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\begin{document}

\title{Research on evolution process of full-layer incision of skin tissue
under different laser incidences}



\author[1]{Kehong Wang}%
\author[1]{Yuxin Chen}%
\author[1]{Jun Huang}%
\author[2]{Yunfeng Rui}%
\author[3]{Wentao Fan}%
\affil[1]{Nanjing University of Science and Technology School of Science}%
\affil[2]{Southeast University Zhongda Hospital Department of Orthopedics}%
\affil[3]{Nanjing Medical University The Fourth School of Clinical Medicine}%


\vspace{-1em}



  
  \date{May 23, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Considering difficulties of achieving vertical incidence of beam in
different positions of skin, it's significant to study potential effects
of incidence angles of laser on incisions. Surgical platform with a
1064nm continuous fiber laser was established. Incident angle was
adopted and real-time temperature fluctuations in laser operating area
could be monitored. The rats were treated with laser at day 0 and day 3
after incision modeling, and H\&E, Masson, Sirius Red and
Immuno-histochemical staining and enzyme-linked immunosorbent assay were
adopted at day 3,7,14 to analyze the performance of healing. Laser with
energy density of 67.54J/mm \textsuperscript{2} can effectively
accelerate wound healing in vivo, in which laser with incident angle
around 60\selectlanguage{ngerman}° can effectively avoid scar hyperplasia. Therefore, the use of
low energy laser with small deflection angle has a good clinical
application prospect in promoting wound healing.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


Research on evolution process of full-layer incision of skin tissue
under different laser incidences

Yuxin Chen\textsuperscript{a*}, Jun Huang\textsuperscript{a*}, Kehong
Wang\textsuperscript{a*}, Xiaopeng Li\textsuperscript{a}, Yunfeng
Rui\textsuperscript{b}, Wentao Fan\textsuperscript{c},

a School of Material Science and Technology, Nanjing University of
Science and Technology, Nanjing, China

b Department of Orthopaedics, Southeast University, Zhongda Hospital,
Nanjing, China

c First school of clinical medicine, Nanjing Medical University,
Nanjing, China

*Corresponding author: Kehong Wang, E-mail:\emph{wkh1602@126.com,} Jun
Huang, E-mail:\emph{huangjun@njust.edu.cn,}Yuxin Chen, Email:
chenyuxin1602@njust.edu.cn

\textbf{Abstract} \textbf{:}Considering difficulties of achieving
vertical incidence of beam in different positions of skin, it's
significant to study potential effects of incidence angles of laser on
incisions. Surgical platform with a 1064nm continuous fiber laser was
established. Incident angle was adopted and real-time temperature
fluctuations in laser operating area could be monitored. The rats were
treated with laser at day 0 and day 3 after incision modeling, and H\&E,
Masson, Sirius Red and Immuno-histochemical staining and enzyme-linked
immunosorbent assay were adopted at day 3,7,14 to analyze the
performance of healing. Laser with energy density of
67.54J/mm\textsuperscript{2} can effectively accelerate wound healing in
vivo, in which laser with incident angle around 60\selectlanguage{ngerman}° can effectively
avoid scar hyperplasia. Therefore, the use of low energy laser with
small deflection angle has a good clinical application prospect in
promoting wound healing.

\textbf{Key words:}Laser tissue welding; Wound healing; Incident angles;
Laser automated surgical platform; Collagen remodeling

Introduction

The skin is the largest organ of the human body, playing a role in
maintaining the fluids and electrolytes homeostasis, resisting physical
and chemical damage, as well as preventing microbial infections
\textsuperscript{{[}1{]}}. Regrettably, full-thickness skin defects are
prone to occur under various injury factors, such as large-scale burns,
traumatic injuries, diabetic ulcers, and vascular lesions, which
seriously harmed not only the life quality of patients, but also the
medical economy. Current therapeutic interventions remain far from ideal
repair\textsuperscript{{[}2{]}}.

