Fig.5 Pathological examination of wound tissue (A) Representative images of evolution of microstructure during healing processes by H&E staining (\(\times 400\)). (B) Quantitative data of width of wound gap after the two stages of laser welding at different time points (n ¼ 3). Data represent mean \(\pm\) SD; *, P < 0.05, **, P < 0.01.
Overall collagen proliferation during wound healing
In Masson’s Trichrome staining, collagen fiber is represented by blue coloration, as depicted in Fig.6. On the third day after the first laser welding, it was observed that collagen deposition in the 90° laser group concentrated mainly in the dermis, becoming more pronounced near the subcutaneous fat layer. Alterations in the structure of some subcutaneous fat were visible, including finer, smaller fat particles and larger fibroblast nuclei. In contrast, the 60° laser group displayed significant collagen deposition distributed within the dermis and epidermis surrounding the wound, with this group demonstrating the most substantial deposition area according to the intensity of the blue shades in Fig.6.
By day 7, there was a noticeable difference from the initial stage of laser welding. The collagen distribution in all three laser groups was finer and more even compared to day 3, signifying that most deposited collagen underwent further remodeling to form a dense network post the second laser welding—an indicative feature of extraordinary healing performances. However, incision marks were still visible in the cross-section provided by Fig.6, indicating that the wound was yet to fully heal and remained in the fibroblast proliferation phase. In conclusion, the densest collagen network was found in the 90° laser group, followed by the 60° and 30° laser groups respectively.
14 days into the healing process, notable changes were observed. Firstly, collagen in both the 90° and 30° laser groups had further proliferated, and fibroblast levels essentially returned to normal. The 90° laser group showed only slight gaps in collagen, and the dermis area affected by the laser exhibited complete newly formed fine collagen tissue. In contrast, the samples in the 60° laser group demonstrated restoration closer to the original collagen texture, closely resembling the dermis collagen of normal skin. This suggests that the samples in the 90° and 30° laser groups may require an extended healing cycle due to their overly fine collagen network and fibroblast levels lower than the pre-treatment state. These conditions could result in decreased elasticity of the collagen network and potential abnormal proliferation post-collagen cracking [43-45]. Such phenomena might be caused by the concentrated energy of the 90° laser, which, besides promoting fibroblast proliferation, also inflicts additional thermal damage, causing the destruction and potential loss of biological activity in some hematopoietic cells and integrins. Although the healing process was significantly enhanced, it remained challenging to ensure the thickening and hardening of the collagen scaffold post the epithelial regeneration stage.