Joris van Dongen

228 Chapter 10 (BD Microlance needle, blue) to each side of the face. As a control, 1 ml of sterile saline was mixed with 2.5 ml of PRP and used as a placebo. For all subjects in this study, 1 ml of tSVF was send to the lab for analysis. All procedures were performed by the senior author (HPS). Immunohistochemical and immunocytochemical analysis of tissue- stromal vascular fraction Samples of tSVF were formalin-fixed and embedded in paraffin. Then, 4 μm slides were cut, deparaffinized and stained following the previously published protocol by van Dongen et al. 19 Samples were stained for α -Smooth Muscle Actin ( α -SMA, 1:200, Abcam, Cambridge, United Kingdom) to stain smooth muscle cells, von Willebrand Factor (vWF, 1:200, DAKO, Glostrup, Denmark) to stain endothelial cells and Perilipin A (1:200, Abcam) to stain adipocytes. As secondary antibodies, polyclonal rabbit anti- mouse for α -SMA, polyclonal swine anti-rabbit for vWF and polyclonal goat anti-rabbit for Perillipin A were used in 1:100 (DAKO). A third antibody was used for the α -SMA staining (polyclonal, swine anti-rabbit, 1:100, DAKO). Masson’s trichrome staining was used to stain extracellular matrix deposition. Measurement outcomes All measurements of every patient were performed by the first author (JAD), who was blinded for treatment, at predetermined time-points: preoperative, 6 weeks postoperative, 3 months postoperative, 6 months postoperative and 12 months postoperative. Subjects did not apply any skin products on the day of surgery as well as on the day of the measurements. Only the right side of the subjects’ face was measured. Skin elasticity and transepidermal water loss measurements Local skin elasticitywas measuredwith the use of the non-invasive validated Cutometer MPA580 elasticity probe (Courage & Khazaka GmbH, Cologne, Germany). 20-23 Local transepidermal water loss (TEWL) was measured using the non-invasive validated TewameterTM300 (Courage&KhazakaGmbH, Cologne,Germany). 24,25 Measurements were done at standardized evaluation zones (Fig. 1). True skin elasticity was defined as ratio of elastic recovery to total deformation (R7 parameter). Also, net elasticity (R5 parameter) and ratio of viscoelasticity to elastic extension (R6 parameter) were measured. TEWL is expressed as in grams per square meter per hour (g/m 2 /h). Data was corrected for humidity, room temperature and age.