Medical laser is known as an epoch-making progress in traditional
surgery, and has become a special and effective means of diagnosis and
treatment in many medical fields\textsuperscript{{[}3-4{]}}. In recent
years, laser has made great progress in the field of biomedicine,
especially in minimally invasive surgery, due to its excellent
monochromatic properties, collimation and flexible energy density
\textsuperscript{{[}5-6{]}}. The thermal effect produced by laser acting
on biological tissue with different wavelength and energy is also
different, so different types of tissue normally need different kinds of
lasers \textsuperscript{{[}7,23-25{]}}. The surgical application and
safety procedures of lasers are related to the degree of thermal damage
\textsuperscript{{[}8-9{]}}. The degree of thermal damage is not only a
function of laser parameters, such as power density, duration, pulse
width, etc., but also depends on the absorption and scattering
properties of tissues and thermophysical parameters such as thermal
conductivity and thermal diffusivity of tissues
\textsuperscript{{[}10-11{]}}. Once the heat source is determined, the
tissue temperature distribution changes can be calculated according to
the heat transport equations and various assumed conduction and
convection boundary conditions \textsuperscript{{[}12-13{]}}. An
increase in temperature can lead to cell inactivation, protein
denaturation, and even vaporization and cutting of tissues
\textsuperscript{{[}14{]}}. Medical laser technology, a non-contact skin
tissue suture has the advantage of high immediate tensile strength
\textsuperscript{{[}15{]}}, fast recovery, low infection and less scar
tissue and so on which has outstanding application potential in surgery,
obstetrics and gynecology and cosmetic
surgery\textsuperscript{{[}16{]}}. However, in practical applications,
the location of the full-layer incision in the skin tissue is relatively
flexible, which cannot guarantee that the laser can be vertically
incident. Therefore, it is of great significance to study the evolution
rule of microstructure, collagen degeneration\textsuperscript{{[}17{]}}
process and content changes of important cytokines in the process of
bonding full-layer incision of skin tissue by different laser incidences
in order to accelerate the prognostic process for achieving the clinical
progress of laser induced full-layer incision connection
technology\textsuperscript{{[}18-19{]}}. Since the thickness of skin
tissue is small and the photo-thermal effect between laser and skin
tissue is sensitive \textsuperscript{{[}20{]}}, the evolution process of
microstructure corresponding to different layers of skin is an important
reference to judge the healing situation of samples. Similarly, the
change of macro morphology is inevitably based on the change of
microstructure morphology. In this study, continuous low-power laser was
performed on the back of living SD rats by different laser incident
angles. The macro-morphology, microstructure morphology, fibroblast
morphology, endothelial growth factor content, and the distribution of
type I and III collagen were continuously characterized at fixed
periods. The laser bonding processes of skin tissue was further
investigated from the microscopic mechanism of healing, also the
potential of laser bonding incisions in different positions have been
verified theoretically and experimentally.

Materials and Methods

On the basis of great researches before, we constructed a comprehensive
welding system which could meet all basic demands during the laser
welding processes, as shown in Fig.1. The skin tissue connection machine
used in this study consists of three parts: a laser working system, a
thermograph, and an overall control system. The laser working system
consists of 1064nm Nd:YAG consecutive fiber lasers, a workbench and
relevant clamping devices. The thermograph consists of a Fortic
near-infrared thermal imager and a control subsystem. The overall
control system consists of the temperature output data processing
subsystems of a laser control system and a thermal imager. The overall
control system in Fig.2.1 can adjust and set the laser parameters in
real time and monitor the temperature changes of skin tissues, and can
regulate tissue injuries and the macroscopic condition of welding. In
order to guarantee the accuracy and scientificity of experimental data,
we also carry out the experiments in an ultra-clean laser surgery area,
as shown in Fig.1.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig.1 Ultra-clean Low energy Nd:YAG 1064nm laser automated surgical
platform

On the basis of previous experiments, we have summarized the optimized
basic laser parameters including laser power, laser scanning path,
defocus amount, wavelength and scanning
speed\textsuperscript{{[}27-28{]}}. Through comprehensive analysis on
both macroscopic and microscopic of skin tissue after laser welding,
combined with the body temperature and thickness of rats, we further
optimized the laser parameters as shown in table.1. And we operated on
the wounds of the rats on day 0 and day 3 of the healing cycle.

