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RESEARCH ARTICLE
Temporal evaluation of efficacy and quality of tissue repair
upon laser-activated sealing
Deepanjan Ghosh
1
| Christopher M. Salinas
2
| Shubham Pallod
1
|
Jordan Roberts
3
| Inder Raj S. Makin
4
| Jordan R. Yaron
1,5
| Russell S. Witte
2,6
|
Kaushal Rege
1,5
1
Biological Design Graduate Program, School
for Engineering of Matter, Transport, and
Energy, Arizona State University, Tempe,
Arizona, USA
2
James C. Wyant College of Optical Sciences,
University of Arizona, Tucson, Arizona, USA
3
School of Life Sciences, Arizona State
University, Tempe, Arizona, USA
4
School of Osteopathic Medicine, A.T. Still
University, Mesa, Arizona, USA
5
Department of Chemical Engineering, School
for Engineering of Matter, Transport, and
Energy, Arizona State University, Tempe,
Arizona, USA
6
Department of Medical Imaging, University of
Arizona, Tucson, Arizona, USA
Correspondence
Kaushal Rege, Biological Design Graduate
Program, School for Engineering of Matter,
Transport, and Energy, Arizona State
University, Tempe, AZ 85287, USA.
Email: rege@asu.edu
Funding information
National Institute of Arthritis and
Musculoskeletal and Skin Diseases,
Grant/Award Number: NIH R01 AR074627;
National Institute of Biomedical Imaging and
Bioengineering, Grant/Award Number: K01
EB031984
Abstract
Injuries caused by surgical incisions or traumatic lacerations compromise the struc-
tural and functional integrity of skin. Immediate approximation and robust repair of
skin are critical to minimize occurrences of dehiscence and infection that can lead to
impaired healing and further complication. Light-activated skin sealing has emerged
as an alternative to sutures, staples, and superficial adhesives, which do not integrate
with tissues and are prone to scarring and infection. Here, we evaluate both shorter-
and longer-term efficacy of tissue repair response following laser-activated sealing of
full-thickness skin incisions in immunocompetent mice and compare them to the effi-
cacy seen with sutures. Laser-activated sealants (LASEs) in which, indocyanine green
was embedded within silk fibroin films, were used to form viscous pastes and applied
over wound edges. A hand-held, near-infrared laser was applied over the incision,
and conversion of the light energy to heat by the LASE facilitated rapid photothermal
sealing of the wound in approximately 1 min. Tissue repair with LASEs was evaluated
using functional recovery (transepidermal water loss), biomechanical recovery (tensile
strength), tissue visualization (ultrasound [US] and photoacoustic imaging [PAI]), and
histology, and compared with that seen in sutures. Our studies indicate that LASEs
promoted earlier recovery of barrier and mechanical function of healed skin com-
pared to suture-closed incisions. Visualization of sealed skin using US and PAI indi-
cated integration of the LASE with the tissue. Histological analyses of LASE-sealed
skin sections showed reduced neutrophil and increased proresolution macrophages
on Days 2 and 7 postclosure of incisions, without an increase in scarring or fibrosis.
Together, our studies show that simple fabrication and application methods com-
bined with rapid sealing of wound edges with improved histological outcomes make
LASE a promising alternative for management of incisional wounds and lacerations.
KEYWORDS
incisional wounds, laser-activated sealing, photoacoustic imaging, skin barrier function recovery,
tissue adhesive, tissue repair, ultrasound
Received: 17 May 2022 Revised: 1 August 2022 Accepted: 8 August 2022
DOI: 10.1002/btm2.10412
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2022 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.
Bioeng Transl Med. 2023;8:e10412. wileyonlinelibrary.com/journal/btm2 1of14
https://doi.org/10.1002/btm2.10412
1|INTRODUCTION
Soft tissue trauma, including lacerations and surgical incisions, require
effective and rapid closure in order to minimize blood loss, prevent
infection and promote healing. Surgical sutures and staples are the
most commonly used devices for approximating soft tissue trauma
including in the skin.
1,2
Although effective in superficial layers of skin,
sutures do not integrate with the tissue, do not lead to immediate clo-
sure, and generally do not demonstrate optimal performance in dee-
per layers of the tissue, including in the hypodermis. In addition, tissue
strength is suboptimal at early times after tissue approximation,
which, by itself and in case of infections, can compromise effective
healing.
Localized conversion of laser light energy to heat energy using
endogenous or exogenous chromophores
3
results in rapid photother-
mal sealing of soft tissues.
3–11
Laser-activated sealants (LASEs) in
which exogenous chromophores are incorporated within a biomaterial
sealant matrix offer promising alternatives to sutures and staples. In
this approach, laser irradiation of the LASE-tissue interface and the
concomitant photothermal response can facilitate interdigitation of
LASE biomolecules and tissue proteins, which results in rapid sealing
and effective repair of soft tissues. We have previously reported the
fabrication and characterization of LASEs as an approach for the rapid
sealing and repair of ruptured tissues.
11–15
The LASE system com-
prises of three components: (i) a matrix consisting of biomaterials,
such as elastin-like polypeptides, collagen, or silk fibroin, which inte-
grate with the tissue upon sealing and act as a scaffold for aiding
repair, (ii) chromophores including gold nanorods (GNRs) or the FDA-
approved dye, indocyanine green (ICG), which convert laser light
energy to heat energy (photothermal energy conversion), thus result-
ing in a local increase in temperature, and (iii) a hand-held near-
infrared (NIR) laser tuned to 808 nm that is used to carry out the
tissue sealing procedure using LASEs. The rapid bonding of wound
edges mediated by interdigitation of tissue proteins, leading to rapid
sealing, has been demonstrated for temperatures ranging from 50–
60C.
16,17
A recent report which investigated the effect of tempera-
ture on tissue sealing observed highest welding strengths of tissue at
55C. Use of elevated temperatures greater than or equal to 65C led
to denaturation of tissue proteins and negatively impacted tissue ten-
sile strength.
18
Our previous results have shown that LASE-mediated
tissue sealing results in improved recovery of tissue biomechanical
properties in live mice, compared to Vetbond, a cyanoacrylate-based
skin glue.
11,14
In addition to facilitating sealing and repair, LASEs can
be loaded with antibacterial drugs in order to combat methicillin-
resistant Staphylococcus aureus infection in at surgical site, thus pro-
tecting the tissue.
15
ICG is an FDA-approved dye that absorbs and emits light in the
NIR region of the wavelength spectrum. Upon irradiation with NIR
lasers, approximately 85% of the energy absorbed by the dye is con-
verted into heat, which makes ICG a good photoconverter for various
applications including photothermal sealing and photodynamic ther-
apy.
19,20
In addition, ICG dye has a relatively short clearance period of
60–80 min from the body and is excreted unchanged via bile.
21
The
biodistribution and toxicity profiles of ICG dye are better understood
compared to that for nanoparticles that are used as chromophores. It
may also be possible to minimize batch-to-batch variation in LASE
properties and performance by using the well-established ICG dye.
In this study, we carried out a detailed investigation into the effi-
cacy of laser tissue sealing using functional, biomechanical, visual
(imaging), and histological evaluation at different time points during
the course of healing following surgery, and compared these out-
comes to those seen with sutures. ICG dye-loaded silk fibroin (“silk”)
films were used for sealing 1 cm, full-thickness incisional wounds in
BALB/c immunocompetent mice and transepidermal water loss
(TEWL), and ultimate tensile strength (UTS) of skin were determined
in order to investigate functional and biomechanical recovery, respec-
tively, following tissue approximation. A combination of ultrasound
(US) and photoacoustic imaging (PAI) along with histological evalua-
tion was carried out in order to further visualize and gain insights into
LASE-mediated tissue repair.
22–27
These findings indicate that LASE-
mediated tissue sealing is significantly more effective at restoring
function and biomechanical properties of skin compared to sutures at
early time points following surgery.
