The outer epidermal skin is a primary barrier that protects the body from extrinsic factors, such as ultraviolet (UV) radiation, chemicals and pollutants. The complete epithelialization of a wound by keratinocytes is essential for restoring the barrier function of the skin. However, age-related alterations predispose the elderly to impaired wound healing. Therefore, wound-healing efficacy could be also considered as a potent function of an anti-aging reagent. Here, we examine the epidermal wound-healing efficacy of the fourth-generation retinoid, seletinoid G, using HaCaT keratinocytes and skin tissues. We found that seletinoid G promoted the proliferation and migration of keratinocytes in scratch assays and time-lapse imaging. It also increased the gene expression levels of several keratinocyte proliferation-regulating factors. In human skin equivalents, seletinoid G accelerated epidermal wound closure, as assessed using optical coherence tomography (OCT) imaging. Moreover, second harmonic generation (SHG) imaging revealed that seletinoid G recovered the reduced dermal collagen deposition seen in ultraviolet B (UVB)-irradiated human skin equivalents. Taken together, these results indicate that seletinoid G protects the skin barrier by accelerating wound healing in the epidermis and by repairing collagen deficiency in the dermis. Thus, seletinoid G could be a potent anti-aging agent for protecting the skin barrier.
Keywords: seletinoid G; wound healing; keratinocyte; human skin equivalents; optical coherence tomography; second harmonic generation
The skin, which comprises the outer epidermis, underlying connective tissue and the dermis, functions as a barrier that protects the body from environmental stressors, such as pathogens and physical stress [[
The term "retinoids" refers to the naturally occurring or synthetic members, precursors or derivatives of vitamin A that bind to nuclear retinoid receptors, such as retinoid X receptors (RXRs) and retinoic acid receptors (RARs). Their key functions in physiology are controlling cellular proliferation and differentiation. In skin, retinoids promote keratinocyte proliferation, strengthen the protective function of the epidermis, protect collagen against degradation and inhibit metalloproteinase activity; thus, they have an anti-aging effect on skin. Topical retinoids have been shown to prevent and repair clinical features of both intrinsic aging and photo-aging [[
In 2005, our institute developed seletinoid G, a novel pyranone derivative, 2-((3E)-4(2H,3H-benzo[3,4-d]1,3-dioxolan-5-yl)-2-oxo-but-3-enyloxy)-5-hydroxy-4H-pyran-4-one, as a novel synthetic retinoid [[
In the current study, we used several advanced protocols to examine the wound-healing efficacy of seletinoid G. Scratch assays and automated time-lapse imaging were used to visualize the real-time migration of HaCaT cells (an immortalized normal human keratinocytes). Optical coherence tomography (OCT) was applied to examine wound healing in the epidermis layer of human skin equivalents treated with and without seletinoid G. Finally, three-dimensional investigation using second harmonic generation (SHG) imaging was used to reveal that seletinoid G realigns collagen deposition in the dermis of human skin equivalents.
Prior to observing the wound-healing efficacy of seletinoid G, we defined the concentration of seletinoid G that was tolerated in the context of the cell viability and proliferation of human keratinocytes (HaCaT cells) and primary normal human dermal fibroblasts (NHDF). We applied various concentrations of seletinoid G to HaCaT cells and NHDF for 24 or 48 h. As shown in Figure 1, seletinoid G treatment at concentrations up to 25 µM had no effect on cell viability at 24 h and clearly increased the number of HaCaT cells up to 48 h. Similar results were obtained in NHDF (Figure 1). Thus, we tested seletinoid G at concentrations up to 25 µM in our in vitro experiments.
Next, we tested the wound-healing efficacy of seletinoid G in HaCaT cells. HaCaT cells were seeded to culture plates and grown for 24 h, and the formed monolayers were scratched in a straight line on the plate. Wound healing was then monitored for 48 h in the presence of various concentrations of seletinoid G. As shown in Figure 2A and the Movie S1, we observed that seletinoid G-treated cells were more proliferative and/or migrated more to close the wounded area, compared to the control cells. When we measured the wound-healing area every hour for 48 h using time-lapse imaging microscopy, we observed that HaCaT cells treated with 6, 12 and 25 µM seletinoid G all covered the wounded area significantly better than the control cells, although 12 µM seletinoid G tended to be more effective than 25 µM seletinoid G (Figure 2B).
