Subcutaneous Injection of Conditioned Medium from Photobiomodulation- Treated Adipose-Derived Stromal Cells Promotes Hair Growth in Mice

Subcutaneous Injection of Conditioned Medium from Photobiomodulation- Treated Adipose-Derived Stromal Cells Promotes Hair Growth in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Subcutaneous Injection of Conditioned Medium from Photobiomodulation- Treated Adipose-Derived Stromal Cells Promotes Hair Growth in Mice In-Su Park This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6110120/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The purpose of this study was to investigate whether subcutaneous injection of conditioned medium from photobiomodulation (PBM) irradiated adipose-derived stromal cells (ASCs) could stimulate hair growth in mice. Human ASCs were plated on cell culture dishes at a concentration of 2 × 10 4 cells/cm 2 and allowed to adhere at 37°C. Light emitting diode (660 nm, 50 mW/cm 2 ) was applied for 10 min daily from day 1 to day 10. Conditioned medium (CM) fractions were collected from PBM-irradiated ASCs to yield (PBM-CM). They were centrifuged at 13,000 g for 10 min at 4°C and stored prior to use for ELISA, protein assay, or in vivo assays. Forty-one growth factors in PBM-CM from cells cultured under PBM or non-PBM conditions were analyzed. Survival, differentiation and secretion of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) of PBM-CM were evaluated by western blot. Secretion of insulin-like growth (IGF), platelet-derived growth factor (PDGF), and insulin-like growth factor binding protein (IGFBP) were significantly increased by PBM. PBM-CM enhanced hair growth in a hair loss model compared to non-irradiated ASC-CM or Phosphate-buffered saline (PBS). PBM is an effective biostimulator of ASC-CM in hair regrowth by enhancing secretion of growth factors. Thus, PBM-CM has the potential to stimulate hair growth and treat alopecia. Photobiomodulation Adipose-derived stem cells Hair growth Conditioned medium Alopecia Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Alopecia is marked by unusual hair loss or thinning. ( 1 ). Although alopecia is not considered a life threatening disease, its negative psychological effects are commonly underestimated ( 2 , 3 ). At present, drugs such as Finastride and Minoxidil are almost the only and most effective treatments. Discontinuation of these drugs risks accelerated hair loss ( 4 ). Thus, effective treatment strategies need to be developed to overcome issues faced by current treatment strategies. Since hair growth efficiency of those using drugs is not sufficient, various attempts have been made using stem cells as a treatment for hair loss ( 5 ). According to previous studies, ASCs, present in various adult tissues, serve as a promising source for cell therapy in tissue regeneration due to their ability to self-renew and differentiate into diverse cell types and tissues ( 6 , 7 ). Transplanting hASCs can induce neovascularization and improve blood flow in animal models ( 8 , 9 ). The current hair loss treatment method using stem cells involves inducing stem cells to differentiate into hair follicle cells by injecting stem cells directly into the site showing symptoms of hair loss or hair loss ( 10 ). However, hair growth cell therapies have certain limitations, such as low cell survival in avascular wounds, the potential risk of cancer development, and challenges in clinical application ( 11 ). To overcome these shortcomings, regenerative potential of stem cells can be induced with a novel approach by administering the secretome released by these cells, including Extracellular matrix (ECM) molecules, cytokines, and growth factors known to play an important role in stimulating molecular and cellular processes that govern hair growth ( 12 ). Moreover, soluble factors derived from hASCs, including immune-modulatory secretions, anti-inflammatory cytokines, and growth factors, differ significantly from those of adult cells, suggesting that expressing these cytokines and growth factors from hASCs may play a role in an anti-inflammatory environment and tissue regeneration in vivo and they function as a viable cell source, particularly for tissue engineering ( 13 , 14 ). The use of stem cell culture-derived CM as a therapeutic approach has been extensively studied in both in vitro and in vivo models, yielding outstanding results ( 15 , 16 ). CM fractions collected from peripheral blood mononuclear cell (PBMC) have also been assessed for their contents containing potentially beneficial cytokines, growth factors, and chemokines, it has been applied to cutaneous wounds in mice ( 15 ). It is widely recognized that PBM enhances both the proliferation ( 17 , 18 ) and growth factor secretion of hASCs ( 19 ). The low-power lasers used in PBM, also known as cryolasers, use optical energy to raise the temperature to less than 0.1 to 0.5 degrees Celsius. PBM can also stimulate the production of growth factors such as HGF, EGF, and bFGF ( 20 ). PBM utilizes red or near-infrared light at low power densities to induce beneficial effects on cells and tissues ( 17 , 19 ). PBM exerts a photobiological effect via stimulation of cytochrome c oxidase (CCO) located in the cytochrome of the mitochondrial respiratory chain complex IV. In this study, we have focused on the activation of mitochondria and associated changes in several cytokines and growth factors. In recent years, PBM therapy has been used to treat alopecia due to its proven safety and effectiveness, with minimal reported side effects ( 21 ). Recently, PBM has gained significant attention for its role in stimulating the growth of new blood vessels ( 22 ). A 660 nm red light-emitting diode (LED) has been shown to promote tissue healing by stimulating angiogenesis in various animal models of ischemia ( 23 ). However, little is known about effects of PBM on subcutaneous injection of CM in animal models of alopecia. Therefore, this study aimed to determine whether CM, a cell-free, paracrine factors rich source with PBM could be an alternative to cell-based therapies for hair regeneration. 2. Materials and Methods 2.1. Culture of ASCs The ASC were supplied by Cell Engineering for Origin, CEFO (Seoul, Korea) under a material transfer agreement. ASC were isolated from the human- adipose tissue and were cultured in low-glucose Dulbecco's modified Eagle's medium F-12 (DMEM/F-12; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, Welgene), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO 2 incubator. The ASC between passage 5 and 8 were used for all experiments (Supplemental Fig. 1). These cells were positive for human MSC markers CD29 (ß1 integrin), CD90 (Thy-1), and CD105 (endoglin), but negative for human endothelial cell markers CD34, CD31, KDR (VEGF receptor), and hematopoietic cell marker CD45 based on flow cytometry analyses (Supplemental Fig. 1). These results indicated that these expanded cells included a large population of hASCs without contamination of endothelial cells. 