2|EXPERIMENTAL
2.1 |Materials
Silkworm (Bombyx mori) cocoons were purchased from Mulberry
Farms as a source of silk fibroin protein (henceforth referred to as
silk). Sodium carbonate (Na
2
CO
3
), and lithium bromide (LiBr) were
purchased from Millipore Sigma for silk fibroin extraction from silk-
worm cocoons. Dialysis bags, 3.5 kDa molecular weight cut-off
(Spectra/Por), were purchased from Fisher Scientific to facilitate puri-
fication of silk fibroin. ICG dye was purchased from MP Biomedicals
(#ICN15502050) and stored at 4C. All solutions were freshly prepared
in nanopure water (NPW; resistivity ~18.2 MΩcm; Millipore Filtration
System). BALB/c mice were purchased at ~10 weeks from Charles River
Laboratories. Commercially available 4-0 Monosof™Monofilament
Nylon Sutures (Medtronic) were purchased from esutures.com.
2.2 |LASE fabrication
Silk fibroin was extracted from B. mori silkworm cocoons using previ-
ously described protocols.
14,28
Briefly, silkworm cocoons were
degummed in a boiling 0.02 M Na
2
CO
3
(Sigma-Aldrich) solution for
30 min, washed in NPW three times, and dried at room temperature
(RT). Degummed silk fibroin was dissolved in 9.3 M LiBr solution at
60C for 4 h, centrifuged to separate insoluble contents, and dialyzed
for 72 h at 4C against a 3.5 kDa membrane in order to remove LiBr
and impurities. Dissolved silk fibroin solution was centrifuged at
14,000 rpm for 20 min to remove remaining impurities. Stock ICG
solution (5 mM dissolved in NPW) was added to aqueous silk fibroin
solution (6 wt%) and homogenously mixed to obtain a final ICG
2of14 GHOSH ET AL.
concentration of 0.1 mM. This solution (500 ml) was poured over
2cm2 cm square plastic coverslips and dried overnight at RT to
obtain silk-ICG LASE films or simply LASE films. The LASE films gener-
ated using this method had approximately 0.31 mg per film and all
films were stored at RT prior to further use.
2.3 |LASE characterization
Absorbance spectra of ICG solution (0.1 mM), as-prepared LASE,
LASE dissolved in saline to form a viscous paste, and LASE after laser
irradiation were recorded from 400 to 999 nm using UV–Vis absorp-
tion spectroscopy (Synergy 2 Multi-Mode Reader; BioTek Instru-
ments). Absorbance spectra of NPW and silk fibroin film (LASE
without ICG) were also recorded as controls for ICG solution and
LASE, respectively. A hand-held, continuous wave NIR laser (LRD-0808;
Laserglow Technologies), coupled with armored optical fiber with
FC/PC connector (#AFF2001X, 1 m in length, and 200 μmincorediam-
eter), and tuned to 808 nm was used for laser irradiation. The fixed laser
spot size was 2 mm. A FieldMate laser power meter was used to mea-
sure the power of the laser beam, and power density of the laser beam
was calculated by dividing the power of the laser beam by the area of
the beam. An A325sc infrared (IR) camera (FLIR), equipped with a
10 mm 45lens, was used to determine the surface temperature of
LASE during laser exposure.
2.4 |Sealing of full-thickness incisional wounds
in mice
BALB/c mice (10–12 weeks, weighing ~22–25 g; Charles River Labo-
ratories) were used in this study and were housed in groups of five
until surgery. All animal care and procedures were performed in strict
compliance with protocols approved by the Institutional Animal Care
and Use Committee (IACUC) at Arizona State University. Before sur-
gery, mice were anesthetized with 120 mg/kg ketamine and 6 mg/kg
xylazine (100 μl cocktail) by intraperitoneal injection. Dorsal hair was
clipped, and the skin was prepped using three alternating swabs of
chlorhexidine gluconate and 70% isopropyl alcohol. Two 1-cm full-
thickness incisions were made side-by-side on the back of each
mouse spaced roughly 2 cm apart using sterile scalpel blades (#15;
Integra Miltex).
29
In case of suture-closed incisions, four evenly
spaced simple interrupted knots were used to close a 1-cm incision
using 4-0 nylon suture (#SN5699G; Medtronic; Monosof Black 1800 P-
13 cutting). For LASE-sealed incisions, 10 μl of phosphate-buffered
saline (PBS; pH 7.4) were topically applied and a 1.2 cm 0.5 cm
LASE film was placed over the incision; contact of LASE with PBS
resulted in quick dissolution of the film to form a viscous paste
between the incision edges. The incision edges were approximated
using a forceps, and the incised edges were aligned prior to laser
sealing. The LASE-tissue interface (incision line) was irradiated at a
rate of 0.5 mm/s with the NIR laser tuned to 808 nm (CW) for
1 min while keeping the incision line approximated using forceps.
The laser was applied at an angle between 60and 80to the skin
at a power density of ~5.1 W/cm
2
(~160 mW power output,
~2 mm laser beam diameter), corresponding to temperatures in the
range of 50–60CattheskinLASE surface.
15
Closure of left and
right incisions with sutures or LASE were randomized. The mice
were allowed to recover on heating pads until mobile and were
housed individually. Incisions were assessed every day for up to
7 days postsurgery for any signs of infection, suture removal, or
wound dehiscence and mice with any of these conditions were
removed from the study.
2.5 |Measurement of TEWL of healing wounds
TEWL is a measurement of change of water vapor density across the
stratum corneum layer and is used to assess the barrier function of
skin. Disruption to skin due to trauma, injury, wounds results in ele-
vated TEWL levels and is indicative of weaker barrier function.
30,31
In
this study, TEWL was measured on Days 2, 4, 7 postsurgery using a
portable, closed chamber VapoMeter device (#SWL5580; Delfin
Technologies). The VapoMeter was fitted with a small adapter
(4.5 mm diameter, ~16 mm
2
surface area) and a closed chamber was
created on skin contact during the duration of measurement (~9–
15 s). Ambient relative humidity and temperature (C) were recorded
during every measurement using a room sensor (#RHD1367) supplied
along with the VapoMeter. For every TEWL reading, three consecu-
tive readings were acquired along nonoverlapping regions over an
incision area and the chamber was passively ventilated between every
measurement.
32,33
TEWL readings of unwounded skin of sham mice
were acquired on the same days. In all cases, TEWL values were
recorded using the DMC software (Delfin Technologies) and values
are displayed as mean ± standard error of mean from six independent
mice (n=6).
2.6 |Biomechanical recovery of skin strength
Following euthanasia, rectangular section of the healed skin
(~2 cm 1 cm) were excised around the incision area on Days 2 and
7 postclosure to investigate the biomechanical recovery of skin fol-
lowing suturing or laser sealing. In case of suture-closed incisions,
sutures were removed prior to tensile strength measurements in order
to obtain strength of the healed skin alone. Excised skin samples were
secured in clamps and stretched until failure stretched at a rate of
2 mm/s under constant tension using a TA.XT Plus texture analyzer
(Texture Technologies Corp.). UTS was determined from the maxi-
mum force of the tissue prior to failure, where the maximum force (F)
and cross-sectional area of the tissue sample (A, length of skin sample
1 cm and tissue thickness 500 μm) determined the UTS (σ, Pa) of
healed skin (σ=F/A). The tensile strength of unwounded skin (~2 cm
length 1 cm) was also tested for comparison. All tensile strengths
are displayed as mean ± standard error of mean from six independent
experiments (n=6).
GHOSH ET AL.3of14
UTS of healed skin in PaðÞ
¼Maximum force at skin ruptre in NðÞ
Cross sectional area of skin in m2, length thicknessðÞ
:
Percentage intact skin strength for healed skin were calculated as a
difference between UTS for each group either on Day 2 or Day 7 post-
closure from unwounded skin strength, with the difference then con-
verted to a percentage.
