Abstract：
OBJECTIVE: The prevalence of chronic wounds continues to be a burden in human medicine. Methicillin-resistant Staphylococcus aureus (MRSA) is commonly isolated from infected wounds. MRSA infections primarily delay healing by impairing local immune cell functions. This study aimed to investigate the potential of mesenchymal stromal cell (MSC)-secreted bioactive factors, defined as the secretome, to improve innate immune responses in vivo. MSCs were isolated from the bone marrow of horses, which serve as valuable translational models for wound healing. The MSC secretome, collected as conditioned medium (CM), was evaluated in vivo using mouse models of acute and MRSA-infected skin wounds.
METHODS: Punch biopsies were used to create two full-thickness skin wounds on the back of each mouse. Acute wounds were treated daily with control medium or bone marrow-derived MSC (BM-MSC) CM. The antibiotic mupirocin was administered as a positive control for the MRSA-infected wound experiments. Wounds were photographed daily, and wound images were measured to determine the rate of closure. Trichrome staining was carried out to examine wound tissue histologically, and immunofluorescence antibody binding was used to assess immune cell infiltration. Wounds in the MRSA-infected model were swabbed for quantification of bacterial load.
RESULTS: Acute wounds treated with BM-MSC CM showed accelerated wound closure compared with controls, as illustrated by enhanced granulation tissue formation and resolution, increased vasculature and regeneration of hair follicles. This treatment also led to increased neutrophil and macrophage infiltration. Chronic MRSA-infected wounds treated with BM-MSC CM showed reduced bacterial load accompanied by better resolution of granulation tissue formation and increased infiltration of pro-healing M2 macrophages compared with control-treated infected wounds.
CONCLUSIONS: Collectively, our findings indicate that BM-MSC CM exerts pro-healing, immunomodulatory and anti-bacterial effects on wound healing in vivo, validating further exploration of the MSC secretome as a novel treatment option to improve healing of both acute and chronic wounds, especially those infected with antibiotic-resistant bacteria.
METHODS: Punch biopsies were used to create two full-thickness skin wounds on the back of each mouse. Acute wounds were treated daily with control medium or bone marrow-derived MSC (BM-MSC) CM. The antibiotic mupirocin was administered as a positive control for the MRSA-infected wound experiments. Wounds were photographed daily, and wound images were measured to determine the rate of closure. Trichrome staining was carried out to examine wound tissue histologically, and immunofluorescence antibody binding was used to assess immune cell infiltration. Wounds in the MRSA-infected model were swabbed for quantification of bacterial load.
RESULTS: Acute wounds treated with BM-MSC CM showed accelerated wound closure compared with controls, as illustrated by enhanced granulation tissue formation and resolution, increased vasculature and regeneration of hair follicles. This treatment also led to increased neutrophil and macrophage infiltration. Chronic MRSA-infected wounds treated with BM-MSC CM showed reduced bacterial load accompanied by better resolution of granulation tissue formation and increased infiltration of pro-healing M2 macrophages compared with control-treated infected wounds.
CONCLUSIONS: Collectively, our findings indicate that BM-MSC CM exerts pro-healing, immunomodulatory and anti-bacterial effects on wound healing in vivo, validating further exploration of the MSC secretome as a novel treatment option to improve healing of both acute and chronic wounds, especially those infected with antibiotic-resistant bacteria.
摘要：
目的：慢性伤口的流行仍然是人类医学的负担。耐甲氧西林金黄色葡萄球菌（MRSA）通常从感染的伤口中分离出来。MRSA感染主要通过损害局部免疫细胞功能来延迟愈合。本研究旨在探讨间充质基质细胞(MSC)分泌生物活性因子的潜能,定义为分泌组，改善体内先天免疫反应。从马的骨髓中分离出MSCs,作为伤口愈合的有价值的转化模型。MSC分泌组，收集为条件培养基（CM），使用急性和MRSA感染的皮肤伤口的小鼠模型进行体内评估。
方法:使用穿孔活检在每只小鼠的背部产生两个全厚度皮肤伤口。每天用对照培养基或骨髓来源的MSC(BM-MSC)CM治疗急性伤口。抗生素莫匹罗星作为MRSA感染的伤口实验的阳性对照。每天拍摄伤口，和测量伤口图像以确定闭合率。进行三色染色以从组织学上检查伤口组织，免疫荧光抗体结合用于评估免疫细胞浸润。擦拭MRSA感染模型中的伤口以定量细菌负荷。
结果：与对照组相比，用BM-MSCCM治疗的急性伤口显示加速的伤口闭合，如肉芽组织形成和分辨率增强所示，增加的脉管系统和毛囊的再生。这种处理还导致嗜中性粒细胞和巨噬细胞浸润增加。与对照治疗的感染伤口相比，用BM-MSCCM治疗的慢性MRSA感染伤口显示出减少的细菌负荷，同时肉芽组织形成的分辨率更高，促愈合M2巨噬细胞的浸润增加。
结论：总的来说，我们的发现表明，BM-MSCCM具有促进愈合的作用，体内对伤口愈合的免疫调节和抗菌作用，验证进一步探索MSC分泌组作为一种新的治疗选择，以改善急性和慢性伤口的愈合，尤其是那些感染了抗生素抗性细菌的人。
方法:使用穿孔活检在每只小鼠的背部产生两个全厚度皮肤伤口。每天用对照培养基或骨髓来源的MSC(BM-MSC)CM治疗急性伤口。抗生素莫匹罗星作为MRSA感染的伤口实验的阳性对照。每天拍摄伤口，和测量伤口图像以确定闭合率。进行三色染色以从组织学上检查伤口组织，免疫荧光抗体结合用于评估免疫细胞浸润。擦拭MRSA感染模型中的伤口以定量细菌负荷。
结果：与对照组相比，用BM-MSCCM治疗的急性伤口显示加速的伤口闭合，如肉芽组织形成和分辨率增强所示，增加的脉管系统和毛囊的再生。这种处理还导致嗜中性粒细胞和巨噬细胞浸润增加。与对照治疗的感染伤口相比，用BM-MSCCM治疗的慢性MRSA感染伤口显示出减少的细菌负荷，同时肉芽组织形成的分辨率更高，促愈合M2巨噬细胞的浸润增加。
结论：总的来说，我们的发现表明，BM-MSCCM具有促进愈合的作用，体内对伤口愈合的免疫调节和抗菌作用，验证进一步探索MSC分泌组作为一种新的治疗选择，以改善急性和慢性伤口的愈合，尤其是那些感染了抗生素抗性细菌的人。