2.2. PBM hASCs were plated on cell culture dishes at a concentration of 2 × 10 4 cells/cm 2 , and allowed to adhere at 37°C. Light emitting diode (LED; WON Technology, Daejeon, Korea) was applied for 10 min daily from day 1 to 10 (Fig. 1 ). The LED was had an emission wavelength peaked at 660 nm. The light source is placed 6 cm 2 away from the cells. The irradiance at the surface of the cell monolayer was measured by a power meter (Orion, Ophir Optronics Ltd., UT). To obtain the energy dose of 30 J/cm 2 , exposure time for LED array was 10 min under power density of 50 mW/cm 2 (1 milliwatt × second = 0.001 joules). Fresh culture media was replaced every 2 days. 2.3. Preparation of CM fractions derived from PBM-ASCs CM were collected every 2 days from the PBM-ASC to yield nor PBM, respectively, centrifuged at 13,000 g at 4°C for 10 min, and stored prior to use. For the in vivo analysis, aliquots of conditioned media were concentrated by further centrifugation and filtration through a centrifugal filter unit (Millipore, Billerica, MA, USA). 2.4. Mitochondria ATP synthesis assay ATP sylnthesis was measured with cellTiter-Glo 2.0 cell viability assay (Promega, Madison, USA). To measure the ATP systhesis of the cells, 5 × 10 3 cells were placed in the 96-Well plate and then mixed at a celltiter-Glo 2.0 Reagent 1: 1 ratio to react at room temperature that blocked the light for 10 minutes. The degree of light emitting was measured using the Synergy HTX Microplate Reader (BIOTEK, Vermont, USA). 2.5. Human protein analysis To analyze the expression profiles of angiogenesis-related proteins as GCSF, IGFBP, IGF, PDGF, we used a Human protein Array Kit (R&D Systems, Ltd., Abingdon, UK). Cell samples (5 × 10 6 cells) were harvested, and 150 µg of protein were mixed with 15 µl of biotinylated detection antibodies. After pre-treatment, the cocktail was incubated with the array overnight at 4°C on a rocking platform. Following washing to remove unbound material, streptavidin–horseradish and chemiluminescent detection reagents are added sequentially. The signals on the membrane film were detected by scanning on an image reader LAS-3000 (Kodak, Rochester, NY) and were quantified using the MultiGauge 4.0 software (Kodak). The positive signals seen on developed film were identified by placing a transparency overlay on the array image and aligning it with the two pairs of positive control spots in the corners of each array. 2.6. Western blot analysis Samples were solubilized in lysis buffer for 1 h at 4°C. The lysates were then clarified by centrifugation at 15,000 g for 30 min at 4°C, were diluted in Laemmli sample buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol, and were heated for 5 min at 90°C. The proteins were separated via SDS polyacrylamide gel electrophoresis (PAGE) using 10% or 15% resolving gels followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies against HGF (Santa cruz), VEGF (Abcam), and EGF (Abcam) for 1h at room temperature (Supplementary table 1 ). This antibody can be used at a concentration of 0.1–0.2 µg/ml. Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) were used as described by the manufacturer for detection. The membranes were scanned to create chemiluminescent images that were then quantified with an image analyzer (Kodak). 2.7. In vivo animal experiment for hair growth Experiments involving mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 − 23, revised 1996). All aspects of the animal care and experimental protocols were approved by the Ajou University Committee on Animal Care (IACUC No. 2020-23). Six-week-old male BALB/c mice (Daehan Biolink, Korea) (27.0 ± 1.4 g) purchased and maintained for 1 week before beginning experiments. Inbred lab mice are not isogenic. Hair growth activity in mice was determined visually according to a slight modification of the method described by Hattori and Ogawa (Hattori and Ogawa, 1983). The backs of the mice were shaved using a hair shaver (ISIS, B. Braun, Melsungen, Germany) and depilated using cold wax bands (Veet Minima). Shaving was performed in a region (2× 3 cm) followed by a hair remover cream (Reckitt Benckiser France, Cedex, France) to avoid wax sticking to grown non-shaved hair. The cold wax stripe was applied once with the skin well stretched and removed at once against the direction of hair growth. BALB/c mice were divided into three groups of seven mice each: PBS (control) group, CM group and PBM-CM experimental group. PBS was topically applied to the shaved skins of mice in the control group. Then, 60 µl of CM solution were applied to the CM group. PBS or CM solution was subcutaneous injected and gently rubbed until it is completely absorbed into the skin. For all groups, 60 µl samples were applied twice daily for 3 weeks. Mice were sacrificed by CO 2 exposure on the 21st day. Their backs were then shaved, visually evaluated, and photographed. 2.8. Determination of hair growth area Hair regrowth and nongrowth areas were photographed and printed. Regrowth and nongrowth regions were cut from the prints. The percentages of hair regrowth areas relative to total shaved skin areas were calculated as follows: Hair growth area (%) = 100 × [regrowth area weight / (regrowth area weight + nongrowth area weight)]. Our analysis of these images quantify by image analysis by ImageJ (free software). 2.9. Histological staining Samples were harvested 21 days after treatment. Specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated in a graded ethanol series, and embedded in paraffin. Specimens were sliced into 4 µm-thick sections and were stained with hematoxylin and eosin (H&E) to examine muscle degeneration and tissue inflammation. The histological parameters considered were re-epithelialization, dermal regeneration, granulation tissue formation, and angiogenesis. All quantitative results were obtained from triplicate samples. Regeneration of skin appendages was assessed by counting the number of hair follicles or sebaceous glands in the skin. 2.10. Immunofluorescence staining Indirect immunofluorescence staining was performed using a standard procedure. In brief, tissues cryosectioned at a 4-µm thickness were fixed with 4% paraformaldehyde, blocked with 5% BSA/PBS (1 h, 24°C), washed twice with PBS, treated with 0.1% Triton X-100/PBS for 1 min, and washed extensively in PBS. The sections were stained with specific primary antibodies and fluorescent-conjugated secondary antibodies (Table 2) using a M.O.M kit according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). The cells were counterstained with DAPI (4,6-diamino-2-phenylindole dihydrochloride; Vector Laboratories). Mouse IgG (Dako, Carpinteria, CA) and rabbit IgG (Dako) antibodies was used as negative controls. The stained sections were viewed with a DXM1200F fluorescence microscope (Nikon, Tokyo, Japan). The processed images were analyzed for fluorescence intensity using the ImageJ software (NIH). 