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Wound healing potential of adipose tissue stem cell extract
Biochemical and Biophysical Research Communications 485 (2017) 30e34
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Wound healing potential of adipose tissue stem cell extract You Kyung Na a, 1, Jae-Jun Ban b, 1, Mijung Lee b, Wooseok Im b, c, *, Manho Kim b, d, ** a
Department of Brain and Cognitive Sciences, Tufts University, MA, USA Department of Neurology, Seoul National University Hospital, Seoul, South Korea c Neuroscience Research Institute, Seoul National University College of Medicine, Seoul, South Korea d Protein Metabolism Medical Research Center, College of Medicine, Seoul National University, Seoul, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2017 Accepted 20 January 2017 Available online 27 January 2017
Adipose tissue stem cells (ATSCs) are considered as a promising source in the field of cell therapy and regenerative medicine. In addition to direct cell replacement using stem cells, intercellular molecule exchange by stem cell secretory factors showed beneficial effects by reducing tissue damage and augmentation of endogenous repair. Delayed cutaneous wound healing is implicated in many conditions such as diabetes, aging, stress and alcohol consumption. However, the effects of cell-free extract of ATSCs (ATSC-Ex) containing secretome on wound healing process have not been investigated. In this study, ATSC-Ex was topically applied on the cutaneous wound and healing speed was examined. As a result, wound closure was much faster in the cell-free extract treated wound than control wound at 4, 6, 8 days after application of ATSC-Ex. Dermal fibroblast proliferation, migration and extracellular matrix (ECM) production are critical aspects of wound healing, and the effects of ATSC-Ex on human dermal fibroblast (HDF) was examined. ATSC-Ex augmented HDF proliferation in a dose-dependent manner and migration ability was enhanced by extract treatment. Representative ECM proteins, collagen type I and matrix metalloproteinase-1, are significantly up-regulated by treatment of ATSC-Ex. Our results suggest that the ATSC-Ex have improving effect of wound healing and can be the potential therapeutic candidate for cutaneous wound healing. © 2017 Elsevier Inc. All rights reserved.
Keywords: Wound healing Adipose derived stem cell Collagen
1. Introduction Skin is the largest organ of the body, which is consists of the epidermis, dermis and subcutaneous layer, and protects us as a barrier from external environment [1e4]. The epidermis mostly consists of keratinocytes and major cell type of the dermis is fibroblast. The fibroblast is a cell that expresses and secretes the extra-cellular matrix (ECM), which is the major component of the dermis of the skin [5]. The ECM works to maintain homeostasis, to prevent skin aging, and to aid wound-healing [6]. Though the ECM is composed of various complex factors, most defining component of ECM is collagens. Most abundant types of the collagen is collagen type 1 (COL-1) [7], and its degrading enzyme is called the matrix
* Corresponding author. Department of Neurology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, South Korea. ** Corresponding author. Department of Neurology, Seoul National University Hospital, Seoul, South Korea. E-mail addresses: [email protected] (W. Im), [email protected] (M. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2017.01.103 0006-291X/© 2017 Elsevier Inc. All rights reserved.