%intact skin strength
¼UTS of healed skin on Day 2 or Day 7ðÞ
UTS of unwounded skin no incision control onDay 2 or Day 7ðÞ
100:
2.7 |US and PAI of LASE-tissue interface
Similar to studies on biomechanical recovery of skin, rectangular sec-
tions of the healed skin (~2 cm 1 cm) were excised around the inci-
sion area on Days 2 and 7 postclosure, collected in biopsy cassettes,
and stored in ice-cold 1X PBS (10 mM sodium phosphate, 1.8 mM
potassium phosphate, 2.7 mM potassium chloride, 137 mM sodium
chloride, pH 7.4) prior to US and PAI. All skin specimens were imaged
within ~4 h of necropsy and skin collection. Skin specimens were
removed from 1X PBS, blotted to remove excess buffer and embed-
ded in a 1.5% agarose gel (Millipore Sigma; #A9539; low EEO) within
an in-house 3D printed tray (Figure 1a). The outer section of the tray
was filled with deionized water in order to submerge the agarose layer
(Figure 1a). High-resolution US and PAI were carried out with the
MX550D (50 MHz) linear array transducer, fiber bundle and motor
setup of the Vevo 3100 +LAZR-X (VisualSonics) at the University of
Arizona, Tucson, AZ. A transducer jacket (VisualSonics; Figure 1bii)
was used to combine the transducer (Figure 1bi) and fiber bundle
(Figure 1biii), allowing the laser light to be directed to a region 7 mm
away from the transducer head. The transducer with jacket was low-
ered into the water bath to achieve opto-acoustic coupling for PAI
with an optimal standoff required for the LAZR-X system of approxi-
mately 7 mm. The spatial resolution of the imaging system is ~30
microns transverse normal, ~50 μm azimuth, and ~ 300 μm slice thick-
ness. For 3D scanning of skin, the transducer with jacket was moved
incrementally at a step size of 150-μm in the elevational direction
across the length of each skin sample (Figure 1biv). For each skin speci-
men, PA data were obtained at eight wavelengths (40-nm increments
from 680 to 960 nm) for each depth/width cross-section of the 3D
scan, along with a standard pulse echo US image. A center slice for each
sample is chosen for full PA spectrum characterization (5-nm incre-
ments from 680 to 960 nm). Furthermore, length/width sample cross-
sections are compiled from the 3D data set in image postprocessing.
Spectral unmixing was performed on the obtained PA signal to
discern the tissue constituents. The VevoLAB software (VisualSonics)
is utilized for such spectral unmixing, where three wavelength compo-
nents (680, 800, 960 nm) are used to discern LASE signal from that of
the weakly absorbing normal skin. Multi-wavelength unmixing for ICG
content carried out using the Vevo system has been shown previously
to produce accurate results for deep tissue imaging in tissue phan-
toms and murine subjects.
34,35
Control skin has a relatively weak and
flat PA spectrum across the wavelength band, implying that the strong
ICG absorption at 800 nm can be used to identify LASE within the
samples (Figure 1c), considering that ICG is mixed with silk to form
the LASE.
2.8 |Tissue collection and processing for histology
analyses
Following euthanasia, healed tissues were carefully excised, flattened
between two foam biopsy sponges in a tissue cassette, and fixed by
submersion in 10% neutral-buffered formalin (#HT501128; Sigma-
Aldrich) for a minimum of 72 h at RT. Tissues were dehydrated
through a graded alcohol series and paraffin embedded with Paraplast
Plus (#19217; EMS Diasum) by manual processing (Table S1). Individual
5-μm thick sections were cut with an Accu-Cut SRM 200 Rotary Micro-
tome (Sakura Finetek USA) and collected on charged glass slides
(Hareta, Springside Scientific) in a floating water bath (XH-1003; IHC
World). Slides were dried overnight at 37C and stored at RT until use.
2.9 |Hematoxylin and eosin staining
Dried sections on charged glass were deparaffinized and rehydrated
thorough xylene and graded alcohols into tap water. Rehydrated sec-
tions were submerged in a solution of Hematoxylin (Gill No. 2;
#GHS232; Sigma-Aldrich) for 3 min, differentiated by 6–12 quick dips
in acid alcohol (0.3% hydrochloric acid in 70% ethanol), and blued in a
solution of ammonium water (0.2% ammonium hydroxide in distilled
water) for 30 s. Slides were further submerged in a solution of 0.5%
Eosin Y (#318906; Sigma-Aldrich) in distilled water (acidified with
0.2% glacial acetic acid vol/vol) for 4 min followed by dehydration
through 90% and absolute ethanol, further dehydrated in two changes
of 100% xylene, dried and mounted in CytoSeal XYL (Richard-Allan/
Thermo Fisher Scientific). Samples were imaged on an Olympus BX43
upright microscope equipped with an Olympus DP74 CMOS camera
operated by cellSens Standard software (Olympus Corporation).
2.10 |Picrosirius red staining
Dried sections on charged glass were deparaffinized and rehydrated
thorough xylene and graded alcohols into tap water. Rehydrated sec-
tions were submerged in a 0.1% solution of Picrosirius Red composed
of Direct Red 80 (#365548; Sigma-Aldrich) in a saturated aqueous
solution of picric acid (#P6744; Sigma-Aldrich) for 1 h at RT to achieve
stain saturation. Slides were washed twice in acidified water (0.5% gla-
cial acetic acid in distilled water) for 2 min each. Slides were dehydrated
through an abbreviated 90% and absolute ethanol series, further dehy-
drated in xylene and mounted in CytoSeal XYL (Richard-Allan/Thermo
Fisher Scientific). Brightfield images were collected on an Olympus
4of14 GHOSH ET AL.
BX43 upright microscope equipped with an Olympus DP74 CMOS
camera operated by cellSens Standard software (Olympus Corporation).
2.11 |Immunohistochemistry
Captured sections were rehydrated and overnight epitope retrieval
was performed in sodium citrate buffer at 60C. Tissue sections were
blocked with 5% BSA in TBS containing 0.2% Tween-20 (TBST) at RT
for 1 h and incubated overnight with primary antibodies for iNOS
(Abcam; ab15323; rabbit polyclonal; 1:50), Arginase-1 (Cell Signaling
Technologies; #93668; rabbit monoclonal; 1:200), or Ly6G
(Invitrogen; #14-5931-82; rat monoclonal; 1:100). Secondary anti-
bodies were probed for 2 h at RT using HRP-(rabbit) or AP-(rat) conju-
gates (Jackson Immunolabs). Tissues were developed with ImmPACT
DAB (HRP) or Vector Red (AP) substrate (Vector Labs). Arginase-1
and Ly6G sections were counterstained with hematoxylin while iNOS
sections were left without counterstain. Tissues were dehydrated
through alcohol and xylene and mounted with Cytoseal XYL. Images
were collected on an Olympus BX43 upright microscope equipped
with an Olympus DP74 CMOS camera operated by cellSens Standard
software. Images were quantified in ImageJ/FIJI.
2.12 |Image analyses
Morphometric features of healing were assessed in ImageJ/FIJI.
Images were calibrated according to magnification. Epidermal gap
was measured as the linear distance between the two epidermal
faces of the wound edges and identified by canonical appearance
of the epidermis via hematoxylin and eosin (H&E) staining on Days
2 and 7 postinjury. Histological scar area was measured as the area
weakly stained by Picrosirius Red and bounded by the basement of
the epidermis and above the hypodermis, and the edges of the
mature collagen in periwound tissue strongly stained by Picrosirius
redonDay7postinjury.
36
Dermal gap was measured as the linear
distance between intact areas of dermis, indicated by bundled col-
lagen fibers.