2.11. Statistical analyses Data were expressed as a mean ± SD, and the statistical analyses were carried out using two-sample t tests to compared two groups of samples and a One-way Analysis of Variance (ANOVA) for the three groups. A value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. ATP synthesis ATP synthesis was evaluated to mitochondrial activity. With the same cell number, the ATP production in PBM cells was 1.63 ± 0.2 times higher than that in non -PBM cells (Fig. 2 A). 3.2. Production of growth factors by PBM cells As shown in Fig. 2 B, most growth factors were up-regulated in PBM with some exceptions. PBM upregulated the expression of growth factors, with PBM cultured cells showing significantly up-regulated expression of GCSF, IGFBP, IGF, and PDGF. hASCs also secreted a wide spectrum of growth factors including HGF, VEGF and EGF (Fig. 2 C). The expression of growth factors in PBM cultured cells was much greater than that in non-PBM treated cells. 3.3. Hair growth promoting effect of PBM-CM in BALB/c mice The hair growth promoting effect of CM was systematically tested using BALB/c mouse. PBM-CM enhanced hair growth activity, increasing hair growth area and length to greater extents than treatment with CM or PBS alone (Fig. 3 A). PBM-CM treatment enhanced the hair growth activity of mice (PBM-CM group: 18.6 mg, 422%; ASC-CM group: 13 mg, 295% per PBS group: 4.4 mg) increase in the hair weight (Fig. 3 B). 3.4. Histological analysis of hair growth promotion by CM in mice HE staining revealed numbers of hair follicles and hair shafts (Fig. 3 C). The number of hair follicles was increased 2.5-fold ( p < 0.05) in the PBM-CM treated group (32.1 hair follicles) compared with that in the CM-treated group (12.8 hair follicles) (Fig. 3 D). Hair follicle cycling was augmented in the PBM-CM treated group compared to that in the CM-treated group. These results indicate that PBM-CM can promote hair follicle cycling, leading to an increased number of hair follicles in the dorsal skin of mice. 3.5. PBM-CM increased a ngiogenesis in mice Many of the CD31 + cells in the PBM-CM group were double stained for smooth muscle actin (SMA). Endotherial cells and smooth muscle cells were detected via human CD31 and αSMA antibodies, respectively (Fig. 4 ). Compared with the control group, blood vessels increased in number in the PBM-CM group on day 21. These findings suggest the greater effectiveness of the PBM-CM treatment for angiogenesis in the hair loss. 4. Discussion Stem cell derived CM is widely explored for its therapeutic potential to replace cell-based therapies for a wide range of diseases (24,25). Recently, for more effective hair regeneration, several methods for preconditioning ASC in various conditions have been suggested (26). Hypoxia conditions can amplify paracrine effects of ASCs, which show hair-regeneration and promotion effects by enhancing the secretion of certain growth factors (27). It has been reported that applying low doses of UVB radiation to ASCs can stimulate hair regeneration by regulating reactive oxidative stress (28). We have studied whether PBM-CM treatment method could lead to effective ways to precondition ASCs for more effective hair growth conditions. PBM, when applied with optimal energy density and wavelengths, effectively enhances stem cell viability and proliferation (15,17,18). PBM may enhance cellular responses, including gene expression, growth factor secretion, and cell proliferation, by increasing mitochondrial ATP synthesis (Fig. 2A). It has been reported that ASC can produce various cytokines known as hair-growth promoting factors (15). We investigated the ability of ASC secretomes to promote hair growth and the effect of PBM on ASC function. We found that CM from PBM culture of ASC increased hair regrowth in an animal model (Figs. 3). To identify the mechanism by which PBM enhanced ASC-stimulated hair growth, we used a growth factor antibody array to analyze 41 growth factors. GCSF, IGFBP, IGF, and PDGF were found to be significantly up-regulated (Fig. 2B). hASCs can also secrete a wide spectrum of growth factors including HGF, VEGF, and EGF. Most importantly, PBM culture increased the expression of growth factors and the release of several hair growth-related growth factors including VEGF, IGF, and PDGF from stem cells. In the present study, PBM-CM demonstrated hair growth-promoting effects, as shown by visible hair regrowth on the shaved backs of BALB/c mice, along with increased hair growth areas and hair length (Fig. 3B, 3C). It is widely recognized that neo-angiogenesis plays a crucial role in stimulating hair growth (11). Angiogenesis is controlled by the balance between pro-angiogenic and anti-angiogenic factors (29). VEGF is well known as a representative proangiogenic factor (30). When incorporated into a collagen hydrogel and subcutaneously implanted into the back of mice, VEGF can accelerate hair follicle entry into the anagen phase and promote hair elongation (31). These results demonstrate that VEGF can promote hair follicle growth by inducing angiogenesis. It has been demonstrated that IGF expressed in the hair follicle can affect follicular proliferation, tissue remodeling, and the hair growth cycle (32). Loss of IGF can induce a delay telogen and retard the onset of the second anagen phase, with guard hairs of transgenic mice skin showing significant elongation (33). PDGF has been shown to induce the anagen phase of the hair growth cycle at the injection site on the dorsal skin of mice by acting through its receptors in the hair follicle epithelium (34). Knockout of the PDGF gene in mice resulted in a thinner dermis, misshapen hair follicles, smaller dermal papillae, abnormal dermal sheaths, and thinner hair compared to their wild-type siblings (35). Previous studies have shown that both cortisol and testosterone affect hair growth, and hair analysis can provide insight into long-term hormone levels. However, these hormones have not yet been studied about the interaction of CM with these hormones. Although hormone levels were not measured in this study, further research is needed to understand cortisol and testosterone in hair (36, 37). The secretion of potent growth factors was altered, which might explain the difference in hair growth between non-PBM and PBM. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6110120","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448884911,"identity":"55008586-91ee-46a9-bff0-92befcef6758","order_by":0,"name":"In-Su Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACefbmAwcS/9jA+AmEtRj2HEt88LEhjQQtDDdyjA1nNhwmQQtjQ4KZNO+O83m67QcYH1cwpOUT1MLOcCBNmvfM7WKzMwnMhmcYciwbCNrS2HBMmoftduK2Gwxskg0MFQaEXXaYsQ2o5RwpWo4xMxvObDsA05JDWIthDxvjgw9nkhO3nUlsNmwwSCOsRV7+/YcDCRV2iduOHz74sKEimQiHIQBjAwMDSRpGwSgYBaNgFOAEAMauPsvsAZ+8AAAAAElFTkSuQmCC","orcid":"","institution":"Ajou University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"In-Su","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2025-02-26 06:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6110120/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6110120/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81983541,"identity":"2c665407-2348-4a26-b153-e8654ef1b237","added_by":"auto","created_at":"2025-05-05 15:23:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS\u003c/strong\u003eimplified schematic of PBM irradiation and PBM-CM collection protocol.