Received: 14 October 2020 Revised: 17 March 2021 Accepted: 22 March 2021

DOI: 10.1002/jbm.a.37177

ORIGINAL ARTICLE

Microneedle based adipose derived stem cells-derived

extracellular vesicles therapy ameliorates UV-induced
photoaging in SKH-1 mice

Zhi Cao1,2 | Shengkai Jin1,2 | Peiru Wang1 | Qiumin He1 | Yuling Yang1 |
2,3,4 1
Zhengliang Gao | Xiuli Wang

1
Institute of Photomedicine, Shanghai Skin
Disease Hospital, School of Medicine, Tongji Abstract
University, Shanghai, 200092, China Extracellular vesicles from adipose derived stem cells (ADSCs-EVs) have shown
2
Yangzhi Rehabilitation Hospital (Shanghai
immunomodulation and anti-photoaging effects; however, the skin barrier prevents
Sunshine Rehabilitation Center), Shanghai,
Tongji Univeirsity School of Medicine, their absorption via skin. Meanwhile, microneedle (MN) is a widely used and mini-
Shanghai, China, China
mally invasive tool for dermal delivery of drugs, it also has neocollagenesis effect by
3
Institute of Geriatrics (Shanghai University),
Affiliated Nantong Hospital of Shanghai creating tiny injuries and initiating wound healing process. To investigate the effect
University (The Sixth People's Hospital of of MN combined with ADSCs-EVs on skin aging, photoaging in SKH-1 mice was
Nantong), School of Medicine, Shanghai
University, Nantong, China induced by chronic exposure to ultraviolet radiation. Then the mice were treated fol-
4
Shanghai Engineering Research Center of lowing a split-dorsal scheme, in which one side had MN alone or MN + EVs treat-
Organ Repair, School of Medicine, Shanghai
ment and the other side was left untreated. For the side treated with MN alone or
University, Shanghai, China
MN + EVs, the epidermal thickness was decreased and the skin barrier function was
Correspondence
enhanced compared with the untreated side. However, MN + EVs group showed the
Xiuli Wang, Institute of Photomedicine,
Shanghai Skin Disease Hospital, Tongji least wrinkles, the highest collagen density and the most organized collagen fibers
University, Shanghai, China.
among the three groups. The level of CD11b + cell infiltration was lower in MN
Email: [email protected]
Zhengliang Gao, Department of Gynecology + EVs group than that in the MN group at 3 day after the treatment. These results
and Obstetrics, Tongji University, Shanghai,
indicated that MN treatment alone could improve epidermal structure and function
China.
Email: [email protected] of photoaging skin, and a combination with ADSCs-EVs would accelerate the restora-
tion of inflammation caused by MN and improve the content of collagen. In all, this
Funding information
Lifeng Institute of Regenerative Medicine of study indicated that a combination of MN and topical applied ADSCs-EVs was a fea-
Tongji University
sible and safe strategy to ameliorate photoaging, providing a new avenue for safe
administration of EVs.

KEYWORDS
extracellular vesicle, inflammation, microneedle, photoaging

1 | I N T RO DU CT I O N the pursuit of healthy body and young appearance has always been
one of the main concerns of people, many treatments have been
Skin provides physical, chemical, and microbiome barrier against developed to protect against or to treat photoaging, such as, the topi-
harmful factors from the environment. Photoaging, which is caused by cal retinoids, antioxidants, and cosmetic lasers.3-5 Besides, biopharma-
chronic exposure to ultraviolet (UV), is one of the most common type ceutical treatment has shown great potential in anti-aging and
of skin aging. It is characterized by coarse wrinkles, roughness, sagging rejuvenated area in recent years. Extracellular vesicle (EV) is important
and the decline of barrier function, and is related to various diseases, vehicle secreted by diverse cells and mediates the intracellular com-
such as, lentigo maligna, actinic keratosis, and skin cancers.1,2 Since munication. It was reported that mesenchymal stem cell (MSC)

J Biomed Mater Res. 2021;1–9. wileyonlinelibrary.com/journal/jbma © 2021 Wiley Periodicals LLC. 1

