Fig.13 (A)Representative images of wound tissue sections stained by Picrosirius Red in different treatment groups on day 3 (Scale bar ¼ 200μm and 10μm). (B) Quantification of the Ratio of collagen I:III in different treatment groups on day 3 (n ¼ 3).(C) Quantification of the content of collagen I in different treatment groups on day 3 (n ¼ 3)
Next, we need to compare collagen distribution behavior and prognosis in samples after the second laser welding on the 7th day and the 14th day. As shown in Fig.14(A), the deposition area of collagen basically coincides with the proliferation area of COL-1 fiber, indicating that after the second laser welding, the proliferation of COL-1 fiber was mainly stimulated. However, for 90° laser group, the outcome was the opposite. After the second laser welding, the content of COL-1 fiber in the 90° laser group was greatly reduced. On the other hand, the COL-3 fiber content was significantly higher than that after the first stage of laser welding, resulting in ratio of collagen type was only 1.62 on the 7th day. This trend was maintained until the 14th day, when the COL-3 fiber type continued to increase and the ratio of collagen type was only 0.86, lower than normal skin. For the samples of 60° laser group, after the second laser welding, the COL-1 fibers of the samples acquired a large proliferation, and the ratio of collagen type reached 5.2 on the 7th day, which was much higher than other groups for all time points, indicating that the strength of samples could be greatly increased after the second laser welding with 60°incident angle, which laid a good foundation for the subsequent healing process. This also explained the reasons for samples of 60° laser group on the 14th day were the most similar to the collagen structure of normal skin tissue, consistent with the analysis results after Masson staining, and also explains the rationality of the argument. The collagen type ratio of 30° laser group on the 7th and the 14th day was between 2-2.5, which on the one hand ensured the healing speed and softness of tissue to a certain extent, but also indicated that the proliferation process of COL-1 fibers was much slower. Although the wounds of the samples were completely closed on day 14, the proliferation of COL-1 fibers in the later stage would take longer time[50].
At the same time, as shown in Fig.14(C), we also calculated the proportion of collagen fiber deposition in samples at these two time points. We found that although the proportion of collagen deposition in the 90° laser group was the highest on the third day, it did not increase significantly after the second laser treatment due to additional thermal damage caused by vertically incident laser. The proportion of collagen deposition in the 60° laser group reached a maximum of 34.6% on day 7, and then continued to increase during the following healing process.
In this section, the distribution of collagen deposition, the proliferation tendency of collagen types and the structure and morphology of collagen network were discussed in detail. In Fig.14(A), it can be seen that the COL-1 fiber distribution of the samples in the 90° laser group is the most significant, and the COL-3 fiber distribution is also very fine. At the same time, the deposition area of collagen network according to Masson staining is also wide and the overall collagen network turned out to be very fine and dense, indicating that the healing process of samples was the fastest under 90° laser welding, but COL-1 collagen was proliferated too much and could be tended to cause scars. The collagen network of the 30° samples also showed fine morphology after Masson staining, but differed from the 90° laser group, the samples of the 30° laser welding showed more COL-3 fibers, and the ratio of two types of collagens were the lowest among all groups, indicating that the skin elasticity was better, but the healing process would be very slow, and there was a tendency of inflammation recurrence in the later stage. On the 14th day, the two staining results of samples under were the closest to normal skin. From the perspective of collagen growth behavior, it can be concluded that 60° laser welding was the most conducive to wound healing.