Before we designed our experimental parameters, we tried to start the incident angles from a very small degree which far below 30°, however, we found that when incident angle is below 30°, the incident angle of laser is too small to focus on the plane of the wound, and the laser beam could not transfer the heat and photon into full layer of tissue. When incident angle was below 30°, only the epidermis and upper dermis can receive laser energy which on the other hand led to severe heat damage of wound surface. So, we started our experiments using incident angels larger than 30°. We designed three different incident angles which vary from 30° to 90° in order to simulate different position of potential wound on the back of rats, the scheme diagram is shown in Fig.4(C). Because of the symmetry of the laser incidence, we performed a deflected incidence of the rat wound on the fixed side.
All SD rats were purchased from Changzhou Covens Laboratory Animal co., LTD and familiarized with their new environment for a week. All rats (180-220g) used were male and fed under SPF (specific pathogen-free animal) facility with a 12:12h light–dark cycle. All surgical procedures were conducted under animal care protocols approved by the Ethics Committee of Nanjing University of Science and Technology.
45 rats were randomly assigned to each experimental group and distributed by three test indexes with four time points. Fifty rats were randomly divided into three different groups [90° (n = 5), 60° (n = 5), and 30° (n = 5)] at each time point:3, 7, 14 and 24 days, and each group had one control rat.
After anaesthetization (1.5–3 vol.% Isoflurane), the dorsal surface hairs were shaved and then incised with a 15mm sterile bistoury, all the operation was carried out in Ultra-clean platform as shown in Fig.2(A). The incision area could be observed in Fig.2 which could also be regarded as welding area in the follow-up laser processing. The length of full-thickness wound is 3cm and the depth of wound is 2mm. The whole processes of animal modeling and laser bonding were conducted in specialized ultra-clean workshop as shown in Fig.2(A) and (B). Incision wound sites after laser treatments were covered with air-permeable Tegarderm™ (3 M, St. Paul, MN) tape as seen in Fig.2(C). Throughout the experiment, rats were housed individually post-operation with access to food and water ad libitum, kept under controlled conditions of humidity (50%-70%), temperature (20–22), light: dark cycle (12 h:12 h), and free access to food and water. The digital photographs were used to monitor the wound closure over time on days 0, 3, 7, 14 and 21 post surgery. The wound healing progress was analyzed using Image Jsoftware and calculated based on the wound reduction area using the following equation Then, the wound area was determined by tracing the wound margins with Image J software and an image analysis program (Adobe Photoshop cs6). The degree of wound healing is expressed as a percentage of original wound size. Rats were sacrificed and their dorsal skin was surgically removed for histological analysis[29-33].