Fig.3 Model of laser treatment in wound of rats. (A) laser automated surgical platform with different incident angles. B) Schematic cross-sectional view of the treatment process of rat skin tissue. C) Schematic of laser scanning path-Zigzag .
We monitored and quantitatively measured the wound healing process at pre-determined intervals post-operation as depicted in Fig.4(A). The observations revealed innate self-healing capabilities in rats as evidenced by the continuous reduction in wound area in the control group, despite wounds remaining unhealed after 24 days without any additional treatment. It appears that low-energy laser treatment at 1064nm can foster wound healing to an extent, as all three laser-treated groups exhibited smaller wound areas compared to the control group at every time-point post-treatment. Notably, complete recovery was achieved in all laser-treated groups within 14 days, marking a healing period 41.7% shorter than that of the control samples. As per Fig.4(C) and Fig.4(B), the 30°Laser samples showed the quickest healing speed during the first stage (initial 3 days) of laser welding, while the 90°Laser samples exhibited the fastest healing speed in the second stage. For the 60°Laser samples, even though the initial healing speed wasn’t particularly prominent, the healing processes post the second laser weld were the most significant. It remains challenging to determine the optimal parameter group based solely on the trend of healing speed and wound size since factors like collagen fiber disposition, distribution of inflammatory factors, and varying types of collagen content are all vital indicators of healing performance. To further scrutinize the differences between incident angles and the mechanism of laser welding tissue, we proceeded with comprehensive characterization and analysis of cells, collagen, and functional protein groups during the healing process. This data has been incorporated into Fig.4(C) and will be detailed in the following sections.
Fig.4 Whole process of wound healing. (A) Representative images of macro appearances of rats during whole healing processes in three groups. (B) Schematic diagram of the wounds managed by different laser treatments in 24 days. (C) Quantitative data of wound sizes at different time points (n ¼ 3). (D) Quantitative data of wound healing ratio at different time points (n ¼ 3). Data represent mean \(\pm\) SD; *, P < 0.05, **, P < 0.01.
Microstructures of rats after laser welding with different incident angles
In trauma treatment, the wound healing process is typically segmented into stages: the inflammatory response stage, the granulation stage, and the epithelial formation stage. These stages often overlap with each other. Figure 5 presents Hematoxylin and Eosin (H&E) stained images of the wound tissue at specific time points, which include days 3, 7, and 14. The figure distinctly reveals noticeable differences in the microstructure of samples throughout the healing process under varied laser incident angles.
On day 3, post the first laser welding, the incision location and gap are still clearly visible at 200 μm magnification. Following the first laser weld, the most pronounced recovery was observed in the 90° laser and 30° laser groups; the wound incision gap in the dermis layer was considerably reduced, and partial remodeling of the collagen in the dermis had occurred through integrin interactions. On the contrary, the samples in the 60° group exhibited significant epidermal gaps and it remained challenging to discern clear collagen deposition in the dermis.
Following the second laser welding on day 3, marked improvements were noted across all groups, with particularly tremendous healing progress in both the 90° and 30° laser groups. At this stage, the incision’s micromorphology was barely noticeable in all three groups, and re-epithelialization of all samples had commenced along with new granulation formation. This re-epithelialization process is pivotal to accelerating dermal healing; the beneficial thickening of the upper epidermis serves as a bio-functional barrier established early in the wound healing process. This barrier plays a crucial role in preventing excessive transdermal water loss and further wound infection.
By the 14th day of the healing process, it was observed that any existing gaps in the wound incision had entirely disappeared. The upper epidermis was completely covered, and a dense new collagen network structure was evident in the dermis.