Abstract
Background: Ultraviolet (UV) exposure causes direct and indirect damages to skin structures. Human adipose stem cell-conditioned medium (hASC-CM) is a collection of several soluble factors, such as cytokines, chemokines, and Growth Factors (GF), secreted by almost all living cells in the extracellular space which support wound healing and skin rejuvenation.
Objective: To determine the effects of human adipose stem cell-conditioned medium (hASC-CM) in photoaged skin and evaluate photoaging improvement after treatment.
Methods: An experimental randomized controlled trial was performed, involving 64 photoaged subjects at the Dermato-Venereology Outpatient Clinic of Jemursari Islamic Hospital Surabaya from March to June 2022. The subjects were divided into the hASC-CM group and vehiculum (control) group. Patients’ transepidermal water loss value, skin tone, and Glogau score in weeks 0, 4, and 8 were evaluated. The data were then analyzed using Mann-Whitney test (p<0.05).
Results: All subjects had Glogau scale of III (89.6%) with mean ± SD (3.121 ± 0.329). hASC-CM group showed higher improvements in the pore, wrinkle, spot polarized, spot UV parameters and skin tone compared to vehiculum group(p<0.05).
Conclusions: hASC-CM effectively improved all parameters observed. The limitation of this research was on the lack of long-term follow-up after treatment.
This research had received an ethical permit from The Ethical Committee Board of Jemursari Islamic Hospital Surabaya under letter 006/KEPK-RSISJS/II/2022.
Keywords: Glogau score, hASC-CM, Photoaging, Skin analyzer, TEWL
Introduction
Indonesia is a tropical country with high UV exposure throughout the year[1]. Repeated and long-term UV exposure will result in photoaged skin referred to as extrinsic ageing which overlaps with intrinsic ageing [2–4]. Photoaging accounts for 80% of facial ageing, characterized by fine wrinkles, dryness, laxity, rough texture, and decreased elasticity which can trigger skin cancer[5,6]. Ultraviolet exposure causes direct and indirect damages to skin structures due to the reactive oxygen species that activates the downstream signalling pathways. The signalling pathways then cause extracellular matrix degradation in the forms of collagen, elastin, and skin barrier damages[6].
The paracrine effect of adipose stem cells (ASC) secretes molecules known as secretomes or conditioned medium (CM) - a collection of several soluble factors, such as cytokines, chemokines, and GF, secreted by almost all living cells in the extracellular space[7–10]. Some of the GFs in ASC-CM include platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) that help wound healing and skin rejuvenation[11]. Adipose stem cell-conditioned medium has been widely used as an anti-wrinkle therapy for skin aging both in-vitro and in-vivo[6] which can reduce cell death in apoptosis, increase the expression of type I collagen protein and decrease the expression of MMP-1 protein in in-vitro studies[12]. Another in vitro research reported that ASC-CM could downregulate the activation of MAPK signaling pathways and proinflammatory target proteins such as IL-6, inhibit AP-1 activation as well as increasing TGF-β[13]. Adipose stem cell conditioned medium improves the skin barrier through decreased TEWL and increased skin hydration[6,9,14,15].
This research was performed to examine the effects of human adipose stem cell-conditioned medium (hASC-CM) on photoaged skin using Tewameter, skin analyzer device, and Glogau score.
Material and methods
Research design and participants
In this experimental randomized controlled trial research, 64 subjects were consecutively selected based on the inclusion criteria. The research took place at the Dermato-Venereology Outpatient Clinic of Jemursari Islamic Hospital Surabaya from March to June 2022. The subjects consented to participating and were divided into the ASC-CM (treatment) group and vehiculum (control) group. Ethical permit had been obtained from the Ethical Committee Board of Jemursari Islamic Hospital Surabaya under letter number 006/KEPK-RSISJS/II/2022
Isolation of Adipose Stem Cell
Adipose tissue sample was obtained from subcutaneous fat using international standardized method of tissue donors and stem cell cultures extraction from humans. The adipose tissue was washed three times with sterile phosphate-buffered saline solution (PBS). The tissue was then cut into pieces and digested by collagenase (type I collagenase, Worthington, USA) for forty minutes at 37°C with gentle agitation. Enzyme activity was neutralized using Minimum Essential Medium Eagle-Alpha Modification (Alpha MEM) supplemented with 2% fetal bovine serum (FBS), and the tissues were filtered with 150 μm filters. After centrifugation at 300 g for 10 minutes, the cell pellet were then resuspended in Alpha MEM containing 2% human platelet lysate (hPL) and 1% penicillin-streptomycin before being cultured and expanded at 37°C in 5% CO2 incubators.