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6110120/v1/17d754deef19fbf4cfeaafd9.png"},{"id":81983577,"identity":"9bb1b107-2748-4238-9a2a-54beacd7a296","added_by":"auto","created_at":"2025-05-05 15:23:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128498,"visible":true,"origin":"","legend":"\u003cp\u003e(A) ATP synthesis levels [nM] of ASCs were increased from PBM-preactivated ASCs. * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 compared with the PBM group, t test, n = 4 in each group. Error bars represent the standard deviation (SD) of the mean.\u003cstrong\u003e \u003c/strong\u003e(B) Angiogenesis-related protein analysis and quantification of PBM-CM. (C) Western blot analysis and quantification of hair growth related growth factors.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6110120/v1/d3c780996a2ef026e255818d.png"},{"id":81984144,"identity":"b0d83df8-0e83-473b-99b0-49badcf79152","added_by":"auto","created_at":"2025-05-05 15:31:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":315446,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effects of PBM-CM on hair re-growth of BALB/c mice. \u003cem\u003en = 7\u003c/em\u003e in each group. (B) Hair weight (mg) on hair re-growth of BALB/c mice. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the PBM-CM group, t test, \u003cem\u003en = 7\u003c/em\u003e in each group. Error bars represent the standard deviation (SD) of the mean. (C) Three weeks after injection, histological analysis of hair-regenerated skin was performed using hematoxylin \u0026amp; eosin (H\u0026amp;E) staining. The PBM-CM group showed a large number of hair follicles and longer length of hair follicles than the CM group. Bar = 20 μm. Data shown represent one of each group. H\u0026amp;E staining Experiments were performed three times independently. (D) Total number of hair follicles. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the PBM-CM group, t test, \u003cem\u003en = 7\u003c/em\u003e in each group. Error bars represent the standard deviation (SD) of the mean.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6110120/v1/2318f263df11608d3161c0e3.png"},{"id":81983486,"identity":"157fd54c-9eee-4d16-9c23-a89aa153d515","added_by":"auto","created_at":"2025-05-05 15:23:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120131,"visible":true,"origin":"","legend":"\u003cp\u003eStimulatory effect of PBM-CM on angiogenesis. (A) The vascularization of the hair regeneration site was analyzed Day 21 after injection. Endotherial cells and smooth muscle cells were detected via human CD31 and αSMA antibodies. The scale bars indicate 200 μm. (B) Vessel number in the hair loss tissue (\u003cem\u003e*p \u0026lt; 0.05 \u003c/em\u003ecompared to the ASC-CM group).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6110120/v1/5e13b56c8c21d9c67446d014.png"},{"id":81984931,"identity":"26bbec56-eef0-47b0-bf39-8d3d1accaa30","added_by":"auto","created_at":"2025-05-05 15:39:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1291600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6110120/v1/671461a3-a5ac-4290-bbf1-ffd009fa84ad.pdf"},{"id":81983692,"identity":"b74906e9-4e3b-4e52-b9d5-4dd250b4e6b0","added_by":"auto","created_at":"2025-05-05 15:23:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":205685,"visible":true,"origin":"","legend":"","description":"","filename":"supplement240813.docx","url":"https://assets-eu.researchsquare.com/files/rs-6110120/v1/edc53c5efb43710811713fe4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Subcutaneous Injection of Conditioned Medium from Photobiomodulation- Treated Adipose-Derived Stromal Cells Promotes Hair Growth in Mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlopecia is marked by unusual hair loss or thinning. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Although alopecia is not considered a life threatening disease, its negative psychological effects are commonly underestimated (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). At present, drugs such as Finastride and Minoxidil are almost the only and most effective treatments. Discontinuation of these drugs risks accelerated hair loss (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Thus, effective treatment strategies need to be developed to overcome issues faced by current treatment strategies.\u003c/p\u003e \u003cp\u003eSince hair growth efficiency of those using drugs is not sufficient, various attempts have been made using stem cells as a treatment for hair loss (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). According to previous studies, ASCs, present in various adult tissues, serve as a promising source for cell therapy in tissue regeneration due to their ability to self-renew and differentiate into diverse cell types and tissues (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Transplanting hASCs can induce neovascularization and improve blood flow in animal models (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The current hair loss treatment method using stem cells involves inducing stem cells to differentiate into hair follicle cells by injecting stem cells directly into the site showing symptoms of hair loss or hair loss (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, hair growth cell therapies have certain limitations, such as low cell survival in avascular wounds, the potential risk of cancer development, and challenges in clinical application (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo overcome these shortcomings, regenerative potential of stem cells can be induced with a novel approach by administering the secretome released by these cells, including Extracellular matrix (ECM) molecules, cytokines, and growth factors known to play an important role in stimulating molecular and cellular processes that govern hair growth (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Moreover, soluble factors derived from hASCs, including immune-modulatory secretions, anti-inflammatory cytokines, and growth factors, differ significantly from those of adult cells, suggesting that expressing these cytokines and growth factors from hASCs may play a role in an anti-inflammatory environment and tissue regeneration \u003cem\u003ein vivo\u003c/em\u003e and they function as a viable cell source, particularly for tissue engineering (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The use of stem cell culture-derived CM as a therapeutic approach has been extensively studied in both in vitro and in vivo models, yielding outstanding results (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). CM fractions collected from peripheral blood mononuclear cell (PBMC) have also been assessed for their contents containing potentially beneficial cytokines, growth factors, and chemokines, it has been applied to cutaneous wounds in mice (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is