metalloproteinases, also known as MMPs [8]. Since the skin is protective barrier against harmful environmental factors, its damage must be mended efficiently and properly. Repair efficiency is reduced by various factors such as aging, stress, diabetes and obesity, and impaired skin healing is a major area of unmet clinical need [9]. Wound healing process is consists of several different and overlapped stages including hemostasis, inflammation, tissue formation and tissue remodeling [9e12]. During inflammatory phase, neutrophils and macrophages infiltrate the wound area and produce cytokines to augment healing process. In the tissue formation and remodeling phase, the fibroblast migration, proliferation and ECM expression contribute to the process of skin repair. The expressions of ECM remodeling factors (COL-1 and MMPs) should be precisely controlled in these stages for perfect wound healing process [13,14]. Unlike other types of stem cells, adipose tissue stem cells (ATSCs) can easily be collected without ethical problems and can be differentiated into specialized cells [15e17]. Thus, they are studied as one of the leading sources for regenerative medicine research. Past research includes stem cell application for aiding tissue
Y.K. Na et al. / Biochemical and Biophysical Research Communications 485 (2017) 30e34
damage along with the most recent research done on paracrine function of stem cells, one of the many functions of stem cells. With the help of different growth factors and extra-cellular vesicles, ATSCs can play a key role in alleviating hostile environments. The secretion factors of stem cells have resulted in modulation of neurodegenerative diseases, ischemic damage, wrinkle-formation, wound-healing and hair growth [18e22]. In addition, a recent study suggests that the cell-free extract from stem cells can regulate various diseases. Though the paracrine function is known to be one of the beneficial effects of fat stem cells, no research has yet been done on functions of fibroblasts for proper wound healing through ATSC extract (ATSC-Ex). In this research, we examined the effects of ATSC-Ex on wound healing in vivo and HDF functions in vitro. 2. Materials and methods 2.1. Ethics statement This study using human samples was performed with approval from the Institutional Review Board (IRB) of the Seoul National University Hospital. All animal experiments were studied with the approval of the Institutional Animal Care and Use Committee (IACUC, Approval number: 13-0058-C2A1) of Seoul National University Hospital. 2.2. Culture of ATSC Subcutaneous fat samples were obtained from normal humans who provided written informed consent to participate in the experiment. Adipose tissues isolated from the volunteers were kept in phosphate buffered saline (PBS) containing antibiotics (Invitrogen, CA, USA) and digested for cell isolation within a day. The adipose samples were digested in 0.075% collagenase type I solution (Invitrogen, CA, USA) with gentle shake for 1 h at 37 C. Mature adipocyte fractions were removed from stromal fractions by centrifugation at 1200 g for 10 min. The remaining stromal fractions were treated with red blood cell lysis buffer (Sigma), and centrifuged at 1200 g for 10 min. The remaining stromal fractions of the samples were resuspended and seeded in endothelial growth mediume2 MV (EGMe2 MV; Clonetics, MD, USA), which contained vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factore1, hydrocortisone, and ascorbic acid with 5% fetal bovine serum (FBS). The cells were used for the generation of ATSC-Ex after 3 or 4 passages.
31
Waltham, MA, USA) containing phosphatase and protease inhibitors (Roche, NJ, USA). Using BCA protein assay kit, the protein contents of cells were analyzed. Forty micrograms of protein samples were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE, 4e12% Novex NuPage Bis-Tris gel, Invitrogen, Mount Waverley, Australia) and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, MA, USA) in transfer buffer after blocking with 5% non-fatdried milk which is dissolved in 1 TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature. The blots were incubated overnight at 4 C with diluted primary antibodies. Blots were again incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (1:3000, GE Healthcare, NJ, USA), and developed using ECL solution (Enhanced chemiluminescence solution, Advansta, CA, USA). Band intensities were measured using IMAGEJ software from three independent results. Western blot figure show the representative one from three separate experiments. 2.5. Cell proliferation assay Cell survival rates were measured by a colorimetric assay using the WST-1 (Roche, Mannheim, Germany) according to manufacturer's instruction. Briefly, cells were seeded in a 96-well plate and incubated with ATSC-Ex with varying concentrations, 0 mg/mL, 5 mg/mL, 100 mg/mL, 200 mg/mL, and 400 mg/mL for 48 h. After the incubation period, WST-1 reagent was added to each well, and the cells were again incubated in 5% CO2 at 37 C for 2 h. Absorbance was measured using a plate reader at 450 nm (reference 650 nm) and the result shown represented the averages of three independent experiments. 2.6. Scratch assay HDF was seeded using DMEM with 10% FBS in 24 well culture plates and maintained in 5% CO2 at 37 C for 24 h. A linear wound was generated using a sterile 100 ml pipette tip and cellular debris was washed with PBS. DMEM with ATSC-Ex or same volume of PBS was added and incubated for 12 h at 37 C with 5% CO2. Three representative images from each wells of the scratched area were taken using microscope to estimate migration ability of HDF. The experiments were repeated three times and migration distance from the wound edge was analyzed using ImageJ software. 2.7. In vivo wound healing assay
For the preparation of extract of ATSC, the cultured ATSCs were harvested and centrifuged at 2000 g for 8 min after washing twice with PBS. The ATSCs (approximately 4 107 cells in 175 Tflask) were suspended with 1 ml PBS and lysed by three cycles of rapid freeze/thawing. The lysate was centrifuged at 14,000 g for 15 min, and the supernatant was passed through a syringe filter unit (0.45 mm) to remove cell debris. ATSC-Ex was freshly prepared just before use. All chemicals were purchased from Sigma. The total protein content of each ATSC-Ex was quantified using a bicinchoninic acid protein (BCA) assay kit (Pierce, IL, USA).