2 CAO ET AL.

derived EVs contained abundant peptides, cytokines, microRNAs, and and it has a mature application in clinical practice. It was shown that
lncRNAs, these molecules could modulate various biological pro- the tiny injuries caused by MN could initiate the wound healing pro-
cesses in target cells, such as, collagen synthesis, immuno- cess and promote the synthesis of collagen and elastin within skin, so
modulation, cell senescence, and DNA repair.6,7 With these the MN treatment has gained much attention in facial rejuvenation.8
bioactive contents, MSC derived EVs have been recognized as However, the inflammation-related adverse effects, such as erythema
potential biological agent with multiple functions in rejuvenation and burning sensation, would be severe if the MN was not applied
and inflammatory diseases. Considering that topical applied EVs appropriately. It is possible that a combination of MN roller and topi-
could not be absorbed directly via skin, intravenous or subcutaneous cal applied EVs would balance the absorption of EVs and the adverse
injection were commonly used in previous studies. However, these effect of MN on a large scale, but limited studies have reported topical
invasive administrations were not optimal selections for clinical delivery of EVs with MN roller, and the effects of the combination of
application because of potential systematic adverse effects or pain MN and EVs treatment on photoaging was still unknown.
and uneven drug-distribution. This study was aimed to investigate the effect of MN roller com-
Microneedle (MN) has been an easily available, minimally invasive bining with EVs adipose derived stem cells-derived EVs (ADSCs-EVs)
and painless tool for transdermal delivery of drugs for many years, on photoaging in vivo. The MN roller and ADSCs-EVs were applied in

F I G U R E 1 Experimental design and the efficacy assessment after the treatment. (a) Schematic diagram of the establishment of UV-induced
photoaging mice and the schedule of repeated MN alone or MN + EVs treatments. (b) A photograph of MN roller applied in this study. (c)
Schematic diagram about the combination of MN roller and EVs treatment. (d) Macroscopic photograph of mice. (e) Dermoscopic photograph of
Control, MN alone and MN + EVs group. White arrows (") indicated coarse wrinkles
CAO ET AL. 3

UV-induced photoaging SKH-1 hairless mice, and the improvements 2.3 | Experimental design
in photoaging were evaluated.
After showing features of photoaging, 36 mice were divided into two
groups randomly and accepted treatments on its left or right side of
2 | MATERIAL AND METHODS dorsal skin randomly. One group of mice accepted MN treatment on
one side of the dorsal skin, following by topical application of PBS (MN
2.1 | Animals and UV irradiation alone grou), and no treatment was applied on the other side (Control).
The other group accepted MN treatment and topical application of
Hairless SKH-1 mice (female, 8 weeks old, immunocompetent) were ADSCs-EVs on one side of the dorsal skin (MN + EVs), and no treat-
obtained from Shanghai Public Health Clinical and housed under con- ment was applied on the other side either (Control) (Figure 1a).
trolled conditions in the animal facility of Shanghai Skin Disease Hos- When the treatment was applied, the mice were firstly anesthe-
pital. All experiments were conducted following the ARRIVE tized by isoflurane and the medium line was marked from the cervical
guidelines and approved by the Animal Ethics Committee in Shanghai vertebra to the tail, along the spine. Then the MN roller (Figure 1b)
Skin Disease Hospital. was applied to the treatment side by rolling over for 100 times, after
To induce the phenotype of photoaging, ultraviolet irradiation which erythema and punctate hemorrhage could be seen on the skin.
device (Solar UV Simulator, SIGMA Shanghai, China) with a mixed Then 200 μl of EVs or PBS were applied topically to the treatment
source of UVA (0.60 mW/cm2) and UVB (3.5 mW/cm2) was used to side of the dorsal skin.
irradiate the mice with repeated cycles. Each irradiating cycle lasted The first treatment began at the last irradiating day of the cycle 6.
for 10 days, which contained five continuous irradiating days and five Another treatment was applied 3 days later, which was in the middle
resting days. The dose of UV in each cycle was increased by extending (the third day) of resting days. The same irradiation-treatment sched-
the irradiation progressively as follows: cycle 1 and 2, 60 s (minimum ule was repeated during the next 3 cycles (cycle 7–9) and each mouse
erythema dose) in each irradiating day; cycle 3 and 4, 70 s in each irra- accepted eight treatments in total (Figure 1a).
diating day; cycle 5–6, 80 s in each irradiating day; cycle 7–10, 90 s in
each irradiating day (Figure 1a).
After the last irradiation in cycle 5, each mouse was anesthetized and 2.4 | Macroscopic and dermoscopic observation
tattooed with green dye on the dorsal skin. Three pairs of green dots
were punctured symmetrically along the median line. The middle pair of Macroscopic photographs were taken by camera (PowerShot G16,
dots located bilaterally at the midpoint of median line and 0.5 cm away Canon, Japan) when the mice were anesthetized before the first treat-
from the median line. The other two pairs of dots were 0.5 cm away from ment. Photographs were taken again after the last irradiation in cycle 10.
the median line and 1.0 cm away from the middle pair of dots. The dermoscopic photographs were taken by a hand-take dermoscope,
After the irradiation days in cycle 6, features of photoaging could and the center of the picture was the green tattoo described above.
be observed on the dorsal skin of mice, including coarse wrinkles, ery-
thema and scales. These mice were then divided into MN group and
MN + EVs group randomly. 2.5 | Detection of stratum corneum hydration and
transepidermal water loss