widely recognized that PBM enhances both the proliferation (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and growth factor secretion of hASCs (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The low-power lasers used in PBM, also known as cryolasers, use optical energy to raise the temperature to less than 0.1 to 0.5 degrees Celsius. PBM can also stimulate the production of growth factors such as HGF, EGF, and bFGF (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). PBM utilizes red or near-infrared light at low power densities to induce beneficial effects on cells and tissues (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). PBM exerts a photobiological effect via stimulation of cytochrome c oxidase (CCO) located in the cytochrome of the mitochondrial respiratory chain complex IV. In this study, we have focused on the activation of mitochondria and associated changes in several cytokines and growth factors. In recent years, PBM therapy has been used to treat alopecia due to its proven safety and effectiveness, with minimal reported side effects (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Recently, PBM has gained significant attention for its role in stimulating the growth of new blood vessels (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). A 660 nm red light-emitting diode (LED) has been shown to promote tissue healing by stimulating angiogenesis in various animal models of ischemia (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, little is known about effects of PBM on subcutaneous injection of CM in animal models of alopecia. Therefore, this study aimed to determine whether CM, a cell-free, paracrine factors rich source with PBM could be an alternative to cell-based therapies for hair regeneration.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Culture of ASCs\u003c/h2\u003e \u003cp\u003eThe ASC were supplied by Cell Engineering for Origin, CEFO (Seoul, Korea) under a material transfer agreement. ASC were isolated from the human- adipose tissue and were cultured in low-glucose Dulbecco's modified Eagle's medium F-12 (DMEM/F-12; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, Welgene), 100 units/ml penicillin, and 100 \u0026micro;g/ml streptomycin at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The ASC between passage 5 and 8 were used for all experiments (Supplemental Fig.\u0026nbsp;1). These cells were positive for human MSC markers CD29 (\u0026szlig;1 integrin), CD90 (Thy-1), and CD105 (endoglin), but negative for human endothelial cell markers CD34, CD31, KDR (VEGF receptor), and hematopoietic cell marker CD45 based on flow cytometry analyses (Supplemental Fig.\u0026nbsp;1). These results indicated that these expanded cells included a large population of hASCs without contamination of endothelial cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. PBM\u003c/h2\u003e \u003cp\u003ehASCs were plated on cell culture dishes at a concentration of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e, and allowed to adhere at 37\u0026deg;C. Light emitting diode (LED; WON Technology, Daejeon, Korea) was applied for 10 min daily from day 1 to 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The LED was had an emission wavelength peaked at 660 nm. The light source is placed 6 cm\u003csup\u003e2\u003c/sup\u003e away from the cells. The irradiance at the surface of the cell monolayer was measured by a power meter (Orion, Ophir Optronics Ltd., UT). To obtain the energy dose of 30 J/cm\u003csup\u003e2\u003c/sup\u003e, exposure time for LED array was 10 min under power density of 50 mW/cm\u003csup\u003e2\u003c/sup\u003e (1 milliwatt \u0026times; second\u0026thinsp;=\u0026thinsp;0.001 joules). Fresh culture media was replaced every 2 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of CM fractions derived from PBM-ASCs\u003c/h2\u003e \u003cp\u003eCM were collected every 2 days from the PBM-ASC to yield nor PBM, respectively, centrifuged at 13,000 g at 4\u0026deg;C for 10 min, and stored prior to use. For the in vivo analysis, aliquots of conditioned media were concentrated by further centrifugation and filtration through a centrifugal filter unit (Millipore, Billerica, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Mitochondria ATP synthesis assay\u003c/h2\u003e \u003cp\u003eATP sylnthesis was measured with cellTiter-Glo 2.0 cell viability assay (Promega, Madison, USA). To measure the ATP systhesis of the cells, 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells were placed in the 96-Well plate and then mixed at a celltiter-Glo 2.0 Reagent 1: 1 ratio to react at room temperature that blocked the light for 10 minutes. The degree of light emitting was measured using the Synergy HTX Microplate Reader (BIOTEK, Vermont, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Human protein analysis\u003c/h2\u003e \u003cp\u003eTo analyze the expression profiles of angiogenesis-related proteins as GCSF, IGFBP, IGF, PDGF, we used a Human protein Array Kit (R\u0026amp;D Systems, Ltd., Abingdon, UK). Cell samples (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) were harvested, and 150 \u0026micro;g of protein were mixed with 15 \u0026micro;l of biotinylated detection antibodies. After pre-treatment, the cocktail was incubated with the array overnight at 4\u0026deg;C on a rocking platform. Following washing to remove unbound material, streptavidin\u0026ndash;horseradish and chemiluminescent detection reagents are added sequentially. The signals on the membrane film were detected by scanning on an image reader LAS-3000 (Kodak, Rochester, NY) and were quantified using the MultiGauge 4.0 software (Kodak). The positive signals seen on developed film were identified by placing a transparency overlay on the array image and aligning it with the two pairs of positive control spots in the corners of each array.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western blot analysis\u003c/h2\u003e \u003cp\u003eSamples were solubilized in lysis buffer for 1 h at 4\u0026deg;C. The lysates were then clarified by centrifugation at 15,000 g for 30 min at 4\u0026deg;C, were diluted in Laemmli sample buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol, and were heated for 5 min at 90\u0026deg;C. The proteins were separated via SDS polyacrylamide gel electrophoresis (PAGE) using 10% or 15% resolving gels followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies against HGF (Santa cruz), VEGF (Abcam), and EGF (Abcam) for 1h at room temperature (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This antibody can be used at a concentration of 0.1\u0026ndash;0.2 \u0026micro;g/ml. Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) were used as described by the manufacturer for detection. The membranes were scanned to create chemiluminescent images that were then quantified with an image analyzer (Kodak).