FIGURE 1 Set up for ultrasound
(US) and photoacoustic imaging (PAI) for
skin incisions closed with sutures of
sealed with LASEs. (a) Prior to imaging,
excised skin samples were removed from
ice-cold 1X PBS and placed over a layer of
1.5% agarose low electroendoosmotic
(EEO) cooled to room temperature in a
3D printed sample tray. Following this,
another layer of agarose solution (~35–
45C) was poured over the skin samples
to completely embed the tissues within
the agarose layers. The sample tray was
then filled with deionized water to form a
layer over the agarose layer.
(b) (i) Scanning of skin samples were
carried out using the Vevo 3100 motor.
(ii) The MX550D (50 MHz) linear array
transducer and jacket consisting of the
fiber bundles (iii) is lowered into the
sample tray submerged in water to
facilitate opto-acoustic coupling with a
7 mm standoff from the skin samples (iv).
(c) Normalized photoacoustic signal of
LASE, suture, and skin sections in the
range of 680–960 nm.
GHOSH ET AL.5of14
2.13 |In vivo live mouse US of sealed wounds
Mice (2-day post-incision) were anesthetized using ketamine/xylazine
cocktail. Once under surgical anesthesia (confirmed by toe pinch),
mice were placed on a heated mat and imaged by US with a GE Logiq
eNextgen ultrasound system equipped with a 10–22 MHz transducer
(Figure S1). Mice were euthanized postimaging and tissues were fixed
and processed as described below. US data were transferred into
ImageJ/FIJI and calibrated according to the length of the transducer's
dimensions (19.3 mm field width). To enhance better feature visualiza-
tion, the US images were normalized by using an ImageJ native
Bandpass Filter function, and epidermal/dermal gap was quantified by
linear measurement. H&E-stained images for matched mice were eval-
uated and epidermal/dermal gap was quantified by linear measure-
ment. Correlation between B-mode US images acquired in vivo and
H&E histology for Day 2 incisions was evaluated by simple linear
regression with 95% confidence interval in GraphPad Prism.
2.14 |Statistical analyses
Data from absorbance, TEWL, and skin UTS are presented as mean
± standard error of the mean. Differences between groups were
assessed using two-way analysis of variance followed by Fisher's LSD
test using GraphPad Prism version 9.2.0 (GraphPad). A p< 0.05 was
considered statistically significant.
3|RESULTS AND DISCUSSION
Laser sealing is an attractive approach for the sutureless approxima-
tion of tissues, including skin, and possesses several potential advan-
tages including fast operation times, low scarring, and faster recovery
of tissue function. However, the temporal dependence of the efficacy
and quality this approach has not been investigated thoroughly. We,
therefore, carried out detailed studies to investigate the efficacy of
laser sealing in live mice in a temporal manner and compared findings
with those seen with sutures. In addition to biomechanical recovery
with UTS, functional recovery of barrier function of skin (using TEWL
measurements), US and photoacoustic visualization and histology
studies were used to develop a more comprehensive investigation
into the quality and efficacy of tissue repair following laser sealing.
3.1 |Generation and characterization of LASE
films
Silk fibroin (“silk”)-ICG LASE films (Figure 2a) were prepared using sol-
vent evaporation methods as described in our previous reports.
15
Briefly, aqueous solutions of silk fibroin (6 wt% or 60 mg/ml) with
0.1 mM ICG were cast and dried overnight at RT resulting in the gen-
eration of LASE films following solvent evaporation. Light absorption
analyses indicated that the LASE films displayed a characteristic
absorbance similar to that of ICG dye (Figure 2b). Upon addition of
saline solution, LASE films dissolve to form an adhesive viscous paste,
which is suitable for sealing incisional wounds. The viscous paste
maintains absorbance properties in the NIR window as seen in case of
dry LASE films. This viscous adhesive paste was irradiated using a
hand-held 808 nm continuous wave laser under conditions (power
output ~100 mW, power density ~3.2 W/cm
2
, 1 min, LASE surface
temperature ~50–60C) similar to those used for in vivo skin sealing.
No significant shifts in absorbance profiles of these irradiated viscous
LASE pastes were seen (Figure 2b). The retention of absorbance prop-
erties by laser-irradiated LASEs warrants the use of optical visualiza-
tion methods for probing LASE following tissue sealing in subsequent
applications.
We also investigated the photothermal response of LASE films
irradiated with a NIR (808 nm), continuous wave hand-held laser
turned on for 15 s (“on”cycle) and off for 15 s (“off”cycle) for a total
of 3 cycles. For photothermal response studies, a LASE section was
applied to an ex vivo porcine skin where the LASE section turned into
a viscous paste upon contact with the skin and the surface tempera-
ture was recorded using an IR camera. Upon irradiation with a laser, a
rapid increase in surface temperature of the LASE-tissue interface
was observed due to efficient photothermal conversion of the embed-
ded ICG dye in the LASE matrix. This photothermal response was repro-
ducible over 3 cycles and varied using different laser power densities
(1.6–2.4 W/cm
2
)(Figure2c). Irradiation of porcine skin alone and LASE
without ICG even at the highest laser power density tested
(2.4 W/cm
2
) did not result in any increase in temperature (red and blue
dotted line, respectively, Figure 2c). Surface temperatures in the range
of 50–60C (shown using blue shaded region) optimal for tissue sealing
were achieved by modulating the laser power density (Figure 2c).
3.2 |In vivo sealing of skin incisions: Barrier
function recovery and healed skin strength
Full-thickness incisional dorsal skin wounds in BALB/c mice, 1-cm in
length, were sealed using LASE or approximated with 4-0 Nylon
sutures. In the case of laser-sealed incisions, the LASE-tissue interface
(incision line) was irradiated for 1 min at a power density of ~5.1 W/
cm
2
(~160 mW power output, ~2 mm laser beam diameter), corre-
sponding to temperatures in the range of 50–60C at the skinLASE
surface (Figure 3a). Mice were allowed to recover and representative
images of incisions on Days 0 (immediately after surgery), 2, 4, and
7 are shown in Figure 3b. Mice without incisions were surgically
prepped, recovered in a similar manner, and were used as controls in
subsequent studies. Following wounding and closure of skin incisions,
barrier function recovery of the healing skin was determined in a non-
invasive manner using measurements for TEWL. TEWL is a marker of
skin permeability which measures water loss thorough the stratum
corneum layer and is one of the standard methods to evaluate barrier
function of skin. Any damage or trauma to the skin barrier leads to an
increase in TEWL levels compared to intact or unwounded skin.
32
TEWL levels of the 1-cm long incisions closed with LASE or sutures
6of14 GHOSH ET AL.
were determined on Days 2, 4, and 7 postsurgery from three nono-
verlapping regions (shown by white arrows) over the incision line
(Figure 3c). The skin in the incisional region closed with LASE or
sutures demonstrated gradual decrease in TEWL values. At Day
2 postwounding, the average TEWL value for incisions closed with
LASE (29.5 ± 1.9 g/m
2
h) was significantly lower than sutured inci-
sions (41.6 ± 4.4 g/m
2
h; p=0.0001). On Days 4 and 7 postwounding,
TEWL values of LASE-sealed incisions were lower compared to those
with sutures (Day 4: 20.6 ± 0.6 vs. 25.5 ± 0.7 g/m
2
h; p=0.0893 and
Day 7: 17.3 ± 1.1 vs. 21.2 ± 1.7 g/m
2
h; p=0.1717), but the differ-
ences were not statistically significant (i.e., pvalues were not <0.05).
Average TEWL values of unwounded skin on Day 2, 4, and 7 post-
wounding were considered as baseline on those corresponding days
(Figure 3d).
In the above TEWL studies, Days 2, 4, and 7 postinjury were cho-
sen in order to investigate early, mid, and later stages of wound repair
following closure by primary intention as in case of incisional wounds.
The time point of Day 2 postinjury is a good temporal representative
of the early inflammatory phase, which helps with clearance of tissue
debris from injury and kickstarts processes that prepare the wound
for subsequent stages of repair, that is, proliferation and remodeling.
