Our findings indicated that the strategy of combining ALA-PDT with hUC-MSCs possessed a significantly enhanced therapeutic effect over either single therapy, providing a promising innovative therapeutic candidate for refractory wounds.
糖尿病溃疡（DU）令人痛苦的问题是其愈合特征不佳，临床解决方案有限。我们之前已经表明，5-氨基酮戊酸光动力疗法（ALA-PDT）是目前治疗 DU 的有限措施的一种有前途的替代方法。间充质干细胞（MSCs）移植被认为对损伤的恢复具有一定的治疗作用。因此，本研究旨在探讨 MSC 与 ALA-PDT 的联合应用是否会对 DU 产生更先进的治疗效果。
通过腹腔注射链脲佐菌素（STZ，60mg/kg/d）连续 5 天诱导糖尿病小鼠。在每只小鼠背部中心创建一个全厚皮肤损伤（直径 6mm），然后加入 10μl 耐甲氧西林金黄色葡萄球菌（MRSA）悬浮液以建立感染性 DU 模型。所有 DU 模型均随机分为四组：未处理组、MSCs 组、ALA-PDT 组和 ALA-PDT 联合人脐带间充质干细胞（hUC-MSCs）（ALA-PDT+MSCs）组。通过数码相机记录伤口大小，并使用 Image J 软件计算愈合率。使用 CFU（集落形成单位）分析测量伤口上的细菌负荷。通过苏木精和伊红（H&E）染色监测上皮化、炎症细胞浸润和肉芽组织形成，并匹配相应的半定量评分。通过酶联免疫吸附试验（ELISA）检测生长和促炎细胞因子。
与未处理组相比，ALA-PDT 或注射 hUC-MSCs 均可导致伤口快速闭合，而两者联合则带来了最显著的愈合效果。在第 12 天，未处理、MSCs、ALA-PDT 和 ALA-PDT+MSCs 组的愈合率分别为 40.56%±7.06%、74.23%±4.83%、84.03%±3.53%和 99.67%±0.49%。细菌负荷减少约 1.58 个对数（97.36%，P<0.05）、2.34 个对数（99.54%，P<0.01）、4.50 个对数（接近 100%，P<0.001）分别为 MSCs、ALA-PDT 和 ALA-PDT+MSCs。与未处理组相比，hUC-MSCs 和 ALA-PDT 治疗后炎症细胞减少，胶原沉淀和血管生成改善。联合治疗导致上皮更完整，类似于健康组织。最后，ELISA 显示，ALA-PDT 刺激转化生长因子-β1（TGF-β1）和血管内皮生长因子（VEGF）以及抑制白细胞介素（IL）-1β和 IL-6 的特性优于 hUC-MSCs，并且这种联合作用的功能超过了任何单一疗法。
我们的研究结果表明，ALA-PDT 联合 hUC-MSCs 的策略比单一疗法具有显著增强的治疗效果，为难治性伤口提供了一种有前途的创新治疗候选物。