Circular cutaneous wounds were made using scissors on the back dermal skin of the mice: one on the left side of the spine, and the other on the right side of the spine. Amongst the two wounds, the right side wound was set as the experimental control group (topical application of ATSC-Ex 200 mg/200ml), whereas the left side wound was set as the control group (200ml of vehicle). After the wounds were made, each of the substances was applied to the corresponding wounds. Wound healing rates were recorded every two days over a period of 8 days. Every two days, the size was traced onto a transparent 3M paper. After the tracing, pictures of the wound size were taken to keep a visual record. After the 10 day experimental period, when the wounds were almost, if not all healed, the experiment ended.
2.4. Protein extraction and western blot analysis
2.8. Statistical analysis
Cultured cells were washed then harvested in PBS using cell scraper. The preparation for protein extracts was done using RIPA buffer (Radio immunoprecipitation assay buffer, Thermo Scientific,
All values indicated in the figures are represented as mean SE. Results of western blot were analyzed using Student's t-test. A twotailed probability value below 0.05 was considered statistically
2.3. Preparation of ATSC-Ex
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Y.K. Na et al. / Biochemical and Biophysical Research Communications 485 (2017) 30e34
significant, Data were analyzed by SPSS version 17.0 (SPSS Inc., USA). 3. Results 3.1. ATSC-Ex enhances wound healing in vivo To confirm the role of ATSC-Ex on cutaneous wound healing, skin wound was made on the back of the mouse and ATSC-Ex or vehicle was topically applied from 0 day of the wounding. Wound measurement and ATSC-Ex treatment was performed for every 2 days. As shown in Fig. 1, wound area treated with ATSC-Ex was smaller compared with vehicle treated wound at day 4, 6, 8, and wound was almost closed within 12 days. This result showed that topical application of ATSC-Ex accelerates wound healing in vivo. 3.2. Augmentation of HDF proliferation by ATSC-Ex Since the proliferation of HDF is an important aspect of proper wound healing, the effect of ATSC-Ex on HDF proliferation was examined. HDF was treated with ATSC-Ex at various concentrations from 0 mg/ml to 400 mg/ml and incubated for 48 h. As a result, ATSCEx augmented proliferation of HDF in a dose-dependent manner without alteration of morphological change (Fig. 2). This data suggest that ATSC-Ex have HDF proliferating molecules. 3.3. ATSC-Ex promotes migration of HDF To examine the migration ability of HDF with or without ATSCEx treatment, in vitro scratch assay of HDF was performed. HDF migration is essential step for proper wound healing in vivo. 12 h after ATSC-Ex treatment, migration distance of HDF from the wound edge was measured using microscopic images. Migration distance of the HDF was significantly enhanced by ATSC-Ex treatment (Fig. 3).
Fig. 2. ATSC-Ex enhances proliferation of HDF. HDF was seeded and various concentration of ATSC-Ex was treated 24 h after seeding. Representative images were taken using light microscope (A). CCK-8 assay was performed and relative cell proliferation was represented using three independent experiments (B). *p < 0.01; **p < 0.001; ***p < 0.0001 vs control, Scale bar ¼ 100 mm.
4. Discussion 3.4. ATSC-Ex modulates ECM protein production in HDF To assess the change of ECM proteins of HDF by ATSC-Ex, HDF was treated with ATSC-Ex and ECM proteins, COL-1 and MMP-1, of the supernatant were measured using western blot. ATSC-Ex treatment resulted in significant up-regulation of COL-1 and MMP-1 production in HDF (Fig. 4). Taken together, ATSC-Ex modulates ECM protein productions of HDF, leading to ECM remodeling of the skin.
ATSC have been showed therapeutic effects on many diseases, and its beneficial effects, in some degree, possibly by paracrine function of the cells [18,19,21]. Although previous studies have revealed that secretory factors of ATSC can enhance wound healing in vivo, the effects of cell-free total extract of ATSC on wound model has not been investigated. In this study, we examined wound healing augmentation in vivo and enhancement of proliferation,
Fig. 1. Wound healing augmentation by ATSC-Ex. Circular wound was made on the back skin of mouse and ATSC-Ex and vehicle lotion was topically applied from the day of operation to 8 days. Representative wound images were take every 2 days (A). Wound area was measured for every 2 days and represented as curved graph (B). *p < 0.05; **p < 0.01 vs control.
