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.