Day 7 postinjury was chosen as a likely representative of the later
remodeling phases, and Day 4 likely captures proliferation and/or
potentially the transition from the proliferation stage to the later
remodeling stage. During proliferation and remodeling stages, deposi-
tion of collagen matrix, angiogenesis, and maturation of granulation
tissue are key events, and newly deposited matrix leads to an
increased tensile strength of healing wounds. This phase can vary in
length based on the wound site, tissue type, and type of injury. Mouse
skin heals by contraction, which is different from skin healing by re-
epithelialization in humans. Contraction-facilitated healing in mice
shows faster kinetics of closure (e.g., over a 4–7-day period) com-
pared to wound healing dynamics seen in humans. To that end, inter-
rogation at an earlier time point, that is, Day 2, can lead to meaningful
insights into the efficacy of different wound approximation devices
including sutures and LASEs.
Mice were euthanized on Days 2 or 7 postwounding to evaluate
biomechanical recovery of the healing skin both at an early and late
time point. At the earlier healing timepoint (Day 2 postwounding),
LASE-sealed incisions had higher UTS (0.87 ± 0.13 MPa) compared to
sutured incisions (0.47 ± 0.08 MPa; p=0.0464). The UTS of
unwounded (no incision control) skin of BALB/c mice of the same age
range was 2.67 ± 0.17 MPa and was used to compare the efficacy of
healing using sutures and LASE. Incisions closed with sutures and
FIGURE 2 Silk-ICG laser-activated
sealants (LASEs). (a) Representative image
of a 2 cm 2 cm LASE film fabricated
from silkworm silk fibroin and indocyanine
green (ICG) dye; the green color of the
LASE is because of the ICG dye.
(b) Absorbance spectra of ICG dye alone
(dashed and dotted purple line), LASE
film—as it is fabricated (dashed blue line),
LASE in a viscous paste form after
addition of saline, which was used to
mimic a moist environment in wound beds
(dashed red line), and post-laser
irradiation in the paste form (dotted black
line). Data shown are mean ± standard
error of the mean of n=4 independent
LASE films. (c) Photothermal response of
LASE on ex vivo porcine skin irradiated
using a continuous wave NIR laser tuned
to 808 nm at varying laser power density
from 1.6 to 2.4 W/cm
2
in a 15 s “on”and
15 s “off”cycle (3 cycles total).
Photothermal responses of silk films (with
no ICG dye added, red dashed line) and
porcine skin (blue dashed line) following
irradiation with the laser at 2.4 W/cm
2
are
also shown. The region shaded in light
blue color (temperature range from ~50C
to ~60C) indicates the optimal
temperature window for laser tissue
sealing. Each photothermal response
curve is a mean of n=3 independent
experiments.
GHOSH ET AL.7of14
LASE resulted in a UTS recovery of approximately 20.4 ± 2.6% and
35.4 ± 3.6%, respectively, relative to that of intact skin on Day 2 post-
closure (secondary axis in Figure 3e). Our results indicate improved
efficacy in recovering the skin tensile strength at an earlier timepoint
with LASE, which is consistent with our previous observations with
silk-GNR gold nanorod sealants for incisional skin repair.
14
At the lat-
est healing time point (Day 7 postwounding), UTS of LASE- and
suture-closed skin increased to 1.15 ± 0.23 and 1.11 ± 0.16 MPa,
respectively (not significant; p=0.8720). At this time point, suture
and LASE closures resulted in a recovery of approximately 41.6
± 4.9% and 43.1 ± 7.1% in UTS, respectively, relative to that of intact
skin (Figure 3e; secondary axis). This is likely because of the contrac-
tile forces in mice skin that aid healing at later durations postsurgery.
Effective functional and biomechanical recovery of skin at early
times following injury or surgery is critical particularly considering that
different pathologies influence the rate of wound healing. For exam-
ple, diabetic humans and mice demonstrate delayed wound healing.
To that end, our approach of following the efficacy of incisional
wound healing using TEWL measurements with time is well-suited to
address temporal progress of healing including in different pathologies
that influence tissue repair. For slower healing wounds, faster closure
and effective tissue repair are imperative in order to prevent
FIGURE 3 Functional and biomechanical recovery of skin following suture closure and LASE sealing in Balb/c mice. (a) Photothermal
response of LASE-skin interface during in vivo sealing irradiated using a continuous wave NIR laser tuned to 808 nm at a laser power density of
~5.1 W/cm
2
. The region shaded in light blue color (temperature range from ~50C to ~60C) indicates the optimal temperature window for laser
tissue sealing. The photothermal response curve shows data that are a mean of n=3 independent experiments. (b) Representative images of
1-cm long skin incisions closed with four, simple interrupted 4-0 nylon sutures or LASE on Days 0 (immediately after closure), 2, 4, and
7 postwounding; control is unwounded skin surgically prepared similarly to incised skin. (c) Representative image showing three approximate
locations at which TEWL measurements were carried out (white arrows) for each type of closure method. (d) Transepidermal water loss (TEWL)
of healed skin and unwounded control skin on Days 2, 4, and 7 postwounding. TEWL value (in g/m
2
h) for each incision type is the average TEWL
measurement from three nonoverlapping spots over the incision line shown in b. Data shown are mean ± standard error of the mean of n=6
mice. (e) Ultimate tensile strength (UTS) and recovery, that is, %UTS of intact skin strength (secondary axis shown in red) of healed skin on Days
2 and 7 postwounding for suture-closed and LASE-sealed incisions. Data shown are mean ± standard error of the mean of n=6 mice. Statistical
significance was determined using two-way ANOVA followed by Fisher's LSD test and individual pvalues are shown; p< 0.05 are considered
statistically significant.
8of14 GHOSH ET AL.
infections and further complications. To that end, the LASE approach,
which shows better barrier function (TEWL) and biomechanical (UTS)
recovery at earlier time points (Day 2), has the potential to also
engender better outcomes in hosts with slower healing wounds.
3.3 |US and PAI
For PAI, a full spectrum scan between wavelengths of 680–980 nm, with
a 5-nm increment, was carried out for a representative center slice of
every skin sample. LASE PA signal shows a maximum at 800 nm, which is
expected given the absorption of ICG. Control skin displays weak PA sig-
nal across all wavelengths, while the black-colored suture produces strong
broadband signal. For PAI and spectral unmixing, signal data must be
acquired at minimum of three wavelengths to distinguish three separate
constituents (i.e., skin, suture, LASE). Observing the PA signal spectrum of
each constituent, wavelengths of 680, 800, and 960 nm are chosen for
the spectral unmixing. A 3D scan is then carried out for each sample with
data being acquired at the three unmixing wavelengths (Figure 4a–c). It
was qualitatively observed that there was a considerable drop in normal-
ized PA signal at 800 nm on Day 7 postclosure compared to Day 2 post-
closure (Figure 4b,c). This is further observed as reduced PA signal from
LASE identified by the spectral unmixing technique on Days 2 and 7 post-
closure. The maximum depth of penetration of LASE signal in the wound
bed from four independent LASE-sealed skin incisions was calculated
from the normalized PA signal overlaid on individual US images at both
the timepoints (Figure 5a). The average depth of the LASE signal in the
wound bed was 1.4 ± 0.2 and 0.6 ± 0.45 mm on Day 2 and Day 7 post-
closure samples, respectively (Figure 5b). The PA signal depth can be
indicative of persistence of LASE in the wound bed as the healing of the
incisional wound progresses over time.
3.4 |Histological evaluation of LASE-sealed and
suture-closed skin sections
During wound healing, re-epithelialization is a crucial step for restor-
ing barrier function and preventing exposure to pathogens that cause
surgical site infections.
37
We visualized the cellular and tissue pro-
cesses that lead to skin healing using a histological analysis (Figure 6a,
b). At Day 2 postclosure, suture-closed skin incisions had a lower epi-
dermal gap compared to LASE-sealed incisions (Figure 6a,c). The
increased epidermal gap seen in LASE-sealed incisions may be attrib-
uted to heat ablation of keratinocytes in the immediate periphery of
the LASE.