Y.K. Na et al. / Biochemical and Biophysical Research Communications 485 (2017) 30e34
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Fig. 3. ATSC-Ex enhances migration of HDF. Monolayer of HDF was scratched using 100 ml pipette tip and washed with PBS, Then, DMEM containing vehicle or ATSC-Ex was treated for 12 h and migrated HDF was imaged using light microscope (A). Migration distance of HDF from the wound edge was calculated and represented by bar graph (B). Scale bar ¼ 100 mm.
producing ECM [29]. Several pathologic conditions such as diabetes, obesity, stress and alcohol consumption impairs proper wound repair, leading to complication of the wound. Previous recent researches showed that topical application of exosomes, extracellular vesicles secreted by cells, showed normalization of healing by fibroblast optimization [30,31]. In addition, growth factors have important roles in wound healing and have therapeutic effects on impaired wound healing [32,33]. We speculated that wound healing augmentation effect of ATSC-Ex can be possibly by extracellular vesicles and/or growth factors in the extract. In this study, we investigated the beneficial effects of ATSC-Ex on the wound healing process of the skin in vivo and HDF proliferation, migration and expression of ECM regulatory factors in vitro. In addition to conditioned medium or direct stem cell injection, cell-free extract of ATSC can be the potential source for augmentation of wound healing. Acknowledgments
Fig. 4. ATSC-Ex modulates ECM expression in HDF. HDS was treated with vehicle or ATSC-Ex for 48 h and representative ECM proteins, COL-1 and MMP-1, in the conditioned medium is analyzed using western blot (A). Relative COL-1 and MMP-1 level from three independent experiments was calculated and represented as bar graph (B), *p < 0.01.
migration and ECM expressions of HDF in vitro by ATSC-Ex. Tissue-derived stem cells have promising implications in tissue engineering and regenerative medicine filed and ATSC is most abundant and easily obtainable source among adult stem cells. ATSC can be differentiated into endothelial cells [23,24], myocyte [25e27] and neuronal cells [28] under appropriate culture conditions. In addition, ATSC isolated from fat tissues have been considered as proper source of paracrine effect-based therapy to ameliorate pathologic conditions. Secretome of ATSC seems to have anti-apoptotic, angiogenic, anti-inflammatory, anti-wrinkle and wound healing effect [18e22]. Our results showed that extract of ATSC also has wound healing enhancing effects. After wounding of adult mammalian skin, intricate and complex healing processes are initiated to rebuild damaged skin [11,12]. Wound healing involves inflammation and formation and remodeling of skin tissue. Healing process such as proliferation, migration and ECM synthesize at the wound edge are examined in the beginning of skin restoration. HDFs proliferate to expand wounded area and are important in the repair of the damaged dermis by
This work was supported by the National Research Foundation of Korea (NRF) (2014R1A2A1A11051520), Korea Health 21 R&D Project (HI14C2348) by the Ministry of Health & Welfare, Republic of Korea, the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2016M3C7A1914002), and National Research Foundation of Korea (2015R1D1A1A01060056). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.01.103. References [1] C. Griffiths, J. Barker, T. Bleiker, R. Chalmers, D. Creamer, in: Rook's Textbook of Dermatology, ninth ed., John Wiley & Sons Inc., Chichester, West Sussex; Hoboken, NJ, 2016. [2] P.M. Elias, L.F. Eichenfield, J.F. Fowler Jr., P. Horowitz, R.P. McLeod, Update on the structure and function of the skin barrier: atopic dermatitis as an exemplar of clinical implications, Semin. Cutan. Med. Surg. 32 (2013) S21eS24. [3] M. Furuse, S. Tsukita, Claudins in occluding junctions of humans and flies, Trends Cell Biol. 16 (2006) 181e188. [4] A. Kubo, K. Nagao, M. Amagai, Epidermal barrier dysfunction and cutaneous sensitization in atopic diseases, J. Clin. Investig. 122 (2012) 440e447. [5] D.J. Prockop, K.I. Kivirikko, L. Tuderman, N.A. Guzman, The biosynthesis of collagen and its disorders (first of two parts), N. Engl. J. Med. 301 (1979) 13e23. [6] A. Gosain, L.A. DiPietro, Aging and wound healing, World J. Surg. 28 (2004) 321e326.