To prepare the wounded skin-equivalent model, a full-thickness skin model comprising human epidermal keratinocytes in an epidermis and human dermal fibroblasts in a dermis was wounded with a 3-mm biopsy punch and topically treated with seletinoid G (12 and 25 µM) every other day. After 3 or 6 days, the re-epithelialized areas were three-dimensionally measured using optical coherence tomography (OCT) and H&E staining. As shown in Figure 3, we observed that topical treatment with 12 μM seletinoid G dramatically accelerated wound closure (blue colors) compared to the control group at day 3. As seen in Table 1, 12 μM seletinoid G significantly showed a wound-healing area of 84.7% while the control group showed a wound-healing area of 51.3%. In the case of 25 μM seletinoid G, there was a tendency for epidermal wound-healing effect on day 3, but no statistical significance. On day 6, both the 12 and 25 μM seletinoid G treated groups showed a slightly faster wound-healing efficacy than the control group, although there was no significance.
Having observed that seletinoid G promoted wound healing in the epidermis, we further elucidated the gene expression level of factors known to affect keratinocyte proliferation and/or migration (two major mediators of wound healing). We treated HaCaT cells with seletinoid G for 24 h and used real-time PCR to detect the gene expression levels of keratinocyte growth factor (KGF), microRNA-31 (miR-31), keratin 1 (KRT1), keratin 10 (KRT10), KI-67 and proliferating cell nuclear antigen (PCNA). As shown in Figure 4, seletinoid G significantly and dose-dependently increased the mRNA levels of KGF, miR-31, KRT1 and KRT10 (all stimulators of keratinocyte proliferation), whereas the mRNA expression of PCNA and KI-67 was not altered under seletinoid G treatment.
A previous study showed that seletinoid G increased the expression of procollagen and reduced that of matrix metalloproteinase (MMP)-1 in skin [[
Skin aging is a combination of biochemical, mechanical and environmental changes that lead to declines in the structure and function of the skin. These changes may be induced by the passage of time (chronological aging) and/or chronic exposure to solar UV irradiation (photo-aging) [[
Previous studies suggested that seletinoid G may be as effective as tretinoin in treating intrinsic and/or photo-aging [[
Our quantitative real-time PCR results to confirm the gene expression of keratinocyte proliferation and migration markers showed that 25 μM seletinoid G was better than 12 μM. However, other results (monolayer scratch assay in Figure 2 and three-dimensional human skin equivalent wound-healing assay in Figure 3) showed that 12 μM is more effective than 25 μM. It is assumed that this discrepancy found in the data with respect to doses results from the presence of mechanical damage to keratinocytes. Wound-healing assays were performed under mechanically induced injury conditions, while RT-PCR studies were performed under normal cell condition. In addition, we supposed that cell culture conditions such as fetal bovine serum (FBS) contents and 2D/3D culture system would affect the effective concentrations of seletinoid G. Given these factors, it is likely that the optimal concentrations of seletinoid G could be slightly changed with in vitro experimental conditions; therefore, we concluded that the effective concentration range of seletinoid G is between 12 and 25 μM in this study.