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. In vivo animal experiment for hair growth\u003c/h2\u003e \u003cp\u003e Experiments involving mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). All aspects of the animal care and experimental protocols were approved by the Ajou University Committee on Animal Care (IACUC No. 2020-23). Six-week-old male BALB/c mice (Daehan Biolink, Korea) (27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 g) purchased and maintained for 1 week before beginning experiments. Inbred lab mice are not isogenic. Hair growth activity in mice was determined visually according to a slight modification of the method described by Hattori and Ogawa (Hattori and Ogawa, 1983).\u003c/p\u003e \u003cp\u003eThe backs of the mice were shaved using a hair shaver (ISIS, B. Braun, Melsungen, Germany) and depilated using cold wax bands (Veet Minima). Shaving was performed in a region (2\u0026times; 3 cm) followed by a hair remover cream (Reckitt Benckiser France, Cedex, France) to avoid wax sticking to grown non-shaved hair. The cold wax stripe was applied once with the skin well stretched and removed at once against the direction of hair growth. BALB/c mice were divided into three groups of seven mice each: PBS (control) group, CM group and PBM-CM experimental group. PBS was topically applied to the shaved skins of mice in the control group. Then, 60 \u0026micro;l of CM solution were applied to the CM group. PBS or CM solution was subcutaneous injected and gently rubbed until it is completely absorbed into the skin. For all groups, 60 \u0026micro;l samples were applied twice daily for 3 weeks. Mice were sacrificed by CO\u003csub\u003e2\u003c/sub\u003e exposure on the 21st day. Their backs were then shaved, visually evaluated, and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Determination of hair growth area\u003c/h2\u003e \u003cp\u003eHair regrowth and nongrowth areas were photographed and printed. Regrowth and nongrowth regions were cut from the prints. The percentages of hair regrowth areas relative to total shaved skin areas were calculated as follows: Hair growth area (%)\u0026thinsp;=\u0026thinsp;100 \u0026times; [regrowth area weight / (regrowth area weight\u0026thinsp;+\u0026thinsp;nongrowth area weight)]. Our analysis of these images quantify by image analysis by ImageJ (free software).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Histological staining\u003c/h2\u003e \u003cp\u003eSamples were harvested 21 days after treatment. Specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated in a graded ethanol series, and embedded in paraffin. Specimens were sliced into 4 \u0026micro;m-thick sections and were stained with hematoxylin and eosin (H\u0026amp;E) to examine muscle degeneration and tissue inflammation. The histological parameters considered were re-epithelialization, dermal regeneration, granulation tissue formation, and angiogenesis. All quantitative results were obtained from triplicate samples. Regeneration of skin appendages was assessed by counting the number of hair follicles or sebaceous glands in the skin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eIndirect immunofluorescence staining was performed using a standard procedure. In brief, tissues cryosectioned at a 4-\u0026micro;m thickness were fixed with 4% paraformaldehyde, blocked with 5% BSA/PBS (1 h, 24\u0026deg;C), washed twice with PBS, treated with 0.1% Triton X-100/PBS for 1 min, and washed extensively in PBS. The sections were stained with specific primary antibodies and fluorescent-conjugated secondary antibodies (Table\u0026nbsp;2) using a M.O.M kit according to the manufacturer\u0026rsquo;s instructions (Vector Laboratories, Burlingame, CA). The cells were counterstained with DAPI (4,6-diamino-2-phenylindole dihydrochloride; Vector Laboratories). Mouse IgG (Dako, Carpinteria, CA) and rabbit IgG (Dako) antibodies was used as negative controls. The stained sections were viewed with a DXM1200F fluorescence microscope (Nikon, Tokyo, Japan). The processed images were analyzed for fluorescence intensity using the ImageJ software (NIH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistical analyses\u003c/h2\u003e \u003cp\u003eData were expressed as a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and the statistical analyses were carried out using two-sample t tests to compared two groups of samples and a One-way Analysis of Variance (ANOVA) for the three groups. A value of \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. ATP synthesis\u003c/h2\u003e \u003cp\u003eATP synthesis was evaluated to mitochondrial activity. With the same cell number, the ATP production in PBM cells was 1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 times higher than that in non -PBM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Production of growth factors by PBM cells\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, most growth factors were up-regulated in PBM with some exceptions. PBM upregulated the expression of growth factors, with PBM cultured cells showing significantly up-regulated expression of GCSF, IGFBP, IGF, and PDGF. hASCs also secreted a wide spectrum of growth factors including HGF, VEGF and EGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The expression of growth factors in PBM cultured cells was much greater than that in non-PBM treated cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Hair growth promoting effect of PBM-CM in BALB/c mice\u003c/h2\u003e \u003cp\u003eThe hair growth promoting effect of CM was systematically tested using BALB/c mouse. PBM-CM enhanced hair growth activity, increasing hair growth area and length to greater extents than treatment with CM or PBS alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). PBM-CM treatment enhanced the hair growth activity of mice (PBM-CM group: 18.6 mg, 422%; ASC-CM group: 13 mg, 295% per PBS group: 4.4 mg) increase in the hair weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Histological analysis of hair growth promotion by CM in mice\u003c/h2\u003e \u003cp\u003eHE staining revealed numbers of hair follicles and hair shafts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The number of hair follicles was increased 2.5-fold (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) in the PBM-CM treated group (32.1 hair follicles) compared with that in the CM-treated group (12.8 hair follicles) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Hair follicle cycling was augmented in the PBM-CM treated group compared to that in the CM-treated group. These results indicate that PBM-CM can promote hair follicle cycling, leading to an increased number of hair follicles in the dorsal skin of mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.5.\u003c/em\u003e \u003cb\u003ePBM-CM increased a\u003c/b\u003e\u003cb\u003engiogenesis in mice\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eMany of the CD31\u0026thinsp;+\u0026thinsp;cells in the PBM-CM group were double stained for smooth muscle\u003c/p\u003e \u003cp\u003eactin (SMA). Endotherial cells and smooth muscle cells were detected via human CD31 and αSMA antibodies, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared with the control group, blood vessels increased in number in the PBM-CM group on day 21. These findings suggest the greater effectiveness of the PBM-CM treatment for angiogenesis in the hair loss.