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[7] L.T. Smith, K.A. Holbrook, J.A. Madri, Collagen types I, III, and V in human embryonic and fetal skin, Am. J. Anat. 175 (1986) 507e521. [8] M. Hernandez-Perez, M. Mahalingam, Matrix metalloproteinases in health and disease: insights from dermatopathology, Am. J. Dermatopathol. 34 (2012) 565e579. [9] S. Guo, L.A. Dipietro, Factors affecting wound healing, J. Dent. Res. 89 (2010) 219e229. [10] R.A.F. Clark, The Molecular and Cellular Biology of Wound Repair, second ed., Plenum Press, New York, 1996. [11] S.A. Eming, T. Krieg, J.M. Davidson, Inflammation in wound repair: molecular and cellular mechanisms, J. Investig. Dermatol. 127 (2007) 514e525. [12] P. Martin, Wound healingeaiming for perfect skin regeneration, Science 276 (1997) 75e81. [13] E. Pirila, J.T. Korpi, T. Korkiamaki, T. Jahkola, A. Gutierrez-Fernandez, C. LopezOtin, U. Saarialho-Kere, T. Salo, T. Sorsa, Collagenase-2 (MMP-8) and matrilysin-2 (MMP-26) expression in human wounds of different etiologies, Wound Repair Regen. 15 (2007) 47e57. [14] A. Nystrom, D. Velati, V.R. Mittapalli, A. Fritsch, J.S. Kern, L. Bruckner-Tuderman, Collagen VII plays a dual role in wound healing, J. Clin. Investig. 123 (2013) 3498e3509. [15] A. Dicker, K. Le Blanc, G. Astrom, V. van Harmelen, C. Gotherstrom, L. Blomqvist, P. Arner, M. Ryden, Functional studies of mesenchymal stem cells derived from adult human adipose tissue, Exp. Cell Res. 308 (2005) 283e290. [16] J.M. Gimble, A.J. Katz, B.A. Bunnell, Adipose-derived stem cells for regenerative medicine, Circ. Res. 100 (2007) 1249e1260. [17] A.J. Katz, A. Tholpady, S.S. Tholpady, H. Shang, R.C. Ogle, Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells, Stem cells 23 (2005) 412e423. [18] W.S. Kim, B.S. Park, S.H. Park, H.K. Kim, J.H. Sung, Antiwrinkle effect of adipose-derived stem cell: activation of dermal fibroblast by secretory factors, J. Dermatol. Sci. 53 (2009) 96e102. [19] E.Y. Lee, Y. Xia, W.S. Kim, M.H. Kim, T.H. Kim, K.J. Kim, B.S. Park, J.H. Sung, Hypoxia-enhanced wound-healing function of adipose-derived stem cells: increase in stem cell proliferation and up-regulation of VEGF and bFGF, Wound Repair Regen. 17 (2009) 540e547. [20] H. Nakagami, K. Maeda, R. Morishita, S. Iguchi, T. Nishikawa, Y. Takami, Y. Kikuchi, Y. Saito, K. Tamai, T. Ogihara, Y. Kaneda, Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells, Arterioscl. Thromb. Vasc. Biol. 25 (2005) 2542e2547. [21] J. Rehman, D. Traktuev, J. Li, S. Merfeld-Clauss, C.J. Temm-Grove,
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Wound healing potential of adipose tissue stem cell extract You Kyung Na a, 1, Jae-Jun Ban b, 1, Mijung Lee b, Wooseok Im b, c, *, Manho Kim b, d, ** a
Department of Brain and Cognitive Sciences, Tufts University, MA, USA Department of Neurology, Seoul National University Hospital, Seoul, South Korea c Neuroscience Research Institute, Seoul National University College of Medicine, Seoul, South Korea d Protein Metabolism Medical Research Center, College of Medicine, Seoul National University, Seoul, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2017 Accepted 20 January 2017 Available online 27 January 2017
Adipose tissue stem cells (ATSCs) are considered as a promising source in the field of cell therapy and regenerative medicine. In addition to direct cell replacement using stem cells, intercellular molecule exchange by stem cell secretory factors showed beneficial effects by reducing tissue damage and augmentation of endogenous repair. Delayed cutaneous wound healing is implicated in many conditions such as diabetes, aging, stress and alcohol consumption. However, the effects of cell-free extract of ATSCs (ATSC-Ex) containing secretome on wound healing process have not been investigated. In this study, ATSC-Ex was topically applied on the cutaneous wound and healing speed was examined. As a result, wound closure was much faster in the cell-free extract treated wound than control wound at 4, 6, 8 days after application of ATSC-Ex. Dermal fibroblast proliferation, migration and extracellular matrix (ECM) production are critical aspects of wound healing, and the effects of ATSC-Ex on human dermal fibroblast (HDF) was examined. ATSC-Ex augmented HDF proliferation in a dose-dependent manner and migration ability was enhanced by extract treatment. Representative ECM proteins, collagen type I and matrix metalloproteinase-1, are significantly up-regulated by treatment of ATSC-Ex. Our results suggest that the ATSC-Ex have improving effect of wound healing and can be the potential therapeutic candidate for cutaneous wound healing. © 2017 Elsevier Inc. All rights reserved.