The present study did not seek to further reveal the molecular mechanism underlying the wound-healing effect of seletinoid G. Instead, we focused on using advanced technology to clearly visualize the wound-healing process in our systems. First, we used time-lapse imaging microscopy to monitor the wound-healing process in real-time. As shown in Movie S1 and Figure 1, our real-time observations of healing monolayers revealed that keratinocytes moved faster in the presence of seletinoid G. Next, we utilized swept-source optical coherence tomography (SS-OCT) to evaluate the wound-healing effect of seletinoid G on an in vitro human skin equivalent. OCT is a near-infrared imaging technique that collects interference signals reflected from the specimen to reconstruct a cross-sectional image. It enables morphological structures to be visualized at up to sub-cellular resolution in a real-time and non-invasive manner. This quantitative tissue monitoring method may be used to evaluate tissue regeneration after skin injury [[
Retinoids are popular cosmetic ingredients for anti-aging. However, they are associated with side effects such as skin problems. For this reason, dermatologists have tried to develop less irritating but comparably effective retinoids. In the previous study, synthetic retinoid seletinoid G has been proposed as a successful retinoid for anti-intrinsic/photo-aging [[
Seletinoid G, which is a retinoid analog previously designed by computer-aided molecular modeling, was synthesized as previously described [[
The human keratinocyte cell line, HaCaT, was purchased from CLS (#300493, Cell Lines Service, Eppelheim, Germany). Normal human dermal fibroblasts, neonatal (NHDF), were purchased from Thermo Fisher Scientific (#C-004-5C, Waltham, MA, USA). HaCaT cells and NHDF were cultured in Dulbecco's modified Eagle's medium (DMEM; #12-604F, Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (#10082-147, Thermo Fisher Scientific), 100 U/mL potassium penicillin and 100 mg/mL streptomycin sulfate (#17-602E, Lonza) at 37 °C in a humidified 5% CO
HaCaT cells were seeded to a 24-well plate and cultured in DMEM containing 10% FBS. After 24 h, the cells were incubated in DMEM containing 1% FBS overnight. Each monolayer was scratched with a P1000 pipet tip and then treated with or without seletinoid G in DMEM containing 1% FBS. The scratched regions (n = 4 per group) were recorded every hour for 48 h using automated time-lapse imaging microscopy (JuLI Stage Real-Time Cell History Recorder, NanoEnTek, South Korea). The wound-healing area (% of the initial wound area at t = 0 h) was calculated with the JuLI-STAT software (NanoEnTek, Seoul, South Korea).
We purchased a human skin equivalent wound model (EpiDermFT
Briefly, image reconstruction was performed as follows. The light was emitted from a swept laser (Axsun Tech., Billerica, MA, USA), which had a 1310-nm center wavelength and a 110-nm tuning range. The light was propagated along the optical fiber and divided by a coupler that moved it toward the sample and reference arms. We designed a Mach–Zehnder interferometer, then the light returned from each arm through a circulator to form an interference signal. The signal was detected by a balanced amplified photodetector (Thorlabs Inc., Newton, NJ, USA) and digitized by a digitizer (AlazarTech Inc., Pointe-Claire, Canada) to create an intensity profile that reflected information on depth. The system had an axial resolution of ~7 µm and a lateral resolution of ~15 µm in the air; each cross-sectional image consisted of 1000 A-lines obtained with an acquisition speed of 20 frames/s.
Total RNA was isolated using an RNeasy Mini Kit (#74104, Qiagen, Hilden, Germany) or an RNeasy Plus Mini Kit (#74134, Qiagen) and cDNA was prepared using a SuperScript III First-Strand synthesis system kit (#51101, Invitrogen, Grand Island, NY, USA). TaqMan primer sets for the KGF (#Hs00940253_m1), miR-31 (#4427975), KI-67 (#Hs04260396_g1), KRT1 (#Hs00196158_m1), KRT10 (#Hs00166289_m1), PCNA (#Hs00427214_g1) and RPLP0 (#4333761F) genes were purchased from Applied Biosystems (Foster City, CA, USA). qRT-PCR was performed using the TaqMan universal PCR master mix (#4304437, Applied Biosystems) and an Applied Biosystems 7500 Fast Real-time PCR system. Relative mRNA expression levels of target genes were normalized to those of the housekeeping gene, RPLP0, and calculated using the comparative ∆∆C
The optical setup for multi-photon microscopy was as described previously [[
We collected the culture medium of human skin equivalents that had been exposed or not to seletinoid G for 48 h. The concentrations of MMP-1 protein in the culture medium were measured using an MMP-1 ELISA kit (#DY901, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.
Data are expressed as the mean ± standard deviations (SDs), and statistical significance was analyzed by the Student's t-test. P-values less than 0.05 were considered statistically significant.