13
However, we observed a continuity of closure in the
LASE-sealed wounds, despite the difference in epidermal gap, due to
the occupancy of the gap by the LASE material itself, analogous to an
eschar. By Day 7 postclosure, no significant difference in epidermal
gap was observed between the two groups. Dermal gap (the distance
between the collagen fronts of the dermis at the wound edge) was
not different between suture of LASE-sealed wounds at Day
2(p=0.2870) or Day 7 (p=0.5216), although as expected there was
a reduction of dermal gap within the treatment groups between Days
2 and 7 for suture (p=0.0867) and LASE (p=0.0331) (Figure 6a,d).
We also evaluated the histological scar area, observed through picro-
sirius staining, to determine if there is a difference in initial scarring
during the healing period in incisions closed with suture or LASE
(Figure 6b,e).
38
Histological scar areas of 0.07 ± 0.01 and 0.08
FIGURE 4 Normalized sample PA
signal. Computed transverse slices of B-
mode scans co-registered with normalized
PA signal at 680, 800, and 960 nm for
(a) control skin without any incision
surgically prepped similarly to skin
samples with incisions (b) skin incisions
sealed using LASE at Day 2 postclosure
and sealing (c) skin incisions sealed using
LASE at Day 7 postclosure and sealing.
Co-registered B-Mode and PA images are
obtained by selecting a slice from the 3D
scan data set that corresponds with
approximately 500 μm subsurface depth
(scale bar in yellow =5 mm).
GHOSH ET AL.9of14
± 0.01 mm
2
(p> 0.05) were seen in case of suture-closed and LASE-
sealed incisions, respectively, at Day 7 postclosure, indicating that
both resulted in similar levels of scarring based on these analyses
(Figure 6e). Nascent collagen deposition was not appreciable on Day
2 postclosure; thus, no scar area was yet present (data not shown).
This is expected considering the longer timeline necessary for
FIGURE 5 Depth profile of LASE in
wound bed. (a) Cross-section of B-mode
scans superimposed with the normalized
PA signal from LASE at 800 nm shown in
green for skin samples at Day
2 postclosure and Day 7 postclosure.
(b) The depth profiles of photoacoustic
signal from LASE in the wound are
represented as mean ± standard error of
mean of n=4 LASE-sealed skin samples
at each timepoint (scale bar in
yellow =5 mm).
FIGURE 6 Histological evaluation of
skin sections during the course of healing
following closure with sutures or sealing
with LASE. (a) Representative hematoxylin
and eosin (H&E) stained micrographs of
the wound sections (4 magnification)
showing the epidermal gap (black arrows)
and dermal gap (red line) on Days 2 and
7 postclosure (scale bar =200 μm).
(b) Representative picrosirius red stained
micrographs of the wound sections (10
magnification) showing the scar area in
the granulation tissue (black dotted line
area) at Day 7 post closure. (c and d)
Quantification of epidermal gap and
dermal gap in skin sections (in μm) closed
with suture or LASE on Days 2 and
7 postclosure. (e) Quantification of
histological scar area (in mm
2
) in skin
sections closed with suture and LASE on
Day 7 postclosure. Data shown are mean
± standard error of mean of n=6 mice
per group. Statistical significance was
determined using two-way ANOVA (for
epidermal gap quantification) and one-
way ANOVA (for scar area quantification)
with Fisher's LSD post hoc analysis.
*p< 0.05 is considered significant.
10 of 14 GHOSH ET AL.
development of scar-like formation. Taken together, these results
indicate that LASE-sealed incisions exhibit rapid and robust sealing
with minimal effect on scarring or tissue integrity, while also providing
an improvement in early barrier function and tissue strength. While
both sutures and LASE-sealed incisions exhibited some degree of epi-
dermal and dermal gap, sutures are an interrupted sealing method
(i.e., sutures have empty space between individual placements) and
tissue puckers and is open between each knot. LASE, on the other
hand, provide a continuous seal across the length and width of the
incision, bridging the tissue space and providing a more complete pro-
tection from the environment, in a manner similar to a natural eschar,
but on-demand and with high strength. This, in part, contributes to
FIGURE 7 Immunohistochemical evaluation of incised skin during the course of healing. (a) Representative micrographs and quantification of
wound sections (4 magnification) stained for Ly6G (pink chromogen) with hematoxylin counterstain at Days 2 and 7 postclosure (scale
bar =200 μm). (b) Representative micrographs and quantification of wound sections (4 magnification) stained for Arginase-1 (brown
chromogen) with hematoxylin counterstain at Days 2 and 7 postclosure (scale bar =200 μm). (c) Representative micrographs and quantification
of wound sections (4 magnification) stained for iNOS (brown chromogen) without nuclear counterstain at Days 2 and 7 post closure (scale
bar =200 μm). Data shown are mean ± standard error of mean of n=5–7 mice per group. Statistical significance was determined using two-way
ANOVA (for epidermal gap quantification) and one-way ANOVA (for scar area quantification) with Fisher's LSD post hoc analysis. Significance
indicated as *p< 0.05; **p< 0.01.
GHOSH ET AL.11 of 14
the higher biomechanical and functional recovery seen with LASE
sealing.
Immunohistochemical analysis of tissue sections indicated that
wounds treated with LASE had a significant reduction in Ly6G-
positive infiltrating neutrophils at 2-day postclosure (p=0.0192)
(Figure 7a). While there was a nonsignificant trend toward increased
iNOS-positive proinflammatory macrophages at 2-day postclosure
(p=0.0940) and Arginase-1-positive proresolution macrophages at
7-day postclosure (p=0.1672) (Figure 7b,c), we observed an
enhancement of Arginase-1 response (proresolution macrophages) at
7-day postclosure versus 2 days with LASE (p=0.0207) which did
not reach significance for sutures (p=0.2693). These data indicate
that sealing wounds with LASE induces an augmentation of immune
cell behavior at early and late stages of wounding, with an enhancing
effect on the arginase-1-positive prohealing macrophage response
and a distinct effect on the dynamics of infiltrative neutrophils. The
role of neutrophils in healing wounds is evolving, with recent evidence
suggesting both positive and negative roles in regulating the healing
process.
39
While neutrophils play an early role in protecting against
infection, they are also drivers of early signals to stimulate repair.
Neutropenia is associated with slower healing and deficiency of sev-
eral signals involved in neutrophil function can result in impaired
healing.
40–43
Conversely, persistence of neutrophils within a wound
can delay healing and an overabundance of neutrophil-derived
PAMPs, such as neutrophil extracellular traps (NETs), can lead to
chronic wounds and have become therapeutic targets to improve
wound healing.
44–47
Here, we show that neutrophils are present in
both sutured and LASE-sealed wounds at Day 2, but in much lower
abundance in LASE-sealed wounds, with levels equalizing by Day
7. Most studies investigating the role of neutrophils in wound healing
utilize excisional wounding models, which proceeds by secondary
intention healing (granulation). Incisional wounding, performed here,
proceeds by primary intention healing and may utilize different biolog-
ical mechanisms. Our finding that reduced neutrophils in LASE-sealed
incisional wounds compared to sutured incisional wounds is in agree-
ment with a recent study by Heuer et al.,
50
which showed that mouse
laparotomy wounds (primary intention healing) treated with DNase I
(to deplete NETs) or with PAD4 knockout (to genetically inhibit NET
formation) exhibited significant improvements in healing quality. Thus,
a controlled or tuned down neutrophil response—as likely induced by
LASE sealing—may positively affect incisional (primary intention)
wound healing.
3.5 |US evaluation of in vivo sealed incisions in
live mice
We sought to evaluate the fidelity of US to interrogate LASE- and
suture-sealed incisions in live mice using a portable, clinical US sys-
tem; the portable nature and clinical application of this system was
considered useful for potential translational applications. Live, anes-
thetized mice were evaluated with a linear probe transducer in
B-mode operating at 22 MHz. Manually collected US images were
compared to matched H&E-stained sections from the same mice and
the linear dimension of the wound width was compared (Figure 8).