Characterization of Adipose Stem Cell
The cells were categorized into osteoblasts, adipocytes, and chondrocytes to confirm the multipotency. Cells were plated at 3 x 104 cells/cm2 density in dishes at passage 5. Once the cells reached 90% confluence, the complete medium was replaced with specific inducing mediums for differentiation. For osteogenic induction, the medium was supplemented with 50 µM ascorbic acid, 0.1 µM dexamethasone, and 10 mM β-glycerophosphate. For adipogenic induction, the medium was supplemented with 1.0 µM dexamethasone, 10 mg/L insulin, 100 µM indomethacin, and 500 µM IBMX. For chondrogenic induction, the medium was supplemented with 1% penicillin-streptomycin, 1% ITS (insulin, transferrin, and selenium), 100 nM dexamethasone, 50 µg/mL L-ascorbic acid 2-phosphate, 10 ng/mL TGFβ1, and 500 ng/mL BMP6. For up to 20 days, the cells were stained with Oil Red O for adipogenic induction, Alizarin Red S for osteogenic induction, or Safranin O for chondrogenic induction. The cultured cell from passage 4 were subjected to CD45 and CD105 immunofluorescence staining to verify the cellular identity.
Extraction of Secretory Protein in Conditioned Medium
The adipose stem cell culture dishes were washed three times with PBS and cultured overnight in a culture medium consisting of Alpha MEM, and 0.2% hPL, resulting in about 6-8 x 106 cells. After 24 hours, the cultured medium was collected and fresh serum-free medium was added. The CM was filtered using a 0.22 μm filter, centrifuged with Amicon Ultra 15 mL (MWCO 3 kD, Millipore) at 4 °C 4000 g for 30 minutes, and then concentrated about five times using an ultrafiltration membrane of 3.5 kD with polyethylene glycol at 4 deg C. Overall, the CM was concentrated about 15 times from 1000 mL of 0.389 ± 0.04 mg/mL to 70 mL of 5.989 ± 0.07 mg/mL. Freeze-dry powder was prepared in sterilized penicillin bottles using Lyophilizer (Boyikang Corp., Beijing, China).
Formulation of topical hASC-CM with SPACE Peptide
The human adipose stem cell-conditioned media was mixed with SPACE Peptide (MedChem) and then made into gel at the Faculty of Pharmacy, Universitas Airlangga.
Procedures
At baseline, skin stabilization was performed for at least 30 minutes to evaluate the research parameters. Transepidermal water loss value was measured using Tewameter. Meanwhile, skin wrinkle, pore, spot UV, spot polarized and skin tone were analyzed using a skin analyzer (Janus) and Glogau score. Glogau scale was chosen considering that it is the most applicable scale in photoaging cases as it is relatively convenient and provides a fair assessment of the photoaging severity. All subjects underwent skin stabilization for 30 minutes after washing their faces. Measurements were taken indoors at temperatures between 20 and 24°C and relative humidity levels between 40 and 60% to minimize environmental effects. There were two groups; the treatment group that received ASC-CM and the control group that received only vehiculum. After parameters were evaluated at baseline, the treatment procedures were initiated. The subjects were instructed to apply sunscreen in the morning and ASC-CM or only vehiculum before going to bed to their faces daily for eight weeks. The vehiculum used in this research contained propylene glycol and carboxymethyl cellulose. Research parameters were reassessed in Week 4 and 8. Side effects after treatment were observed; if the subject experienced severe side effects, the treatment was discontinued, and the subjects were treated.