Table.1 Experimental parameters\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
No. & Defocus amount & Power/w & Speed/mm. s\textsuperscript{-1} &
Scanning mode\textsuperscript{{[}26{]}} & Energy
density/J.mm\textsuperscript{-2} & Incident angle/\selectlanguage{ngerman}°\tabularnewline
\midrule
\endhead
1 & -1 & 7 & 100 & Simulated Stich & 67.543 & 90\tabularnewline
2 & & & & & & 60\tabularnewline
3 & & & & & & 30\tabularnewline
\bottomrule
\end{longtable}

Before we designed our experimental parameters, we tried to start the
incident angles from a very small degree which far below 30\selectlanguage{ngerman}°, however,
we found that when incident angle is below 30°, the incident angle of
laser is too small to focus on the plane of the wound, and the laser
beam could not transfer the heat and photon into full layer of tissue.
When incident angle was below 30°, only the epidermis and upper dermis
can receive laser energy which on the other hand led to severe heat
damage of wound surface. So, we started our experiments using incident
angels larger than 30°. We designed three different incident angles
which vary from 30° to 90° in order to simulate different position of
potential wound on the back of rats, the scheme diagram is shown in
Fig.4(C). Because of the symmetry of the laser incidence, we performed a
deflected incidence of the rat wound on the fixed side.

All SD rats were purchased from Changzhou Covens Laboratory Animal co.,
LTD and familiarized with their new environment for a week. All rats
(180-220g) used were male and fed under SPF (specific pathogen-free
animal) facility with a 12:12h light--dark cycle. All surgical
procedures were conducted under animal care protocols approved by the
Ethics Committee of Nanjing University of Science and Technology.

45 rats were randomly assigned to each experimental group and
distributed by three test indexes with four time points. Fifty rats were
randomly divided into three different groups {[}90° (n = 5), 60° (n =
5), and 30° (n = 5){]} at each time point:3, 7, 14 and 24 days, and each
group had one control rat.

After anaesthetization (1.5--3 vol.\% Isoflurane), the dorsal surface
hairs were shaved and then incised with a 15mm sterile bistoury, all the
operation was carried out in Ultra-clean platform as shown in Fig.2(A).
The incision area could be observed in Fig.2 which could also be
regarded as welding area in the follow-up laser processing. The length
of full-thickness wound is 3cm and the depth of wound is 2mm. The whole
processes of animal modeling and laser bonding were conducted in
specialized ultra-clean workshop as shown in Fig.2(A) and (B). Incision
wound sites after laser treatments were covered with air-permeable
Tegarderm\selectlanguage{english} (3 M, St. Paul, MN) tape as seen in Fig.2(C). Throughout the
experiment, rats were housed individually post-operation with access to
food and water ad libitum, kept under controlled conditions of humidity
(50\%-70\%), temperature (20--22), light: dark cycle (12 h:12 h), and
free access to food and water. The digital photographs were used to
monitor the wound closure over time on days 0, 3, 7, 14 and 21 post
surgery. The wound healing progress was analyzed using \emph{Image
J}software and calculated based on the wound reduction area using the
following equation Then, the wound area was determined by tracing the
wound margins with \emph{Image J} software and an image analysis program
(Adobe Photoshop cs6). The degree of wound healing is expressed as a
percentage of original wound size. Rats were sacrificed and their dorsal
skin was surgically removed for histological
analysis\textsuperscript{{[}29-33{]}}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig.2 Representative images of surgical preparation. (A)Ultra-clean
surgical preparation area. (B) After the animal model operation (C)
Treatment after laser welding.

The wound healing progress was analysed using ImageJ software and
calculated based on the wound reduction area using the following
equation(1):

\(Healing\ Percentage(\%)=\left(\frac{\text{WA}_{0}-\text{WA}_{t}}{\text{WA}_{0}}\right)\times 100\)\% (1)

where WA\textsubscript{0}is the wound area at the wounding day (day 0)
and WA\textsubscript{t} is the wound area at each respective day.

Results

Macroscopic healing performances in vivo

Figure 3 depicts the laser welding model used in our animal experiment.
The procedure begins with making a full-thickness incision on the rats'
back. Following this, we created a solution by combining 5\% Bovine
Serum Albumin (BSA) and 10\% normal saline solution which was then
applied to the wound every 30 seconds throughout the laser operation. We
adopted a Zigzag pattern (as represented in Fig.4(B)) to mimic the
Suture technique commonly used in surgical operations. As seen in
Fig.3(C), we administered two rounds of laser treatment during the
healing process, with the initial procedure on day 0 and the subsequent
one on day 3, each session lasting for 3 minutes. We monitored the wound
recovery at intervals: 3-, 7-, 14-, and 24-days post-treatment. Although
the healing stages were generally completed by the 14th day, we
continued observations until day 24 to comprehensively document and
analyze all aspects of the healing and prognostic cycles. To probe the
laser's potential in accelerating wound healing in rats and to examine
the impacts of varying laser incident angles, we established four
groups: control (no treatment), and groups exposed to lasers angled at
90\selectlanguage{ngerman}°, 60°, and 30° respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

