Klar Agnes S, Biedermann Thomas, Simmen-Meuli Claudia, Reichmann Ernst, Meuli Martin
Tissue Biology Research Unit, University Children's Hospital Zurich, Zurich, Switzerland.
Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland.
Pediatr Surg Int. 2017 Mar;33(3):377-382. doi: 10.1007/s00383-016-4031-x. Epub 2016 Dec 20.
Autologous bio-engineered dermo-epidermal skin substitutes (DESS) represent an alternative therapeutic option for a definitive treatment of skin defects in human patients. Largely, the interaction of host immune cells with transplanted DESS is considered to be essential for the granulation tissue formation, graft take, and its functionality. The aim of this study was to compare the spatiotemporal distribution and density of host-derived monocytes/macrophages and granulocytes in vascularized (vascDESS) versus non-vascularized DESS (non-vascDESS) in a rat model.
Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or human adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal progenitors was used to develop a vascularized collagen type I-based dermal component in vitro. The donor-matched, monolayer-expanded adipose stromal cells lacking endothelial cells were used as a negative control. Subsequently, human keratinocytes were seeded on top of hydrogels to build dermo-epidermal skin grafts. After transplantation onto full-thickness skin wounds on the back of immuno-incompetent rats, grafts were excised and analyzed after 1 and 3 weeks. The expression of distinct inflammatory cell markers specific for host-derived monocytes/macrophages (CD11b, CD68) or granulocytes (HIS48) was analyzed by immunofluorescence microscopy.
All skin grafts were infiltrated by host-derived monocytes/macrophages (CD11b, CD68) and granulocytes (HIS48) between 1-3 week post-transplantation. When compared to non-vascDESS, the vascDESS showed an increased granulocyte infiltration at all time points analyzed with the majority of cells scattered throughout the whole dermal part. Whereas a moderate number of rat monocytes/macrophages (CD11b, CD68) were found in vascDESS at 1 week, only a few cells were detected in non-vascDESS. We observed a time-dependent decrease of monocytes/macrophages in all transplants at 3 weeks.
These results demonstrate a distinct spatiotemporal distribution of monocytes/macrophages as well as granulocytes in our transplants that closely resemble the one observed during physiological wound healing. The differences identified between vascDESS and non-vascDESS may indicate that human endothelial cells lining blood capillaries of vascDESS accelerate infiltration of monocytes and leukocytes.
自体生物工程真皮 - 表皮皮肤替代物（DESS）是人类患者皮肤缺损最终治疗的一种替代治疗选择。在很大程度上，宿主免疫细胞与移植的DESS之间的相互作用被认为对肉芽组织形成、移植物存活及其功能至关重要。本研究的目的是在大鼠模型中比较血管化DESS（vascDESS）与非血管化DESS（non - vascDESS）中宿主来源的单核细胞/巨噬细胞和粒细胞的时空分布及密度。
角质形成细胞和基质血管成分（SVF）分别来源于人皮肤或人脂肪组织。含有内皮细胞和间充质/基质祖细胞的人SVF用于在体外构建基于I型胶原蛋白的血管化真皮成分。缺乏内皮细胞的供体匹配的单层扩增脂肪基质细胞用作阴性对照。随后，将人角质形成细胞接种在水凝胶上以构建真皮 - 表皮皮肤移植物。移植到免疫缺陷大鼠背部的全层皮肤伤口上后，在1周和3周后切除移植物并进行分析。通过免疫荧光显微镜分析宿主来源的单核细胞/巨噬细胞（CD11b、CD68）或粒细胞（HIS48）特异性的不同炎症细胞标志物的表达。
在移植后1 - 3周内，所有皮肤移植物均被宿主来源的单核细胞/巨噬细胞（CD11b、CD68）和粒细胞（HIS48）浸润。与non - vascDESS相比，vascDESS在所有分析时间点的粒细胞浸润均增加，大多数细胞散布在整个真皮部分。在1周时，vascDESS中发现中等数量的大鼠单核细胞/巨噬细胞（CD11b、CD68）细胞，而在non - vascDESS中仅检测到少数细胞。我们观察到在3周时所有移植物中的单核细胞/巨噬细胞均呈时间依赖性减少。
这些结果表明，在我们的移植物中，单核细胞/巨噬细胞以及粒细胞具有独特的时空分布，这与生理伤口愈合过程中观察到的情况非常相似。vascDESS和non - vascDESS之间的差异可能表明，vascDESS中毛细血管内衬的人内皮细胞加速了单核细胞和白细胞的浸润。