We found a high degree of correlation between US and histopathol-
ogy measurements (linear regression y=1.140x182.5; R
2
=0.985,
N=4 each group). Thus, in vivo US, using a clinically relevant system,
provides an accurate representation of wound properties in mice with
incisions sealed by both sutures and LASE.
4|CONCLUSION
Tissue adhesives are an alternative and effective method of skin clo-
sure following surgical incisions or traumatic lacerations. Here, we
comprehensively evaluated the efficacy and quality of silk fibroin-ICG
based LASE for rapid sealing of skin incisional wounds in mice com-
pared to conventional suturing using a temporal study of functional,
biomechanical, and histological evaluation in addition to US and PAI.
We evaluated healing outcomes at different timepoints in the repair
process and our results show LASE-sealed incisions had earlier recov-
ery of skin barrier function compared to suture-closed incisions as
indicated by lower TEWL rate. At the same timepoint, significant
0 1000 2000 3000 4000
0
1000
2000
3000
4000
Ultrasound (microns)
Histology (microns)
Sutures
ICG-LASE
R
2
= 0.9849
SuturesICG-LASE
Photo Ultrasound H&E
(B)(A)
FIGURE 8 Live animal ultrasound evaluation of suture- and LASE-sealed wounds. (a) Representative photographs, ultrasound imaging data,
and H&E images for suture- and LASE-sealed linear incisions imaged by ultrasound in the live animal at Day 2 postclosure. (b) Linear correlation
with 95% confidence intervals for the epidermal/dermal gap of wounds sealed by sutures (red) or ICG LASE materials (blue) measured by
ultrasound (x-axis) or histology (y-axis). N=4 per group.
12 of 14 GHOSH ET AL.
increase in biomechanical recovery of skin was observed in case of
LASE-sealed incisions compared to suture-closed incisions. Higher
biomechanical and functional recovery of skin can prevent dehiscence
of wounds early in the healing period and also protect against surgical
site infections. US and PAI of skin incisions closed with sutures and
sealed with LASE demonstrated that these structures can be identi-
fied by their unique optical absorption properties and help quantify
and track their presence within a sample volume at least several days
postsurgery. The noninvasive dual modality platform can potentially
be applied in vivo to track these changes at the skin interface over
time. Histological analyses of skin at the end of the healing period in
our study (Day 7 postclosure) showed no difference in epidermal gap and
scar area compared to suture-closed incisions which can be indicative of
no excessive scarring or fibrosis in using LASE as a skin closure method.
However, it is important to note that mouse skin heals by contraction,
which poses significant limitations in using mouse models as indicators of
scarring. Further studies in relevant animal models (e.g., porcine models)
will be key to further compare scarring caused by sutures and LASEs.
Evaluation of translational potential of LASEs for application in humans
will also require detailed studies in porcine models, studies in animal
models of specific pathologies including slow-healing wounds (e.g., in dia-
betes) and wounds that are susceptible to infection. To that end, future
work will involve a comprehensive investigation into bioactives that can
accelerate tissue repair following laser sealing and into delivery of effec-
tive antimicrobial drugs for combating infections. In all these studies, a
comprehensive picture of functional, biomechanical, and histological per-
formance of LASEs will be obtained in order to investigate the potential
for translating this technology for clinical use.
AUTHOR CONTRIBUTIONS
Deepanjan Ghosh: Conceptualization (equal); data curation (lead);
formal analysis (lead); investigation (lead); methodology (lead); writing –
original draft (lead); writing –review and editing (lead). Christopher
M. Salinas: Data curation (equal); formal analysis (equal); methodology
(equal); writing –original draft (equal); writing –review and editing
(supporting). Shubham Pallod: Data curation (supporting); writing –
review and editing (supporting). Jordan Roberts: Data curation (sup-
porting); formal analysis (supporting). Inder Raj S. Makin: Data cura-
tion (equal); formal analysis (equal); investigation (equal); methodology
(equal); resources (supporting); validation (equal); writing –review and
editing (equal). Jordan R. Yaron: Data curation (lead); formal analysis
(lead); methodology (lead); writing –original draft (equal); writing –
review and editing (supporting). Russell S. Witte: Formal analysis
(equal); investigation (equal); methodology (equal); resources (equal);
validation (supporting); writing –review and editing (equal).
ACKNOWLEDGMENTS
The authors are grateful to the National Institutes of Health (NIH R01
AR074627) for funding this research. Jordan R. Yaron would like to
acknowledge K01 EB031984 for partial support. The authors would
like to thank Jacquelyn Kilbourne, Dr. Juliane Dagget-Vondras, and
Kenneth Lowe for their invaluable technical assistance with in vivo
experiments.
CONFLICT OF INTEREST
Kaushal Rege is affiliated with a start-up company, Synergyan, LLC.
Other authors declare no conflict of interest.
PEER REVIEW
The peer review history for this article is available at https://publons.
com/publon/10.1002/btm2.10412.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Deepanjan Ghosh https://orcid.org/0000-0002-5948-6995
Christopher M. Salinas https://orcid.org/0000-0001-5038-096X
Jordan R. Yaron https://orcid.org/0000-0002-4133-474X
REFERENCES
1. MacRae HM, McLeod RS. Handsewn vs. stapled anastomoses in
colon and rectal surgery - a meta-analysis. Dis Colon Rectum. 1998;
41(2):180-189.
2. Beuran M, Chiotoroiu AL, Chilie A, et al. Stapled vs. hand-sewn colo-
rectal anastomosis in complicated colorectal cancer—a retrospective
study. Chirurgia. 2010;105(5):645-651.
3. Urie R, Flake T, Rege K. Laser tissue welding in wound healing and
surgical repair, in bioengineering in wound healing. In: Golberg A,
Yarmush ML, eds. Bioengineering in Wound Healing. World Scientific
Publishing; 2017:303-324.
4. Asencio-Arana F, Garcia-Fons V, Torres-Gil V, et al. Effects of a low-
power He-Ne-laser on the healing of experimental colon anastomo-
ses: our experience. Opt En. 1992;31(7):1452-1457.
5. Cilesiz I, Springer T, Thomsen S, Welch AJ. Controlled temperature
tissue fusion: argon laser welding of canine intestine in vitro. Lasers
Surg Med. 1996;18(4):325-334.
6. Chikamatsu E, Sakurai T, Nishikimi N, Yano T, Nimura Y. Comparison
of laser vascular welding, interrupted sutures, and continuous sutures
in growing vascular anastomoses. Lasers Surg Med. 1995;16(1):34-40.
7. Gennaro M, Ascer E, Mohan C, Wang S. A comparison of CO
2
laser-
assisted venous anastomoses and conventional suture techniques:
patency, aneurysm formation, and histologic differences. J Vasc Surg.
1991;14(5):605-613.
8. Grubbs PE, Wang S, Marini C, Basu S, Rose DM, Cunningham JN Jr.
Enhancement of CO
2
-laser microvascular anastomoses by fibrin glue.
J Surg Res. 1988;45(1):112-119.
9. Capon A, Iarmarcovai G, Gonnelli D, Degardin N, Magalon G,
Mordon S. Scar prevention using laser-assisted skin healing (LASH) in
plastic surgery. Aesthetic Plast Surg. 2010;34(4):438-446.
10. Urie R, Ghosh D, Ridha I, Rege K. Inorganic nanomaterials for soft tis-
sue repair and regeneration. Annu Rev Biomed Eng. 2018;20:353-374.
11. Urie R, Quraishi S, Jaffe M, Rege K. Gold nanorod-collagen nanocom-
posites as photothermal nanosolders for laser welding of ruptured
porcine intestines. ACS Biomater Sci Eng. 2015;1(9):805-815.