Keywords: Wound healing Adipose derived stem cell Collagen
1. Introduction Skin is the largest organ of the body, which is consists of the epidermis, dermis and subcutaneous layer, and protects us as a barrier from external environment [1e4]. The epidermis mostly consists of keratinocytes and major cell type of the dermis is fibroblast. The fibroblast is a cell that expresses and secretes the extra-cellular matrix (ECM), which is the major component of the dermis of the skin [5]. The ECM works to maintain homeostasis, to prevent skin aging, and to aid wound-healing [6]. Though the ECM is composed of various complex factors, most defining component of ECM is collagens. Most abundant types of the collagen is collagen type 1 (COL-1) [7], and its degrading enzyme is called the matrix
* Corresponding author. Department of Neurology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, South Korea. ** Corresponding author. Department of Neurology, Seoul National University Hospital, Seoul, South Korea. E-mail addresses: [email protected] (W. Im), [email protected] (M. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2017.01.103 0006-291X/© 2017 Elsevier Inc. All rights reserved.
metalloproteinases, also known as MMPs [8]. Since the skin is protective barrier against harmful environmental factors, its damage must be mended efficiently and properly. Repair efficiency is reduced by various factors such as aging, stress, diabetes and obesity, and impaired skin healing is a major area of unmet clinical need [9]. Wound healing process is consists of several different and overlapped stages including hemostasis, inflammation, tissue formation and tissue remodeling [9e12]. During inflammatory phase, neutrophils and macrophages infiltrate the wound area and produce cytokines to augment healing process. In the tissue formation and remodeling phase, the fibroblast migration, proliferation and ECM expression contribute to the process of skin repair. The expressions of ECM remodeling factors (COL-1 and MMPs) should be precisely controlled in these stages for perfect wound healing process [13,14]. Unlike other types of stem cells, adipose tissue stem cells (ATSCs) can easily be collected without ethical problems and can be differentiated into specialized cells [15e17]. Thus, they are studied as one of the leading sources for regenerative medicine research. Past research includes stem cell application for aiding tissue
Y.K. Na et al. / Biochemical and Biophysical Research Communications 485 (2017) 30e34
damage along with the most recent research done on paracrine function of stem cells, one of the many functions of stem cells. With the help of different growth factors and extra-cellular vesicles, ATSCs can play a key role in alleviating hostile environments. The secretion factors of stem cells have resulted in modulation of neurodegenerative diseases, ischemic damage, wrinkle-formation, wound-healing and hair growth [18e22]. In addition, a recent study suggests that the cell-free extract from stem cells can regulate various diseases. Though the paracrine function is known to be one of the beneficial effects of fat stem cells, no research has yet been done on functions of fibroblasts for proper wound healing through ATSC extract (ATSC-Ex). In this research, we examined the effects of ATSC-Ex on wound healing in vivo and HDF functions in vitro. 2. Materials and methods 2.1. Ethics statement This study using human samples was performed with approval from the Institutional Review Board (IRB) of the Seoul National University Hospital. All animal experiments were studied with the approval of the Institutional Animal Care and Use Committee (IACUC, Approval number: 13-0058-C2A1) of Seoul National University Hospital. 2.2. Culture of ATSC Subcutaneous fat samples were obtained from normal humans who provided written informed consent to participate in the experiment. Adipose tissues isolated from the volunteers were kept in phosphate buffered saline (PBS) containing antibiotics (Invitrogen, CA, USA) and digested for cell isolation within a day. The adipose samples were digested in 0.075% collagenase type I solution (Invitrogen, CA, USA) with gentle shake for 1 h at 37 C. Mature adipocyte fractions were removed from stromal fractions by centrifugation at 1200 g for 10 min. The remaining stromal fractions were treated with red blood cell lysis buffer (Sigma), and centrifuged at 1200 g for 10 min. The remaining stromal fractions of the samples were resuspended and seeded in endothelial growth mediume2 MV (EGMe2 MV; Clonetics, MD, USA), which contained vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factore1, hydrocortisone, and ascorbic acid with 5% fetal bovine serum (FBS). The cells were used for the generation of ATSC-Ex after 3 or 4 passages.