Statistical analysis
The data of this research were analyzed using Mann-Whitney test on the Statistical Product for the Social Sciences (SPSS) data format version 25.0 (SPSS, Inc., Chicago, Illinois) at level of significance of p<0.05.
Results
The distribution of subjects in both groups was non-homogeneous (p<0.05). All subjects were female, with a mean age of 55.21 ± 8.276 years and a mean Glogau score of 3.121 ± 0.329. 28 subjects (48.3%) had indoor activities, while 30 subjects (51.7%) had outdoor activities, with a significant non-homogeneous distribution between the two groups (p < 0.05). Baseline characteristics are detailed in Table 1. The severity of photodamage was measured based on Glogau score and classified based on the wrinkles and age-specific degenerative effects of photoaging on the epidermis and dermis. Skin analyzer (Janus) was also used to measure the wrinkles, spot UV, spot polarized, and skin tone. Whereas, skin barrier parameter was asssed using Tewameter to gain the TEWL value.
Table 1: Demography and baseline characteristics of the respondents.
| Variable | Gel (n=29) | hASC-CM (n=29) | Total |
|---|---|---|---|
| Sex | |||
| Male, n (%) | 0 (0) | 0 (0) | 0 (0) |
| Female, n (%) | 29 (100) | 29 (100) | 58 (100) |
| Age in years | |||
| 40-44, n (%) | 1 (3,.5) | 4 (13.8) | 5 (8.6) |
| 45-49, n (%) | 8 (27.6) | 4 (13.8) | 12 (20.7) |
| 50-54, n (%) | 5 (17.2) | 7 (24.1) | 12 (20.7) |
| 55-59, n (%) | 6 (20.7) | 6 (20.7) | 12 (20.7) |
| 60-64, n (%) | 5 (17.2) | 0 (0) | 5 (8.6) |
| 65-69, n (%) | 3 (10.3) | 8 (27.6) | 11 (19) |
| 70-74, n (%) | 1 (3.5) | 0 (0) | 1 (1.7) |
| Mean ± SD | 55.536 ± 7.637 | 54.896 ± 8.973 | 55.21 ± 8.278 |
| Glogau | |||
| Category III, n (%) | 25 (86.2) | 27 (93.1) | 52 (89.6) |
| Category IV, n (%) | 4 (13.8) | 2 (6.9) | 6 (10.4) |
| Mean ± SD | 3.138 ± 0.351 | 3.103 ± 0.31 | 3.121 ± 0.329 |
| Activity | |||
| Indoor, n (%) | 15 (51.7) | 13 (44.8) | 28 (48.3) |
| Outdoor, n (%) | 14 (48.3) | 16 (55.2) | 30 (51.7) |
The mean differences in pore, wrinkle, spot polarized, spot UV, and skin tone before and after treatment between the control and treatment groups are shown in Table 2 and the significant differences are shown in Table 3. Compared with the vehiculum group, the hASC-CM group showed more signficant improvements in the pore, wrinkle, spot polarized, spot UV parameters, and skin tone. Figure 1 shows patients’ Glogau score improvement. In this study, a majority of the subjects had a Glogau scale of III (89.6%; Table 1) with a mean of 3.121 ± 0.329. A a non-homogeneous distribution was identified in the variables of pores, wrinkles, spot polarized, spot UV, and skin tone (p<0.05).
Table 2: Comparison of clinical photoaging before and after treatment.