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-\emph{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\selectlanguage{ngerman}°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 \selectlanguage{english}< 0.05,
**, P \selectlanguage{english}< 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 \selectlanguage{greek}μ\selectlanguage{english}m magnification. Following the first
laser weld, the most pronounced recovery was observed in the 90\selectlanguage{ngerman}° 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

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 \selectlanguage{ngerman}¼ 3). Data
represent mean \(\pm\) SD; *, P \selectlanguage{english}< 0.05, **, P \selectlanguage{english}< 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 90deg 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 60deg 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 90deg laser
group, followed by the 60deg and 30deg laser groups respectively.

14 days into the healing process, notable changes were observed.
Firstly, collagen in both the 90deg and 30deg laser groups had further
proliferated, and fibroblast levels essentially returned to normal. The
90deg 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 60deg 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 90deg and 30deg 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 90deg 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Fig.6 Pathological examination of wound tissue --- Masson's Trichrome
staining of Tissue

Type I and Type III proteins have their main distribution in the dermis,
while Type IV and Type VII are chiefly located within the subcutaneous
tissue \textsuperscript{{[}47-50{]}}. Before delving into a specific
analysis of the collagen types and their distribution in samples at
various time points as represented in Fig. 7, an elementary examination
was conducted on the Sirius Red-stained microstructure under a 400x
magnification. Initially, on the third day subsequent to the initial
laser welding, sporadic deposition of COL-1 fibers around the wound was
observed, with the most considerable being in the 90\selectlanguage{ngerman}° laser group. After
the application of the second laser welding, the groups subjected to
deflected laser incident angles presented a significantly greater COL-I
fiber deposition compared to the 90° laser group. This observation
suggests that following two instances of laser welding, the wound area
in the 90° laser group had become more supple and softer. COL-1 fibers,
which are densely aligned and relatively rigid, offer structural
support, whereas COL-3 fibers contribute to the flexibility of the
tissue, primarily affecting the skin's softness. In normal tissues, the
Type I to Type III collagen ratio typically fluctuates between 1:1 and
2:1. Any significant deviation from this range may suggest instability
in the skin's collagen structure \textsuperscript{{[}51-52{]}}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Fig.7 Pathological examination of wound tissue (A) Representative images
of evolution of microstructure during healing processes by Sirius Red
staining (\(\times 400\)). (B) Quantitative data of Content of COL-1
fiber after the two stages of laser welding at different time points (n
\selectlanguage{ngerman}¼ 3). Data represent mean \(\pm\) SD; *, P \selectlanguage{english}< 0.05, **, P \selectlanguage{english}<
0.01.

Overall cell proliferation during wound healing in rats

Immunofluorescence staining was utilized to perform immune expression of
Fibronectin antibodies. As depicted in Fig.8(A), it showcases the
progressive evolutions in fibroblast expression throughout the entire
healing process---from day 3 up until day 14---following two laser
welding sessions. On the third day after the initial laser welding, it
was noted that fibroblast proliferation was most prominent in the 60deg
laser group, reaching a fluorescence intensity of 1.28 (as demonstrated
in Fig.8(B)) after normalization, with substantial proliferation
observed within the dermis. Post the second laser welding, the
fluorescence expressions within the groups stabilized around a value of
1.25, and noteworthy fibroblast clustering was detected in the 60deg laser
group. By day 14, the fibroblast content within the laser groups
returned to normal levels, with the primary fluorescence expression
concentrated within the middle and lower regions of the dermis
\textsuperscript{{[}53-58{]}}. The fibroblast expression in the 30deg
laser group was still densely concentrated, indicating a slower healing
process compared to the other two groups.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Fig.8 Representative images of wound tissue sections stained by FITC in
different treatment groups on different days (Scale bar \selectlanguage{ngerman}¼ 100 \selectlanguage{greek}μ\selectlanguage{english}m). (B)
Quantification of the Fluorescence intensity of FITC staining in
different treatment groups on different days. (n \selectlanguage{ngerman}¼ 3). Data represent
mean \(\pm\)SD; *, P \selectlanguage{english}< 0.05, **, P \selectlanguage{english}< 0.01, ***, P \selectlanguage{english}< 0.001.