12. Huang HC, Walker CR, Nanda A, Rege K. Laser welding of ruptured
intestinal tissue using plasmonic polypeptide nanocomposite solders.
ACS Nano. 2013;7(4):2988-2998.
13. Mushaben M, Urie R, Flake T, Jaffe M, Rege K, Heys J. Spatiotempo-
ral modeling of laser tissue soldering using photothermal nanocompo-
sites. Lasers Surg Med. 2018;50(2):143-152.
14. Urie R, Guo C, Ghosh D, et al. Rapid soft tissue approximation and
repair using laser-activated silk nanosealants. Adv Funct Mater. 2018;
28(42):1802874.
GHOSH ET AL.13 of 14
15. Urie R, McBride M, Ghosh D, et al. Antimicrobial laser-activated seal-
ants for combating surgical site infections. Biomater Sci. 2021;9(10):
3791-3803.
16. Matteini P, Sbrana F, Tiribilli B, Pini R. Atomic force microscopy and
transmission electron microscopy analyses of low-temperature laser
welding of the cornea. Lasers Med Sci. 2009;24(4):667-671.
17. Small W IV, Celliers PM, Kopchok GE, et al. Temperature feedback
and collagen cross-linking in argon laser vascular welding. Lasers Med
Sci. 1998;13(2):98-105.
18. Li C, Wang K. Effect of welding temperature and protein denaturation
on strength of laser biological tissues welding. Opt Laser Technol.
2021;138:106862.
19. Philip R, Penzkofer A, Bäumler W, Szeimies RM, Abels C. Absorption
and fluorescence spectroscopic investigation of indocyanine green.
J Photochem Photobiol A Chem. 1996;96(1-3):137-148.
20. Bass LS, Treat MR. Laser tissue welding: a comprehensive review of
current and future. Lasers Surg Med. 1995;17(4):315-349.
21. Desmettre T, Devoisselle J, Mordon S. Fluorescence properties and
metabolic features of indocyanine green (ICG) as related to angiogra-
phy. Surv Ophthalmol. 2000;45(1):15-27.
22. Iyengar S, Makin IR, Sadhwani D, et al. Utility of a high-resolution
superficial diagnostic ultrasound system for assessing skin thickness:
a cross-sectional study. Dermatol Surg. 2018;44(6):855-864.
23. Catalano O, Wortsman X. Dermatology ultrasound. Imaging technique, tips
and tricks, high-resolution anatomy. Ultrasound Q. 2020;36(4):321-327.
24. Pola
nska A, Jenerowicz D, Paszy
nska E, _
Zaba R, Adamski Z, Da
nczak-
Pazdrowska A. High-frequency ultrasonography—possibilities and perspec-
tives of the use of 20 MHz in teledermatology. Front Med. 2021;8:619965.
25. Corvino A, Sandomenico F, Corvino F, et al. Utility of a gel stand-off
pad in the detection of Doppler signal on focal nodular lesions of the
skin. J Ultrasound. 2020;23(1):45-53.
26. Gnyawali SC, Sinha M, el Masry MS, et al. High resolution ultrasound
imaging for repeated measure of wound tissue morphometry, biome-
chanics and hemodynamics under fetal, adult and diabetic conditions.
PLoS One. 2020;15(11):e0241831.
27. Gnyawali SC, Barki KG, Mathew-Steiner SS, et al. High-resolution har-
monics ultrasound imaging for non-invasive characterization of wound
healing in a pre-clinical swine model. PLoS One. 2015;10(3):e0122327.
28. Rockwood DN, Preda RC, Yücel T, Wang X, Lovett ML, Kaplan DL.
Materials fabrication from Bombyx mori silk fibroin. Nat Protoc. 2011;
6(10):1612-1631.
29. Ghosh D, Urie R, Chang A, et al. Light-activated tissue-integrating
sutures as surgical nanodevices. Adv Healthc Mater. 2019;8:e1900084.
30. Alexander H, Brown S, Danby S, Flohr C. Research techniques made
simple: transepidermal water loss measurement as a research tool.
J Invest Dermatol. 2018;138(11):2295-2300.e1.
31. Akdeniz M, Gabriel S, Lichterfeld-Kottner A, Blume-Peytavi U, Kottner J.
Transepidermal water loss in healthy adults: a systematic review and meta-
analysis update. Br J Dermatol. 2018;179(5):1049-1055.
32. Fluhr JW, Feingold KR, Elias PM. Transepidermal water loss reflects
permeability barrier status: validation in human and rodent in vivo
and ex vivo models. Exp Dermatol. 2006;15(7):483-492.
33. Dini V, Barbanera S, Romanelli M. Quantitative evaluation of macera-
tion in venous leg ulcers by transepidermal water loss (TEWL) mea-
surement. Int J Low Extrem Wounds. 2014;13(2):116-119.
34. Ratto F, Cavigli L, Borri C, et al. Hybrid organosilicon/polyol phantom
for photoacoustic imaging. Biomed Opt Express. 201 9;10(8):3719-3730.
35. Forbrich A, Heinmiller A, Zemp RJ. Photoacoustic imaging of lym-
phatic pumping. J Biomed Opt. 2017;22(10):106003.
36. Gurtner GC, Dauskardt RH, Wong VW, et al. Improving cutaneous
scar formation by controlling the mechanical environment: large ani-
mal and phase I studies. Ann Surg. 2011;254(2):217-225.
37. Pastar I, Stojadinovic O, Yin NC, et al. Epithelialization in wound heal-
ing: a comprehensive review. Adv Wound Care. 2014;3(7):445-464.
38. Khorasani H, Zheng Z, Nguyen C, et al. A quantitative approach to
scar analysis. Am J Pathol. 2011;178(2):621-628.
39. Peiseler M, Kubes P. More friend than foe: the emerging role of neu-
trophils in tissue repair. J Clin Invest. 2019;129(7):2629-2639.
40. Liu M, Chen K, Yoshimura T, et al. Formylpeptide receptors mediate
rapid neutrophil mobilization to accelerate wound healing. PLoS One.
2014;9(3):e90613.
41. Nishio N, Okawa Y, Sakurai H, Isobe KI. Neutrophil depletion delays
wound repair in aged mice. Age. 2008;30(1):11-19.
42. Gutiérrez-Fernández A, Inada M, Balbín M, et al. Increased inflamma-
tion delays wound healing in mice deficient in collagenase-2 (MMP-
8). FASEB J. 2007;21(10):2580-2591.
43. Ashcroft GS, Lei K, Jin W, et al. Secretory leukocyte protease inhibitor
mediates non-redundant functions necessary for normal wound heal-
ing. Nat Med. 2000;6(10):1147-1153.
44. Wong SL, Demers M, Martinod K, et al. Diabetes primes neutrophils
to undergo NETosis, which impairs wound healing. Nat Med. 2015;
21(7):815-819.
45. Zhu S, Yu Y, Ren Y, et al. The emerging roles of neutrophil extracellu-
lar traps in wound healing. Cell Death Dis. 2021;12(11):984.
46. Yang S, Gu Z, Lu C, et al. Neutrophil extracellular traps are markers of
wound healing impairment in patients with diabetic foot ulcers trea-
ted in a multidisciplinary setting. Adv Wound Care. 2020;9(1):16-27.
47. Heuer A, Stiel C, Elrod J, et al. Therapeutic targeting of neutrophil
extracellular traps improves primary and secondary intention wound
healing in mice. Front Immunol. 2021;12:614347.
SUPPORTING INFORMATION
Additional supporting information can be found online in the Support-
ing Information section at the end of this article.
How to cite this article: Ghosh D, Salinas CM, Pallod S, et al.
Temporal evaluation of efficacy and quality of tissue repair
upon laser-activated sealing. Bioeng Transl Med. 2023;8(2):
e10412. doi:10.1002/btm2.10412
14 of 14 GHOSH ET AL.
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