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Waltham, MA, USA) containing phosphatase and protease inhibitors (Roche, NJ, USA). Using BCA protein assay kit, the protein contents of cells were analyzed. Forty micrograms of protein samples were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE, 4e12% Novex NuPage Bis-Tris gel, Invitrogen, Mount Waverley, Australia) and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, MA, USA) in transfer buffer after blocking with 5% non-fatdried milk which is dissolved in 1 TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature. The blots were incubated overnight at 4 C with diluted primary antibodies. Blots were again incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (1:3000, GE Healthcare, NJ, USA), and developed using ECL solution (Enhanced chemiluminescence solution, Advansta, CA, USA). Band intensities were measured using IMAGEJ software from three independent results. Western blot figure show the representative one from three separate experiments. 2.5. Cell proliferation assay Cell survival rates were measured by a colorimetric assay using the WST-1 (Roche, Mannheim, Germany) according to manufacturer's instruction. Briefly, cells were seeded in a 96-well plate and incubated with ATSC-Ex with varying concentrations, 0 mg/mL, 5 mg/mL, 100 mg/mL, 200 mg/mL, and 400 mg/mL for 48 h. After the incubation period, WST-1 reagent was added to each well, and the cells were again incubated in 5% CO2 at 37 C for 2 h. Absorbance was measured using a plate reader at 450 nm (reference 650 nm) and the result shown represented the averages of three independent experiments. 2.6. Scratch assay HDF was seeded using DMEM with 10% FBS in 24 well culture plates and maintained in 5% CO2 at 37 C for 24 h. A linear wound was generated using a sterile 100 ml pipette tip and cellular debris was washed with PBS. DMEM with ATSC-Ex or same volume of PBS was added and incubated for 12 h at 37 C with 5% CO2. Three representative images from each wells of the scratched area were taken using microscope to estimate migration ability of HDF. The experiments were repeated three times and migration distance from the wound edge was analyzed using ImageJ software. 2.7. In vivo wound healing assay
For the preparation of extract of ATSC, the cultured ATSCs were harvested and centrifuged at 2000 g for 8 min after washing twice with PBS. The ATSCs (approximately 4 107 cells in 175 Tflask) were suspended with 1 ml PBS and lysed by three cycles of rapid freeze/thawing. The lysate was centrifuged at 14,000 g for 15 min, and the supernatant was passed through a syringe filter unit (0.45 mm) to remove cell debris. ATSC-Ex was freshly prepared just before use. All chemicals were purchased from Sigma. The total protein content of each ATSC-Ex was quantified using a bicinchoninic acid protein (BCA) assay kit (Pierce, IL, USA).
Circular cutaneous wounds were made using scissors on the back dermal skin of the mice: one on the left side of the spine, and the other on the right side of the spine. Amongst the two wounds, the right side wound was set as the experimental control group (topical application of ATSC-Ex 200 mg/200ml), whereas the left side wound was set as the control group (200ml of vehicle). After the wounds were made, each of the substances was applied to the corresponding wounds. Wound healing rates were recorded every two days over a period of 8 days. Every two days, the size was traced onto a transparent 3M paper. After the tracing, pictures of the wound size were taken to keep a visual record. After the 10 day experimental period, when the wounds were almost, if not all healed, the experiment ended.