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eStem cell derived CM is widely explored for its therapeutic potential to replace cell-based therapies for a wide range of diseases (24,25). Recently, for more effective hair regeneration, several methods for preconditioning ASC in various conditions have been suggested (26). Hypoxia conditions can amplify paracrine effects of ASCs, which show hair-regeneration and promotion effects by enhancing the secretion of certain growth factors (27). It has been reported that applying low doses of UVB radiation to ASCs can stimulate hair regeneration by regulating reactive oxidative stress (28). We have studied whether PBM-CM treatment method could lead to effective ways to precondition ASCs for more effective hair growth conditions. PBM, when applied with optimal energy density and wavelengths, effectively enhances stem cell viability and proliferation (15,17,18). PBM may enhance cellular responses, including gene expression, growth factor secretion, and cell proliferation, by increasing mitochondrial ATP synthesis (Fig. 2A).\u003c/p\u003e\n\u003cp\u003eIt has been reported that ASC can produce various cytokines known as hair-growth promoting factors\u0026nbsp;(15). We investigated the ability of ASC secretomes to promote hair growth and the effect of PBM on ASC function. We found that CM from PBM culture of ASC increased hair regrowth in an animal model (Figs. 3). To identify the mechanism by which PBM enhanced ASC-stimulated hair growth, we used a growth factor antibody array to analyze 41 growth factors. GCSF, IGFBP, IGF, and PDGF were found to be significantly up-regulated (Fig.\u0026nbsp;2B). hASCs can also secrete a wide spectrum of growth factors including HGF, VEGF, and EGF. Most importantly, PBM culture increased the expression of growth factors and the release of several hair growth-related growth factors including VEGF, IGF, and PDGF from stem cells. In the present study, PBM-CM demonstrated hair growth-promoting effects, as shown by visible hair regrowth on the shaved backs of BALB/c mice, along with increased hair growth areas and hair length (Fig.\u0026nbsp;3B, 3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is widely recognized that neo-angiogenesis plays a crucial role in stimulating hair growth (11). Angiogenesis is controlled by the balance between pro-angiogenic and anti-angiogenic factors (29). VEGF is well known as a representative proangiogenic factor (30). When incorporated into a collagen hydrogel and subcutaneously implanted into the back of mice, VEGF can accelerate hair follicle entry into the anagen phase and promote hair elongation (31). These results demonstrate that VEGF can promote hair follicle growth by inducing angiogenesis. It has been demonstrated that IGF expressed in the hair follicle can affect follicular proliferation, tissue remodeling, and the hair growth cycle (32). Loss of IGF can induce a delay telogen and retard the onset of the second anagen phase, with guard hairs of transgenic mice skin showing significant elongation (33). PDGF has been shown to induce the anagen phase of the hair growth cycle at the injection site on the dorsal skin of mice by acting through its receptors in the hair follicle epithelium (34). Knockout of the PDGF gene in mice resulted in a thinner dermis, misshapen hair follicles, smaller dermal papillae, abnormal dermal sheaths, and thinner hair compared to their wild-type siblings (35). Previous studies have shown that both cortisol and testosterone affect hair growth, and hair analysis can provide insight into long-term hormone levels. However, these hormones have not yet been studied about the interaction of CM with these hormones. Although hormone levels were not measured in this study, further research is needed to understand cortisol and testosterone in hair (36, 37).\u003c/p\u003e\n\u003cp\u003eThe secretion of potent growth factors was altered, which might explain the difference in hair growth between non-PBM and PBM. In conclusion, this is the first report on CM from PBM preactivated ASCs displaying stimulatory effects on hair growth in a hairless-induced mouse model. Our results suggest that CM from PBM pre-activated ASCs could serve as a promising and enhanced approach that warrants further investigation for treating hair loss.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatement of Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study protocol was reviewed and approved by [Ajou University Committee on Animal Care], approval number [IACUC No. 2020-23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ajou University research fund and National medical center (grant number: NMC I-2024-002, NMC2023-MS-06), Republic of Korea.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eQi J, Garza LA. An Overview of Alopecias. 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Chondrogenic potential of multipotential cells from human adipose tissue. Plast Reconstr Surg 2004; 113:585\u0026ndash;594.\u003c/li\u003e\n \u003cli\u003eGimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 2003; 5:362\u0026ndash;369.\u003c/li\u003e\n \u003cli\u003eSumi M, Toya N, Yanaga K, Ohki T, Nagai R. Transplantation of adipose stromal cells, but not mature adipocytes, augments ischemia-induced angiogenesis. Life Sci 2007; 80:559-565.\u003c/li\u003e\n \u003cli\u003eHarada Y, Tsujimoto S, Matsugami H, Yoshida A, Hisatome I. Transplantation of freshly isolated adipose tissue-derived regenerative cells enhances angiogenesis in a murine model of hind limb ischemia. Biomed Res 2013; 34:23-29.\u003c/li\u003e\n \u003cli\u003eJi S, Sun X, Fu X. Functional hair follicle regeneration: an updated review. signal transduction and targeted therapy 2021; 6(1):66.\u003c/li\u003e\n \u003cli\u003eGentile P, Garcovich S. Advances in Regenerative Stem Cell Therapy in Androgenic Alopecia and Hair Loss: Wnt Pathway, Growth-Factor, and Mesenchymal Stem Cell Signaling Impact Analysis on Cell Growth and Hair Follicle Development. Cells 2019; 8(5):466.\u003c/li\u003e\n \u003cli\u003eDaneshmandi L, Jafari T, Bhattacharjee M, Momah D, Saveh-Shemshaki N, Lo KW, Laurencin CT. Emergence of the Stem Cell Secretome in Regenerative Engineering Trends Biotechnol 2020 38(12):1373-1384.\u003c/li\u003e\n \u003cli\u003eCeccarelli S, Pontecorvi P, Anastasiadou E, Marchese C. Immunomodulatory Effect of Adipose-Derived Stem Cells: The Cutting Edge of Clinical Application. Front Cell Dev Biol 2020; 8:236.\u003c/li\u003e\n \u003cli\u003eCosette M. Rivera-Cruz, Manoel Figueiredo Neto, Marxa L. Figueiredo. The Immunomodulatory Effects of Mesenchymal Stem Cell Polarization within the Tumor Microenvironment Niche. Stem Cells Int 2017:17.\u003c/li\u003e\n \u003cli\u003ePark I-S. Enhancement of Wound Healing by Conditioned Medium of Adipose-Derived Stromal Cell with Photobiomodulation in Skin Wound. Int J Stem Cells 2021; 14(2):212-220\u003c/li\u003e\n \u003cli\u003ePawitan JA. Prospect of Stem Cell Conditioned Medium in Regenerative Medicine. Biomed Res Int 2014; 2014:14.