Throughout the healing process, Transforming Growth Factor-\selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english})
serves as a key regulator closely associated with overall cell
proliferation. As depicted in Fig.9(A), following the initial laser
welding, TGF-\selectlanguage{greek}β \selectlanguage{english}secretion peaked in the 60\selectlanguage{ngerman}° laser group, approximating
300 pg/ml. This suggests that a low-energy laser at a 60° incident angle
could effectively activate the inflammatory response stage, stimulate M2
macrophage activity, and trigger an ample secretion of TGF-\selectlanguage{greek}β\selectlanguage{english}2 factor,
thereby catalyzing fibroblast proliferation. Although the healing
process in the 60\selectlanguage{ngerman}° laser group showed no significant improvements after
the first laser treatment, it established a solid foundation for
collagen skeleton regeneration in subsequent stages. The TGF content in
the 90° laser group was almost equivalent to that in the 30° laser group
(\textasciitilde{}200pg/ml). Following the second laser welding, the TGF
levels across all groups displayed similar rankings, albeit decreased,
indicating the onset of the re-epithelialization stage of healing. By
day 14, the TGF level within the control group began to rise gradually,
while the remaining three groups nearly returned to their normal levels.
However, the TGF level in the 90° laser group remained at around
200pg/ml, suggesting a potential recurrence of scarring.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

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\selectlanguage{ngerman}° 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-\selectlanguage{greek}β \selectlanguage{english}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\selectlanguage{ngerman}° 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

Fig.10 Representative images of upper epidermis around wound tissue
sections in different treatment groups stained by H\&E on day 3 (Scale
bar \selectlanguage{ngerman}¼ 1 mm). (B) Quantification of the length of neo-epithelial tongue
in different treatment groups on day 7 (n ¼ 3). (C) Quantification of
the area of neo-epithelium in different treatment groups on day7 ( n ¼
3). Data represent mean \(\pm\) SD; *, P \selectlanguage{english}< 0.05, **, P
\selectlanguage{english}<0.01, ***, P \selectlanguage{english}< 0.001.

Following second laser welding, we proceeded with the extraction of
samples on the seventh day as shown in Fig.11. Our aim was to
investigate the relationship between healing efficacy and the thickness
of the upper epidermis. The regeneration site of the upper epidermis was
clearly marked, subsequently expanded to a 10\selectlanguage{greek}μ\selectlanguage{english}m scale, and the thickness
of the upper epidermis was calculated. Our findings revealed that seven
days post-wounding, the least degree of upper epidermal thickness was
observed in the control group. Conversely, the most significant
thickness was noted in the 60\selectlanguage{ngerman}° laser group, followed by the 90° and 30°
laser groups respectively. A prior analysis concluded that the upper
epidermis exhibited optimal regenerative capacity after the initial
phase of 90° laser treatment. Additionally, on the seventh day following
the second round of laser welding, the thickness of the upper epidermis
reached its zenith within the 60° laser group. This suggests that the
first round of laser welding at a 90° incident angle effectively
triggers an inflammatory response, thus hastening the healing process
\textsuperscript{{[}47-49{]}}. However, the secondary laser welding at a
90° incident angle results in relatively higher thermal damage, which
paradoxically hinders epithelial regeneration. In contrast, although the
laser with a 60° incident angle didn't yield the most noticeable effect
after the first stage of laser welding, it did ensure reasonable thermal
damage based on the initial laser welding. Consequently, the healing
process was expedited following the second laser procedure.