2.2 | The isolation of ADSCs-EVs The stratum corneum hydration and the transepidermal water loss
(TEWL) on the dorsal skin of mice was measured by Corneometer
Adipose tissue from plastic surgeries was collected according to proto- (CM 825, CK, Germany) and Tewameter (TM 300 w, CK, Germany) in
cols approved by the Ethical Committee of the Tenth People's Hospital the last irradiation day of cycle 10.
Affiliated with Tongji University. Then the human ADSCs were isolated
and cultured as described previously.7 After passaging for several times,
the P4-P5 generation of ADSCs were seeded at a density of 2 × 105/ml 2.6 | Histological assessments
(Corning, 55 cm2). When the ADSCs were 80% confluent, the culture
medium was replaced with 10 ml serum-free DF12 (containing bFGF). Methods of histological assessments can be found in supplementary
The culture medium of ADSCs was collected after 72 hr and the EVs information.
7
were isolated as described before. Briefly, the culture medium was
centrifuged at 3000 × g for 15 min to remove dead cells and cell debris.
Then the ExoQuick-TC (system biosciences) was added to the superna- 2.7 | Statistical methods
tant and mixed up thoroughly. The mixture was then kept upright at
4 C for 14 hr and centrifuged at 1500 × g for 30 min. Then the super- The experimental data was presented as mean ± standard error. The
natant was deserted and the white pellet at the bottom of the tube was one-way analysis of variance was performed to compare the differ-
suspended with 500 ul PBS. ences of stratum corneum hydration, TEWL, epidermal thickness,
4 CAO ET AL.

dermal thickness, the collagen density per field in dermis, the elastin irradiation (Figure 1d). Wrinkle formation and other photo-injury
density per field in dermis and the number of CD11b + cells per related phenotypes were examined macroscopically in the dorsal
2
0.1 mm among different groups. The difference of epidermal cell region after eight treatments. Comparing with untreated skin that did
layer and the grade of collagen fiber disorganization among groups not accepted any treatment, the MN + EVs treated skin showed sig-
were analyzed with Kruskal-Wallis test. An overall alpha-level of 0.05 nificant improvements in coarse wrinkles, erythema, scales, and thick-
was used to determine statistical significance and all tests were two- ening. Though the MN alone treated skin also showed slight
sided. All data were analyzed with the SPSS 19.0 software. improvements in scales and erythema, no significant improvement in
wrinkles was found comparing with untreated skin (Figure 1d).
Dermoscopic examination showed less wrinkles and scales in MN
3 | RESULTS + EVs treated skin compared with untreated Control group. Scales
and area of erythema in MN alone group were also slightly decreased
3.1 | MN combined with EVs treatment reduced compared to the control (Figure 1e). These results indicated that
UV-induced wrinkle formation in photoaging skin repeated MN treatment alone could slightly reduce scales and ery-
thema caused by UV, and a combination with topical applied EVs
The dorsal skin of mice showed coarse wrinkles, erythema, dryness, would further enhance the efficacy of MN and reduce the wrinkle
scales, thickening and even leathery appearance after 6 cycles of UV- formation.

F I G U R E 2 Histological and skin barrier function assessment of epidermis. (a) Image of H&E staining, black arrows (") indicate hypokeratosis in
epidermis. (b) Statistics of epidermal thickness. (c) Image of DAPI staining. (d) Statistics of epidermal cell layer. (e) Statistics of stratum corneum
hydration. (f) Statistics of transepidermal water loss. *, p < 0.05 and indicated a significant difference between groups; **, p < 0.01; ***, p < 0.001
CAO ET AL. 5