\u003c/li\u003e\n \u003cli\u003ePark IS, Ahn JC. Adipose-derived stem cell spheroid treated with low-level light irradiation accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia Cytotherapy 2017; 19(9):1070-1078.\u003c/li\u003e\n \u003cli\u003ePark IS, Chung PS, Ahn JC. Enhanced angiogenic effect of adipose-derived stromal cell spheroid with low-level light therapy in hind limb ischemia mice. Biomaterials 2014; 35(34):9280-9289.\u003c/li\u003e\n \u003cli\u003eB. Mvula, Moore T, H. Abrahamse Effect of low-level laser irradiation and epidermal growth factor on adult human adipose-derived stem cells. 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Lasers Med Sci 2013; 28(3):981-987.\u003c/li\u003e\n \u003cli\u003eMannino G, Longo A, Anfuso CD, Lupo G, Furno D, Giuffrida R, Giurdanella G. Potential therapeutic applications of mesenchymal stem cells for the treatment of eye diseases. World J Stem Cells 2021; 13(6):632\u0026ndash;644.\u003c/li\u003e\n \u003cli\u003eSatija NK, Verma YK, Gupta P, Sharma S, Afrin F, Sharma M, Sharma P, Tripathi PT, Gurudutta GU. Mesenchymal stem cell-based therapy: a new paradigm in regenerative medicine. J Cell Mol Med 2009; 13(11):4385-4402.\u003c/li\u003e\n \u003cli\u003eNepal S, Mysore V. The Role of Adipose Tissue in Hair Regeneration: A Potential Tool for Management? J Cutan Aesthet Surg 2021; 14(3):295-304.\u003c/li\u003e\n \u003cli\u003eJeon SH, Sung JH. Hypoxia enhances the hair growth-promoting effects of embryonic stem cell-derived mesenchymal stem cells via NADPH oxidase 4. Biomed Pharmacother 2023; 159:114303.\u003c/li\u003e\n \u003cli\u003eJeong YM, Kim WK, Kim JH, Kwack MH, Yoon IS, Kim DD, Sung JH. Ultraviolet B preconditioning enhances the hair growth-promoting effects of adipose-derived stem cells via generation of reactive oxygen species. Stem Cells Dev 2013; 22(1):158-168.\u003c/li\u003e\n \u003cli\u003eItaliano JE, Richardson JL, Patel-Hett S, Battinelli E, Zaslavsky A, Short S, Ryeom S, Folkman J, Klement GL. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood 2008 111(3):1227-1233.\u003c/li\u003e\n \u003cli\u003eJohnson KE, Wilgus TA. Vascular Endothelial Growth Factor and Angiogenesis in the Regulation of Cutaneous Wound Repair. Adv Wound Care (New Rochelle) 2014; 3(10):647-661.\u003c/li\u003e\n \u003cli\u003eOzeki M, Tabata Y. Promoted growth of murine hair follicles through controlled release of vascular endothelial growth factor. Biomaterials 2002; 23(11):2367-2373.\u003c/li\u003e\n \u003cli\u003eWeger N, Schlake T. Igf-I signalling controls the hair growth cycle and the differentiation of hair shafts. J Invest Dermatol 2005; 125(5):873-882.\u003c/li\u003e\n \u003cli\u003eDong L, Xia L, Liu J, Ti D , Han W. Treatment of MSCs with Wnt1a-conditioned medium activates DP cells and promotes hair follicle regrowth. Sci Rep 2014; 25(4):5432.\u003c/li\u003e\n \u003cli\u003eTomita Y, Shimizu MA. PDGF isoforms induce and maintain anagen phase of murine hair follicles. J Dermatol Sci 2006; 43(2):105-115.\u003c/li\u003e\n \u003cli\u003eKarlsson L, Betsholtz C, Betsholtz C. Roles for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle. Development 1999; 126(12):2611-2621.\u003c/li\u003e\n \u003cli\u003eOwecka B, Tomaszewska A, Dobrzeniecki K, Owecki M. The Hormonal Background of Hair Loss in Non-Scarring Alopecias. Biomedicines 2024;12(3):513.\u003c/li\u003e\n \u003cli\u003eNatarelli N, Gahoonia N, Sivamani RK. Integrative and Mechanistic Approach to the Hair Growth Cycle and Hair Loss. J Clin Med 2023;12(3):893.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Photobiomodulation, Adipose-derived stem cells, Hair growth, Conditioned medium, Alopecia","lastPublishedDoi":"10.21203/rs.3.rs-6110120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6110120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe purpose of this study was to investigate whether subcutaneous injection of conditioned medium from photobiomodulation (PBM) irradiated adipose-derived stromal cells (ASCs) could stimulate hair growth in mice. Human ASCs were plated on cell culture dishes at a concentration of 2 × 10\u003csup\u003e4 \u003c/sup\u003ecells/cm\u003csup\u003e2\u003c/sup\u003e and allowed to adhere at 37°C. Light emitting diode (660 nm, 50 mW/cm\u003csup\u003e2\u003c/sup\u003e) was applied for 10 min daily from day 1 to day 10. Conditioned medium (CM) fractions were collected from PBM-irradiated ASCs to yield (PBM-CM). They were centrifuged at 13,000 g for 10 min at 4°C and stored prior to use for ELISA, protein assay, or \u003cem\u003ein vivo\u003c/em\u003e assays. Forty-one growth factors in PBM-CM from cells cultured under PBM or non-PBM conditions were analyzed. Survival, differentiation and secretion of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) of PBM-CM were evaluated by western blot. Secretion of insulin-like growth (IGF), platelet-derived growth factor (PDGF), and insulin-like growth factor binding protein (IGFBP) were significantly increased by PBM. PBM-CM enhanced hair growth in a hair loss model compared to non-irradiated ASC-CM or Phosphate-buffered saline (PBS). PBM is an effective biostimulator of ASC-CM in hair regrowth by enhancing secretion of growth factors. Thus, PBM-CM has the potential to stimulate hair growth and treat alopecia.\u003c/p\u003e","manuscriptTitle":"Subcutaneous Injection of Conditioned Medium from Photobiomodulation- Treated Adipose-Derived Stromal Cells Promotes Hair Growth in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 15:23:20","doi":"10.21203/rs.3.rs-6110120/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-02T19:41:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-02T03:20:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-01T08:34:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161336412112014096810938360712929613706","date":"2025-04-25T06:37:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337049371296009000601248362376632216243","date":"2025-04-25T04:40:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20515603462494319004523784714049492859","date":"2025-04-24T10:58:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312054916956493540981819335521022813833","date":"2025-04-23T11:24:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33184356343076246316748170583175053845","date":"2025-04-22T15:03:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-21T05:28:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-16T08:27:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Dermatological Research","date":"2025-04-15T08:53:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1c1e938f-fa4f-4244-b074-2a157bbda8e2","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-30T21:38:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 15:23:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6110120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6110120","identity":"rs-6110120","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}