Graph: Figure 1 Cell viability test of seletinoid G on a human keratinocyte cell line (HaCaT) and normal human dermal fibroblasts (NHDF). The cell viability of HaCaT cells and NHDF treated with seletinoid G at different concentrations for 24 and 48 h was measured by CCK-8 assay. (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. the untreated group).
Graph: Figure 2 In vitro wound-healing effect of seletinoid G on wounded HaCaT keratinocyte monolayers. (A) HaCaT cells were line-scratched and then treated with seletinoid G (SG) at concentrations of 6, 12, and 25 μM in Dulbecco's modified Eagle's medium (DMEM) containing 1% fetal bovine serum (FBS) for 48 h Scale bars indicate 500 μm. (B) Each line-scratched area was automatically measured every hour for 48 h using time-lapse imaging microscopy.
Graph: Figure 3 In vitro wound-healing effect of seletinoid G on a human skin equivalent wound model (MatTek, EFT-400-WH). Seletinoid G (12 or 25 μM) was applied every other day; on days 3 and 6, the skin equivalents were fixed with 4% formaldehyde (n = 3 per group). Three-dimensional imaging was performed using optical coherence tomography (OCT) microscopy. Blue color indicates the regenerated epidermal area. Scale bars indicate 1 mm.
Graph: Figure 4 mRNA expression levels of proliferation- and migration-related genes in seletinoid G-treated HaCaT cells. The ribosomal protein lateral stalk subunit P0 (RPLP0) gene was used as an internal control for quantitative real-time PCR. (n.s.: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).
Graph: Figure 5 (A) Label-free multi-photon images of collagen fibrils (green) at different z-depths of the dermis in human skin equivalents (EFT-400) that were irradiated with UVB (30 mJ/cm2), irradiated and treated with SG (20 μM) in the culture medium for 48 h, or left non-irradiated and untreated (control). Scale bars indicate 40 μm. (B) ELISA of matrix metalloproteinase (MMP)-1 secreted to the culture medium of human skin equivalents (n = 3 per group). (***; p < 0.001).
Table 1 The quantification of wound-healing area, as shown in Figure 3. In a human skin equivalent wound model, we quantify wound-healing area (% of initial wound area at day 0, n = 3 per group) by measuring the regenerated lengths in epidermis via an Image J software from individual OCT XZ-axis images. ("p value" vs. control group).
After Treatment Wound Healing Area (%) Seletinoid G (μM) Control (0) 12 25 Day 3 Mean ± SD 51.3 ± 13.5 84.7 ± 14.9 72.4 ± 23.7 - 0.045 0.250 Day 6 Mean ± SD 89.7 ± 12.8 100.4 ± 5.9 103.3 ± 2.7 - 0.2562 0.1445
Conceptualization, E.-S.L., N.H.P., Y.D.H., W.S.P. and C.S.L.; Data curation, E.-S.L. and C.S.L.; Investigation, E.-S.L.; Methodology, E.-S.L., Y.A., I.-H.B., D.M., W.J. and S.-H.K.; Supervision, Y.D.H., W.S.P. and C.S.L.; Writing—original draft, E.-S.L. and C.S.L.; Writing—review and editing, C.S.L. All authors have read and agreed to the published version of the manuscript.
This research was funded and supported by Amorepacific R&D Center.
The authors declare no conflict of interest.
NHDF normal human dermal fibroblasts UVB ultraviolet B SG seletinoid G RPLP0 ribosomal protein lateral stalk subunit P0 OCT optical coherence tomography SHG second harmonic generation KGF keratinocyte growth factor miR-31 microRNA-31 PCNA proliferating cell nuclear antigen KRT keratin MMP matrix metalloproteinase ECM extracellular matrix
Supplementary materials can be found at https://
By Eun-Soo Lee; Yujin Ahn; Il-Hong Bae; Daejin Min; Nok Hyun Park; Woonggyu Jung; Se-Hwa Kim; Yong Deog Hong; Won Seok Park and Chang Seok Lee
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