3.2 | MN combined with EVs treatment alleviated both of them were reduced compared with untreated side, which was
the epidermal hyperplasia and function decline of 51.4 ± 7.7 μm (p = 0.048 < 0.05 and p = 0.001 < 0.01 in respective).
photoaging skin Although the epidermis of MN + EVs group was slightly thinner than
that of MN alone group, no statistical difference was found between
To evaluate the effect of MN alone and MN + EVs treatment on the them (Figure 2b). The cell layer of epidermis was further assessed.
epidermis of photoaging skin, we histologically analyzed the mouse Consistently, the epidermal cell layer of untreated skin was mostly
dorsal skin. It was shown in H&E staining that the epidermis of photo- maintained at 4, and the overall number of cell layers reduced to 3 in
aging skin was hyperplastic and thickened in untreated control. Partial MN alone and MN + EVs group (Figure 2c, d).
hypokeratosis could also be observed in some of the epidermal area The stratum corneum hydration and TEWL were also detected to
(Figure 2a). The thickness of epidermis in the MN alone and MN assess the change of skin barrier function after 8 treatments. Stratum
+ EVs treated side were 43.9 ± 9.3 and 37.8 ± 6.1 μm, respectively, corneum hydration test indicated that there was no statistical

F I G U R E 3 Histological assessment of
dermis. (a) Image of Masson staining. (b)
Image of Weigert staining. (c) Statistics of
dermal thickness. (d) Statistics of the grade
of collagen fiber disorganization. (e)
Statistics of collagen density per field in
dermis. (f) Statistics of elastin density per
field in dermis. *, p < 0.05 and indicated a
significant difference between groups; **,
p < 0.01; ns, no significant difference
among three groups
6 CAO ET AL.

F I G U R E 4 Infiltration of CD11b + cells at different time points after the treatment. (a) Image of histochemistry staining of CD11b. (b,c)
Statistics of number of CD11b + cells per 0.1 mm2 at 12, 24 hr, and 3 day after the first treatment. *, p < 0.05 and indicated a significant
difference between groups; **, p < 0.01; ***, p < 0.001

difference between MN alone and MN + EVs group of the dermis at the end of irradiation days in cycle 10. There was no
(p = 0.107 > 0.05), however, both of the groups had significantly significant difference in dermal thickness among the three groups
higher stratum corneum hydration value than Control group (p = 0.338 > 0.05, Figure 2a, 3c), however, the dermal components in
(p = 0.017 < 0.05 and p = 0.001 < 0.01 in respective, Figure 2e). Simi- the untreated photoaging skin and the MN alone treated skin were
larly, the TEWL in both treatment groups were significantly lower more disorganized comparing with the MN + EVs group (Figure 2a).
than that of untreated Control group (both p < 0.001), and no differ- Therefore, the collagen and elastic fibers were further assessed
ence was found between the two treatment groups (p = 0.893 > 0.05, respectively. It was shown in Masson staining that the collagen fibers
Figure 2f). in untreated and MN alone treated skin were more scattered and dis-
organized than the MN + EVs treated skin. Some collagen fibers even
became fractured and fragmented in the dermis of MN alone treated
3.3 | MN + EVs improved the organization of skin. By contrast, the MN + EVs treated skin had the densest and the
collagen fiber in the dermis of photoaging skin most organized collagen fibers (Figure 3a). Semi-quantitative grading
for the disorganization of collagen fiber indicated that the degradation
To investigate the changes of the dermal components after MN alone of collagen in MN alone treated skin was more prominent than that of
and MN + EVs treatment, we next performed histological assessments MN + EVs treated skin (p = 0.001 < 0.01, Figure 3d). The collagen
CAO ET AL. 7

