Predominance of CD14 + Cells in Burn Blister Fluids Szu-Han Chen, MD,*† Tak-Wah Wong, MD, PhD,‡ Chou-Hwei Lee, MS,* Chung-Lin Chen, MD,* Li-Wha Wu, PhD,§ and Shin-Chen Pan, MD, PhD* Background: Burn blister fluid contains several angiogenic factors to promote wound neovascularization. In our previous study, we found that deep partial- thickness burn (DPTB) wounds showed higher expression levels of angiogenin to enhance vascularization compared with superficial partial-thickness burn wounds. Neovascularization is a complex process that involves an interaction be- tween circulating angiogenic cells and mediators. We hypothesized that in addi- tion to angiogenic factors burn blisters may contain specific cell types. The aim of the present study was to characterize the specific cells present in burn blisters. Methods: Twenty-four burn blister fluid samples were obtained with informed consent from patients with superficial partial-thickness burn (n = 16) or DPTB (n = 8) wounds. Blister cells were isolated from individual intact blisters and char- acterized with flow cytometry analysis using CD14, CD34, vascular endothelial growth factor receptor 2, and CD133 markers. CD14 + and CD34 + blister cells were also isolated using a magnetic-activated cell sorting system to examine their potential for endothelial differentiation. Angiogenin levels in the burn blister fluids were evaluated with enzyme-linked immunosorbent assay. Results: CD14 + cells were the most highly represented cell type in the burn fluids of both groups, although a significantly greater percentage of CD14 + cells were observed in DPTB fluids. CD14 + blister cells had a higher potency to differenti- ate into functional endothelial cells as compared with CD34 + cells. The propor- tion of CD14 + cells gradually increased after burn injury. In contrast to CD14 + cells, angiogenin showed the highest expression levels at day 1 postburn. With re- gard to burn wound neovascularization, angiogenin expression was partially cor- related with CD14 + blister cells in the burn fluids. Conclusions: We provide the first report on the characterization of blister cells in burn fluids. Our data suggest that CD14 + blister cells may play a role in burn wound neovascularization. Measurement of CD14 + blister cells serves as a possi- ble tool for assessing burn wound status. Key Words: angiogenin, burn blister, CD14 + cells (Ann Plast Surg 2018;80: S70–S74) N eovascularization, which occurs in the early stage of wound healing, is a complex process that involves interactions between different angiogenic factors and many types of angiogenic cells. Our previous study showed that the burn blister fluid contains numerous an- giogenic factors that promote burn wound neovascularization. 1 A burn blister usually occurs in a partial-thickness burn wound. Superficial partial-thickness burn (SPTB) wounds generally heal within 2 weeks with a good cosmetic outcome. However, deep partial-thickness burn (DPTB) wounds tend to require more than 2 weeks to heal and ulti- mately result in a hypertrophic scar. Although burn blisters are present in both types of burn wounds, the factors present in the blister fluid dif- fer between SPTB and DPTB wounds, which might have different ef- fects on their neovascularization and healing processes. We previously found that DPTB blister fluids show significantly enhanced vasculari- zation components compared with those from SPTB wounds. 2 Analysis of the angiogenic factors in blister fluids showed that angiogenin plays a key role in regulating the differential neovasculogenic process between SPTB and DPTB wounds. 1 Vasculogenesis, a component of the neovascularization process, involves the formation of vessels via the recruitment and differentiation of circulating angiogenic cells (CACs) in the peripheral blood. 3 Previ- ous studies showed that burn injury results in the mobilization of CACs from the bone marrow into the peripheral blood. 4,5 Circulating angio- genic cells were shown to recruit to the site of the burn wound to par- ticipate in neovascularization by attracting chemokines that are released because of consequent changes in the microenvironment of the local tissue. 6 However, the involvement of angiogenic factors in attracting CACs to the burn blister to participate in burn wound neo- vascularization is not fully understood. Although the existence of blister cells in burn blisters is well established, their specific functions in the healing process have not been well described. 7 We hypothe- sized that a burn blister may contain not only many angiogenic factors but also specific cell types to exert the angiogenic function of wound healing. To test this hypothesis, the aim of the present study was to characterize burn blister cells and investigate their role in burn wound neovascularization. MATERIALS AND METHODS Patient Samples Twenty-four burn blister fluid samples were obtained with in- formed consent from patients with second-degree burn wounds. The blister fluid contents were collected as previously described. 2 In brief, the fluids were blindly collected from individual blisters at different time points within the first few days postburn prior to burn depth assess- ment. The blister fluid samples were classified into superficial (SPTB) or deep (DPTB) groups according to review of the patient charts indi- cating burn depth after the burn wounds healed. Based on our previous definition, 2 patients were classified in the SPTB group when the wounds healed within 14 days; other patients were classified in the DPTB group. No samples were harvested from patients with local wound infection or other medical problems such as coronary artery disease, diabetes mellitus, and end-stage renal disease. The institutional review board of the National Cheng Kung University Hospital approved the study. Isolation and Identification of Blister Cells To study the potential role of CACs in the burn fluid, blister cells were isolated and characterized from individual intact blisters. After the blister cells were centrifuged, potential stem cell markers, including CD14 (Becton Dickinson, San Jose, Calif), CD34 (MiltenyiBiotec, Bergisch Gladbach, Germany), vascular endothelial growth factor re- ceptor 2 (VEGFR2; R&D Systems, Minneapolis, Minn), and CD133 Received October 16, 2017, and accepted for publication, after revision October 22, 2017. From the *Section of Plastic and Reconstructive Surgery, Department of Surgery, National Cheng Kung University Hospital, †Institute of Clinical Medicine, ‡Department of Dermatology, National Cheng Kung University Hospital, and §Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Conflicts of interest and sources of funding: This work was supported by grants from National Cheng Kung University Hospital (NCKUH-10203034) and the Ministry of Science and Technology, Republic of China (MOST 105-2314-B-006-071- MY2). The authors have no conflicts of interest to declare. Reprints: Shin-Chen Pan, MD, PhD, Section of Plastic and Reconstructive Surgery, Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, No. 138 Sheng Li Road, Tainan 70428, Taiwan. E-mail: pansc@mail.ncku.edu.tw. Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/18/8002–S070 DOI: 10.1097/SAP.0000000000001305 RESEARCH P APERS S70 www.annalsplasticsurgery.com Annals of Plastic Surgery • Volume 80, Supplement 1, February 2018 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.