As demonstrated in Fig.9(B), the Vascular Endothelial Growth Factor (VEGF) levels at the wound site for all groups were quantified using ELISA methods. The data reveals that in comparison to the control group, the VEGF content experienced a significant increase following the first laser welding. Most notably, the VEGF level in the 90° laser group peaked at an impressive 276pg/ml on the third day, indicating that the laser at a 90° incidence had the most profound effect after a single laser welding session, stimulating an abundant production of VEGF. Such an observation also implied that this group had the most rapid healing process. Conversely, the 60° and 30° laser groups exhibited inferior VEGF levels, as shown in Fig.9(A). Following the second laser welding session, the 60° laser group demonstrated the highest VEGF level, whereas the VEGF content decreased in the 90° laser group. This pattern suggests that high-energy concentration at a vertical incident angle might inflict additional thermal damage to the wound tissues, whereas a 60° laser effectively promotes continued VEGF secretion by vasculogenic cells. By day 14, all laser groups registered lower VEGF levels compared to the control group. This trend indicates that while the wound had fully closed, collagen growth and metabolism within the dermis persisted beyond this point. Of note, the VEGF level in the control group was seen to be approximately 200pg/ml on the 14th day—higher than any other group—suggesting that the samples from the control group might need at least 14 additional days for complete wound closure compared to the laser groups. Throughout the entire process, the 30° laser group exhibited minimal fluctuations in VEGF levels and consequently displayed the lowest healing efficiency among the laser groups. Despite the lack of pronounced changes in the VEGF content of the 30° laser group, it consistently maintained significantly higher levels than the control group, demonstrating the capacity of low-energy 1064nm lasers to effectively expedite wound healing.

Fig.9 Cell proliferation and differentiation of wound tissue. (A) Quantitative analysis of TGF-β content in wound tissue by the ELISA method. (B) Quantitative analysis of VEGF content in wound tissue by the ELISA method. Data represent mean \(\pm\)SD; *, P ＜ 0.05, **, P ＜ 0.01, ***, P ＜ 0.001.

Discussions

Wound healing study in vivo

Regarding the 60° laser samples, although initial healing progress wasn’t particularly noticeable, the most significant healing responses were observed following the second laser welding process. However, it is challenging to categorically conclude on the optimal parameter group based solely on the trends in healing speed and wound size, as factors such as collagen fiber disposition, inflammatory factor distribution, and the concentration of different collagen types all play crucial roles in characterizing healing performance. To meticulously analyze the differences between incident angles and decipher the mechanism behind laser-welded tissue healing, we subsequently performed comprehensive characterization and analysis of cells, collagen, and protein functional groups throughout the healing process. These findings have been encapsulated in Fig.4(C), with further details provided in the subsequent sections.

Histology of wound healing site in rats

To provide a more quantitative assessment of the wound healing process at different time points, we evaluated the regenerated epithelium by measuring the length of neo-epithelial tongue and the area of neo-epithelium, as depicted in Fig.10(A). Firstly, we marked the position and length of the epithelial tongues in various groups on the third day post-healing. Following two laser welding treatments, all samples—with the exception of the control group—exhibited some degree of re-epithelialization. This indicated progression to the third stage of healing, characterized by vigorous proliferation of fibroblasts and angiogenic cells. Partial formation of collagen skeletons in the tissue’s dermis was also observed, interconnected via integrin. Concurrently, keratinocytes and adipose-derived stem cells gradually increased in number while the inflammatory response subsided. Examination of Fig.10(A) revealed an abnormal upper epidermis shape in the control group, resembling a gully. Data from Fig.10(B) and (C) confirmed that the length and area of the upper epidermis in this group were the smallest among all test groups. In comparison, laser-welded samples displayed more regular epidermal regeneration. Specifically, specimens from the 90° laser group had the longest upper epidermis and largest surface area, approaching approximately 45 mm2. However, it is worth noting that the wound gap distribution pattern depicted in Fig.10(B) contradicts the upper epidermis growth pattern for the 60° and 30° laser samples. This discrepancy suggests that wound gap reduction primarily indicates the status of dermal collagen healing, whereas the regeneration process of the upper epidermis may not necessarily occur synchronously with collagen remodeling.