density per field in the dermis of MN + EVs group was 62.0% ± 9.8%, into the dermis, causing the senescence of fibroblasts and the degra-
which was higher than that of Control (54.8% ± 12.2%) and MN alone dation of dermal extracellular matrix.1,17 Solutions for photoaging
(53.4% ± 11.4%) group (p = 0.015 < 0.05 and p = 0.040 < 0.05 in mainly focused on promoting the matrix remodeling in dermis,
respective, Figure 3e). Weigert staining was also applied to evaluate improving the skin barrier function, reducing the oxidative stress and
the containing and distribution of elastic fibers. It could be observed eliminating the inflammation in skin.
that most elastic fibers located below the dermo-epidermal junction It was reported that MN treatment could ameliorate the epider-
and surrounding of pilosebaceous units; there was no significant dif- mal dysfunction of aging skin,18 while some other studies reported
ference in the distribution or organization of elastic fiber among three that MN treatment would not disturb long-term skin barrier func-
groups (Figure 3b). Moreover, no difference in elastin density per field tion.19,20 In a previous study where MN arrays was applied repeatedly
was found among these groups (p = 0.074 > 0.05, Figure 3f). on the dorsal skin of SKH-1 mice, it was found that the skin appear-
ance, the skin barrier function and the skin inflammation did not
change significantly regardless of the density of MN or the frequency
3.4 | EVs promoted the recovery from MN- of application21 . However, our study showed that repeated MN roller
induced injury and accelerate the subsidence of treatment alone could reduce the epidermal thickness and the epider-
inflammation mal cell layer, and improve skin barrier function of photoaging SKH-1
mice. One possible reason could be that the animal used in previous
CD11b + cells are the earliest responsive inflammatory cells in acute study was healthy mice without any UV exposure, which means no
inflammation and early phase of wound healing. To evaluate the preceding disturbance in epidermal structure or skin barrier function.
emergence and the resolution of inflammation, histochemistry Another possible reason may lie in that the inflammation induced by
staining of CD11b was performed to evaluate the infiltration of MN arrays was too mild and not sufficient to induce significant
CD11b + cells in the skin (Figure 4a). At 12 hr after the first treat- change in skin barrier function, whereas the barrier disruption caused
ment, CD11b + cells were significantly increased in MN alone and by MN roller in our study was intenser, and the subsequent restora-
MN + EVs treated skin compared with the untreated skin (both tion of skin barrier function was more obvious.
p < 0.001, Figure 4b). Similar amounts of CD11b + cells were found at Previous studies also reported that MN treatment alone has
24 hr (Figure 4c). Later at 3 day after the treatment, the count of infil- neocollagenesis effect. Hong and colleagues reported that the wrin-
trated CD11b + cells decreased in all three groups. It was interesting kles and the skin roughness value of photoaging mice were reduced
to notice that only a small amount of CD11b + cells were detectible in after MN treatment, histology examination indicated that the collagen
MN + EVs group, which was nearly the same as untreated skin. The and the elastic fibers were slightly increased.22 Another clinical study
amount of CD11b + cells in MN alone group was significantly higher reported that MN treatment was effective in reducing the wrinkles on
than the other groups (p = 0.004 < 0.01 and p = 0.003 < 0.01 in aging neck.22 However, no obvious evidence in our study indicated
respective), which indicated a quick recovery from the MN-induced that MN treatment alone could improve the content or the organiza-
injury in MN + EVs treated skin (Figure 4c). tion of collagen fibers. This was possibly because that the effect of
repeated UV exposure was too strong that MN treatment alone was
not sufficient to protect against the damage. Another reason account-
4 | DISCUSSION ing for the disorganization of collagen may be that the frequent appli-
cation of MN treatment and UV exposure evoked sustained
Stratum corneum plays an important role in the physical barrier func- inflammation and disturbed the process of wound healing as well as
tion of skin. While the stratum corneum prevents the entry of harmful extracellular matrix remodeling. It can be inferred that recurring tiny
factors, it also refuses the transdermal delivery of drugs with high wounds activated inflammation-relating signaling pathways, such as
molecular weight (>500 Da).9 Many chemical or physical methods NF-κB, Toll-like receptors and MAPK signaling in tissue cells. The acti-
have been reported to increase the permeability of the skin, among vation of these pathways upregulated a series of down-stream genes,
them MN is an idea option.10 A variety of MN devices has been intro- including matrix-metalloproteinases (MMPs) that promote the degra-
duced in recent years, for instance, the MN roller, MN patchs and MN dation of collagen, and downregulated the genes relating to the syn-
arrays.11,12 With these novel designs, the absorption of target sub- thesis of collagen.23 Besides, inflammatory cells, such as neutrophil
stances was significantly improved through topical application. These and macrophage, were also important sources of MMPs.24 It was
MN-based therapies had shown great potential in many areas, such shown in our study that the resolution of CD11b + cell infiltration in
13-16
as, facial rejuvenation, atopic dermatitis and skin infection. MN alone treated skin was the slowest among the three groups. The
Photoaging is the most common type of extrinsic skin aging; both result also indicated the importance of proper frequency of MN treat-
UVA and UVB participate in this process. UVB which has shorter ment to avoid prolonged inflammation and adverse effects.
wavelength can increase the production of ROS and cause DNA dam- It was interesting to find that MN + EVs treated skin showed a
age, these changes will further induce the senescence and apoptosis significant improvement in content and organization of collagen,
of skin cells. Though most of the influences caused by UVB are limited which indicated that a combination of ADSCs-EVs has profound
in epidermis, UVA with a longer wavelength can penetrates deeper effect on collagen metabolism compared with MN treatment alone. In
8 CAO ET AL.

the past decade, MSC-derived EVs has gained great attention in skin RE FE RE NCE S
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