Regarding laser welding at 30° incident angle, less epithelial
regeneration was observed in comparison to the other groups (as shown in
Fig.10(A) and Fig.11(A)). This implies that the healing process was more
convoluted and slower for these samples. More specifically, epithelial
hyperplasia was accompanied by inflammation, and there was a failure to
achieve 100\% heat transfer in the wound area at the level of the
dermis.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}

Fig.11 Representative images of thickness of upper epidermis around
wound tissue sections in different treatment groups stained by H\&E on
day 7 (Scale bar \selectlanguage{ngerman}¼ 200\selectlanguage{greek}μ\selectlanguage{english}m). (A)Representative images of
wound tissue sections stained by H\&E in different treatment groups on
day 14 (Scale bar \selectlanguage{ngerman}¼ 200\selectlanguage{greek}μ\selectlanguage{english}m and 10 \selectlanguage{greek}μ\selectlanguage{english}m).
(B) Quantification of the epidermis thickness in different treatment
groups on day 14 (n \selectlanguage{ngerman}¼ 3). Data represent mean \(\pm\) SD; *,
P \selectlanguage{english}< 0.05, **, P \selectlanguage{english}<0.01, ***, P \selectlanguage{english}< 0.001.

\par\null

However, the performance of epithelial regeneration cannot directly
correspond to the overall length of healing cycle, it can only reflect
the situation of re-epithelialization in the process of wound healing.
In general, there are crosses and intersections in all 4 stages of
healing. Therefore, in the next section, this paper discussed the
indexes including collagen deposition, collagen types and content during
the healing process combined with microscopic structures stained by
Masson in detail.

Collagen distribution during wound healing

In order to analyze the healing behavior of collagen on the
3\textsuperscript{rd} day after the first laser treatment in detail, it
can be seen from the enlarged figure in Fig.12(A) that there were
obvious collagen fractures in all groups on the third day, among which
the control group had the most significant collagen deposition, the 90deg
laser group had the most significant collagen deposition, and the 30deg
laser group had the denser morphology of healing. The red fracture is
not obvious, while the 60deg laser group has a uniform distribution of
fractures \textsuperscript{{[}48{]}}. We calculated the collagen content
and deposition in the wound area, as shown in Fig.12(B) and (C). The
collagen content in the laser affected area of the 90deg laser group was
as high as 42.6\%, and there was also significant deposition area, which
reached 36\% of the laser affected area, second only to the 60deg laser
group. Although the collagen content of the 30deg laser group reached
34.5\%, the deposition area only accounted for 26.3\%, indicating that
under the laser with large incident angle, there was a tendency to form
a dense collagen network structure at this time point, which also
explained that the laser affected area of this group of samples was
abnormally fine 14 days after healing in Figure 9. In the samples of
laser group, the proportion of collagen fractional area was the largest
in 60deg laser group, which exceeded 40\%. However, according to the above
analysis, from the collagen distribution behavior in
14\textsuperscript{th} day of healing (as shown in Fig.13(C)), the
collagen network of samples of this group was dense, which was not an
optimal healing performance. While the collagen fractional area of 60deg
laser group only occupies 34.8\%, at 14\textsuperscript{th} day,
however, the collagen network structure and fibroblast level of samples
in this group were most similar to normal tissues, indicating that the
follow-up outstanding healing state could be anticipated by the range of
the collagen fractional area, the range could be between
26.3\%\textasciitilde{}40\%.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}

Fig.12(A)Representative images of wound tissue sections stained by
Masson in different treatment groups on day 3 (Scale bar \selectlanguage{ngerman}¼ 1 mm and 50
\selectlanguage{greek}μ\selectlanguage{english}m). (B) Quantification of the collagen content around laser effect zone
in different treatment groups on day 3 (n \selectlanguage{ngerman}¼ 3). (C) Quantification of
the collagen fractional area around laser effect zone in different
treatment groups on day 3 (n ¼ 3).

As shown in Fig.13(A), the wound direction of the four groups of samples
on the 3\textsuperscript{rd} day of healing and the distribution of
different types of collagens around the wound were separately labeled.
It can be observed that the proportion of two types of collagens in the
90° laser group was the most prominent, up to 2.68, and the COL-1 fiber
content was significantly higher than that in the other three groups (as
shown in Fig.13(C)). This indicated that the tissue of the samples in
this group was relatively stiff after the first laser welding, but the
strength was higher, which met the standard of good healing. According
to Fig.13(B), the ratio of COL-1 fibers and COL-3 fibers of samples
under 90turned was also the highest. However, the ratio of COL-1 and
COL-3 fibers in the samples in two laser groups with incident angles
were significantly lower than that in the control group, indicating that
although the collagen proliferation of the samples in these two groups
was higher than that in the control group without any operation, while
COL-3 fiber was the collagen that prolifically proliferated in 60 laser
and 30 laser groups, the tissue was softer, and the elasticity was
significantly higher than that of the 90° laser group at the
3\textsuperscript{rd} day. After the first laser welding, the higher
elasticity of wound tissue indicated that the samples were not prone to
scar, and the thermal damage caused by laser was also relatively low
under 60 laser and 30 laser.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