| Week | ||||
|---|---|---|---|---|
| 0 | 4 | 8 | ||
| TEWL value | Gel (N=29) Mean ± SD | 11.29 ± 4.064 | 11.691 ± 3.603 | 13.304 ± 4.554 |
| hASC-CM (N=29) Mean ± SD | 15.124 ± 1.222 | 11.72 ± 0.634 | 9.171 ± 0.504 | |
| Pore | Gel (N=29) Mean ± SD | 55.482 ± 8.830 | 55.379 ± 8.575 | 55.8621 ± 8.387 |
| hASC-CM (N=29) Mean ± SD | 57.965 ± 2.196 | 54.172 ± 2.237 | 49.552 ± 2.248 | |
| Wrinkle | Gel (N=29) Mean ± SD | 58.31 ± 8.965 | 58.069 ± 9.75 | 58.379 ± 9.124 |
| hASC-CM (N=29) Mean ± SD | 55.965 ± 3.47 | 55.965 ± 3.47 | 34.172 ± 2.981 | |
| Spot (polarized) | Gel (N=29) Mean ± SD | 57.483 ± 8.448 | 57.965 ± 8.377 | 58.138 ± 8.522 |
| hASC-CM (N=29) Mean ± SD | 55.551 ± 3.245 | 46 ± 2.509 | 35.965 ± 2.241 | |
| Spot (UV) | Gel (N=29) Mean ± SD | 51.68 ± 7.7 | 52.517 ± 7.5 | 52.552 ± 7.562 |
| hASC-CM (N=29) Mean ± SD | 37.448 ± 2.84 | 27.172 ± 2.655 | 19.276 ± 2.273 | |
| Skin tone | Gel (N=29) Mean ± SD | 57.069 ± 9.145 | 56.931 ± 9.031 | 57.138 ± 9.164 |
| hASC-CM (N=29) Mean ± SD | 46.345 ± 1.01 | 44.345 ± 1.073 | 42.724 ± 1.078 | |
Table 3: p values of significance of the improvement in clinical photoaging before and after treatment.
| Clinical Photoaging | Visit 1 and 2 | Visit 2 and 3 | Visit 1 and 3 |
|---|---|---|---|
| hASC-CM | |||
| Glogau Score | 0.199 | 0.007* | 0.001* |
| TEWL | 0.014* | 0.002* | 0.000* |
| Pore | 0.191 | 0.157 | 0.019* |
| Wrinkle | 1.000 | 0.000* | 0.000* |
| Spot (Polarized) | 0.027* | 0.003* | 0.000* |
| Spot (UV) | 0.004 | 0.019* | 0.000* |
| Skin Tone | 0.126 | 0.215 | 0.012* |
| Vehiculum only | |||
| Glogau Score | 1.000 | 1.000 | 1.000 |
| TEWL | 0.549 | 0.104 | 0.049* |
| Pore | 0.950 | 0.767 | 0.815 |
| Wrinkle | 0.963 | 0.975 | 0.969 |
| Spot (Polarized) | 0.773 | 0.882 | 0.708 |
| Spot (UV) | 0.726 | 0.944 | 0.761 |
| Skin Tone | 0.963 | 0.957 | 0.950 |
Figure 1. Glogau score improvement; (A) hASC-CM week 0; (B) hASC-CM week 8;(C) Gel week 0; (D) Gel week 8.
Discussion
The effects of human adipose stem cell-conditioned medium (hASC-CM) in photoaged skin were examined using Tewameter, skin analyzer device, and Glogau score to evaluate photoging improvement after treatment. This research only involved female subjects since women tend to be strongly concerned about their skin condition and regularly apply skin care which lowered the possibility of dropping out. This research reported that the number of photoaged women aged 45-49 was equal to those in the 50-54 and 55-59 age groups. Malvy et al. (2000) reported that women aged 55-60 were the most prevalent group for photoaging[16]. In another research, Chung et al. (2001) reported that women in the 50-69 years old group were more exposed to photoaging than other age groups[17]. The photoaging process can be influenced by skin type, ethnicity, geographic location, occupation, and lifestyle-related UV exposure, and by photoprotective activities; sunscreen application, the use of photoprotective clothing, and the avoidance of sun exposure. This research found that women with photoaging were more prevalent among those engaging in outdoor activities (51.7%) compared to indoor activities (48.3%). Likewise, Sartorelli et al., reported that farmers who mostly worked outdoors showed higher degree of photoaging than indoor workers[18]. Furthermore, Grandahl et al., identified a significant correlation between outdoor work and the prevalence of facial wrinkles and actinic keratosis in photoaged skin, noting that it was twice as common among outdoor workers compared to indoor workers[19]. We attempted to minimize the effects of indoor and outdoor activities by recommending patients to use sunscreen and to avoid being out in the sun.