Fig.13 (A)Representative images of wound tissue sections stained by
Picrosirius Red in different treatment groups on day 3 (Scale bar \selectlanguage{ngerman}¼
200\selectlanguage{greek}μ\selectlanguage{english}m and 10\selectlanguage{greek}μ\selectlanguage{english}m). (B) Quantification of the Ratio of collagen I:III in
different treatment groups on day 3 (n \selectlanguage{ngerman}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 7\textsuperscript{th} day
and the 14\textsuperscript{th} 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 7\textsuperscript{th} day.
This trend was maintained until the 14\textsuperscript{th} 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 7\textsuperscript{th} 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 14\textsuperscript{th} 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 7\textsuperscript{th} and the 14\textsuperscript{th} 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\textsuperscript{{[}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
14\textsuperscript{th} 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}

Fig.14 Masson and Picrosirius red staining for determination of collagen
synthesis in rat model on the 7\textsuperscript{th} and the
14\textsuperscript{th} day. (A) Typical photographs of MTS and
Picrosirius red staining at Day 7 and 14 of all the groups. (Scale
bar:200 \selectlanguage{greek}μ\selectlanguage{english}m). (B) The quantitative results of MTS on collagen deposition.
(C) The quantitative results of Picrosirius red staining on the ratio of
COL I/III. Data are expressed as the mean\(\pm\)SEM; n \selectlanguage{ngerman}¼ 3;
*P \textless{} 0.05; **P \textless{} 0.01; ***P \textless{} 0.001; ****P
\textless{} 0.0001.

Conclusion

\begin{enumerate}
\tightlist
\item
  In conclusion, given that a wound occurs at the same position, varying
  incident angles can significantly influence collagen remodeling
  behavior around the wound under uniform laser energy conditions. A
  low-energy 1064nm fiber laser, operating at an energy density of
  67.543J/mm2, can effectively expedite wound healing in rats. Among
  various angles tested, a 60° incident angle proved to be the most
  effective, achieving the fastest healing rate while ensuring optimal
  collagen network densification and minimized scarring.
\item
  In vivo observations revealed that low-energy continuous lasers with
  an incident angle around 60° exhibited superior performance in
  accelerating wound healing and limiting thermal damage. Furthermore,
  histological and immunohistochemical analyses corroborated that a 60°
  laser with an energy density of 67.543 J/cm\textsuperscript{2} could
  enhance wound cell proliferation, mitigate inflammation, stimulate
  angiogenesis, and facilitate optimal re-epithelialization.
\item
  Consequently, low-energy continuous lasers with incident angles around
  60° show considerable promise for promoting wound healing in clinical
  settings. However, it is important to note that the clinical healing
  of chronic wounds can often be significantly influenced by a patient's
  underlying health conditions. As such, our laboratory will continue
  conducting comprehensive research into the effects of low-energy
  lasers on wound healing in diabetic rats, with the ultimate goal of
  transitioning this technology into clinical trials in the near future.
\end{enumerate}

Statements \& Declarations

\textbf{Funding}

\emph{This work was supported by {[}} \emph{Qinlan Project of Jiangsu
Provienc{]} (Grant numbers {[}AD41069{]} ).}

\textbf{Competing Interests}

\emph{The authors have no relevant financial or non-financial interests
to disclose.}

\textbf{Data availability statement}

\emph{The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.}

\textbf{Acknowledgements}

\emph{All authors contributed to the study conception and design.
Material preparation, data collection and analysis were performed by
{[}Yuxin Chen{]}, {[}Jun Huang{]}, {[}Kehong Wang{]}. Animals'
experiments were performed by {[}WenTao Fan{]}. The first draft of the
manuscript was written by {[}Yuxin Chen{]} and all authors commented on
previous versions of the manuscript. All authors read and approved the
final manuscript.}

\textbf{Code, Data, and Materials Availability}

No, the manuscript does not have associated code. No datasets were
generated or analyzed during the present study. No materials were used
for the analysis.

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