Ultraviolet exposure activates downstream signalling, which upregulates the expression of matrix metalloproteinase (MMP) and proinflammatory cytokines[20]. Chronic exposure to UVB results in skin ageing, which causes wrinkle formation, acute erythema, and loss of hydration and elasticity[21]. UV exposure also causes alterations in epidermal barrier function when the lamellar body-deficient keratinocytes arrive at the stratum granulosum/ stratum corneum interface[22]. Transepidermal water loss (TEWL) and skin capacitance are the most commonly used indices for assessing skin barrier function. Therefore, maintaining skin hydration is crucial for preventing skin aging. High TEWL and low capacitance levels are indicative of skin barrier disruption[21]. Previous research in photoaging animal models reported that UVB exposure increased TEWL value[21,23–26], where epidermal thickness and ceramide synthase 3 increased but filaggrin was decreased[23]. A Similar research also reported decreased expression of filaggrin, involucrin, and hyaluronic synthase which are important factors for skin hydration[24].
Adipose stem cell is a type of mesenchymal stem cell which easily obtained from adipose tissue through non-invasive procedures[10]. Adipose stem cell-conditioned medium (ASC-CM) is easy to extract, transport, and store. ASC-CM penetrates the skin barrier through lasers or microneedles to promote skin regeneration[11,20]. This conditioned medium contains active substances such as growth factors, cytokines, and exosomes, which can repair tissue damage by promoting the proliferation and migration of skin fibroblasts and increasing the production of ECM and collagen in photoaging skin[9,11,20]. Xu et al., identified that ASC-CM consisted of active proteins, including connective tissue growth factor (CTGF), fibronectin (FN), tissue inhibitor metalloproteinase (TIMP)-1, and protease inhibitor (PAI)[9]. These proteins are important in cell adhesion, migration, wound healing, and tissue remodeling. Li et al. (2020) and Guo et al. (2020) also showed that ASC-CM protected dermal fibroblasts[13,27]. Li et al. (2020) found that ASC-CM downregulated MAPK pathways which decreased MMP-1 production and IL-6 secretion[13]. Similar to Guo et al., Kim et al., also reported that ASC-CM increased the expression of procollagen and collagen in human dermal fibroblast[28].
Significant differences in all parameters (p<0.05) were observed. Significant decrease in TEWL was found on the second visit in Week 4 and Week 8. Adipose stem cell-conditioned medium improves the skin barrier through decreased TEWL and increased skin hydration[6]. Alligning with our research, Xu et al., and Kim et al., reported that hASC-CM significantly improved skin moisture and TEWL value[9,28]. Putri et al., also found that hASC-CM decreased TEWL, although the decrease was not statistically significant[15].
Other parameters, such as spot polarized and spot UV significantly improved on the second visit. Almost similar to our study, Xu et al., reported that ASC-CM combined with injectable hyaluronic acid (HA) significantly reduced the melanin index compared to the control group (p<0.01) within 15 days[9]. Their melanin index was also lower than placental stem cell-conditioned medium. In previous research, Wang et al., also reported an improvement in melanin index in Week 10 and 12 (p<0.01) compared with baseline[8]. Other parameters showed improvement on the second visit, including Glogau score, pores, wrinkles, and skin tone, with higher significance on the third visit.
The use of gels can lead to reduced skin hydration, and alcohol-based gels may cause stinging sensations when applied to broken skin[29]. In this present research, several side effects such as erythema and skin dryness were found in six patients which could be triggered by damaged skin barrier in photoaged skin from the gel application. Limitations in this study were the absence of anatomical pathology examination and there was no follow-up after treatment was carried out, also we did not conduct a patient satisfaction assessment.
Conclusion
In conclusion, compared with the control group, the treatment group (hASC-CM) exhibited more significant improvements in all parameters. Unfortunately, long-term follow-up after treatment was not conducted in this research.
Acknowledgments
The authors would like to thank all the patients and investigators who participated in this study.
Authors Contribution
All of the authors have equally contributed.
Conflict Of Interests
There are no potential conflicts of interest to declare by the authors.
Sponsor/grants
This study was funded by L’Oréal as part of the Hair and Skin Research Grant 2021 program.
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