2.4. Protein extraction and western blot analysis
2.8. Statistical analysis
Cultured cells were washed then harvested in PBS using cell scraper. The preparation for protein extracts was done using RIPA buffer (Radio immunoprecipitation assay buffer, Thermo Scientific,
All values indicated in the figures are represented as mean SE. Results of western blot were analyzed using Student's t-test. A twotailed probability value below 0.05 was considered statistically
2.3. Preparation of ATSC-Ex
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significant, Data were analyzed by SPSS version 17.0 (SPSS Inc., USA). 3. Results 3.1. ATSC-Ex enhances wound healing in vivo To confirm the role of ATSC-Ex on cutaneous wound healing, skin wound was made on the back of the mouse and ATSC-Ex or vehicle was topically applied from 0 day of the wounding. Wound measurement and ATSC-Ex treatment was performed for every 2 days. As shown in Fig. 1, wound area treated with ATSC-Ex was smaller compared with vehicle treated wound at day 4, 6, 8, and wound was almost closed within 12 days. This result showed that topical application of ATSC-Ex accelerates wound healing in vivo. 3.2. Augmentation of HDF proliferation by ATSC-Ex Since the proliferation of HDF is an important aspect of proper wound healing, the effect of ATSC-Ex on HDF proliferation was examined. HDF was treated with ATSC-Ex at various concentrations from 0 mg/ml to 400 mg/ml and incubated for 48 h. As a result, ATSCEx augmented proliferation of HDF in a dose-dependent manner without alteration of morphological change (Fig. 2). This data suggest that ATSC-Ex have HDF proliferating molecules. 3.3. ATSC-Ex promotes migration of HDF To examine the migration ability of HDF with or without ATSCEx treatment, in vitro scratch assay of HDF was performed. HDF migration is essential step for proper wound healing in vivo. 12 h after ATSC-Ex treatment, migration distance of HDF from the wound edge was measured using microscopic images. Migration distance of the HDF was significantly enhanced by ATSC-Ex treatment (Fig. 3).
Fig. 2. ATSC-Ex enhances proliferation of HDF. HDF was seeded and various concentration of ATSC-Ex was treated 24 h after seeding. Representative images were taken using light microscope (A). CCK-8 assay was performed and relative cell proliferation was represented using three independent experiments (B). *p < 0.01; **p < 0.001; ***p < 0.0001 vs control, Scale bar ¼ 100 mm.
4. Discussion 3.4. ATSC-Ex modulates ECM protein production in HDF To assess the change of ECM proteins of HDF by ATSC-Ex, HDF was treated with ATSC-Ex and ECM proteins, COL-1 and MMP-1, of the supernatant were measured using western blot. ATSC-Ex treatment resulted in significant up-regulation of COL-1 and MMP-1 production in HDF (Fig. 4). Taken together, ATSC-Ex modulates ECM protein productions of HDF, leading to ECM remodeling of the skin.
ATSC have been showed therapeutic effects on many diseases, and its beneficial effects, in some degree, possibly by paracrine function of the cells [18,19,21]. Although previous studies have revealed that secretory factors of ATSC can enhance wound healing in vivo, the effects of cell-free total extract of ATSC on wound model has not been investigated. In this study, we examined wound healing augmentation in vivo and enhancement of proliferation,
Fig. 1. Wound healing augmentation by ATSC-Ex. Circular wound was made on the back skin of mouse and ATSC-Ex and vehicle lotion was topically applied from the day of operation to 8 days. Representative wound images were take every 2 days (A). Wound area was measured for every 2 days and represented as curved graph (B). *p < 0.05; **p < 0.01 vs control.
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Fig. 3. ATSC-Ex enhances migration of HDF. Monolayer of HDF was scratched using 100 ml pipette tip and washed with PBS, Then, DMEM containing vehicle or ATSC-Ex was treated for 12 h and migrated HDF was imaged using light microscope (A). Migration distance of HDF from the wound edge was calculated and represented by bar graph (B). Scale bar ¼ 100 mm.
producing ECM [29]. Several pathologic conditions such as diabetes, obesity, stress and alcohol consumption impairs proper wound repair, leading to complication of the wound. Previous recent researches showed that topical application of exosomes, extracellular vesicles secreted by cells, showed normalization of healing by fibroblast optimization [30,31]. In addition, growth factors have important roles in wound healing and have therapeutic effects on impaired wound healing [32,33]. We speculated that wound healing augmentation effect of ATSC-Ex can be possibly by extracellular vesicles and/or growth factors in the extract. In this study, we investigated the beneficial effects of ATSC-Ex on the wound healing process of the skin in vivo and HDF proliferation, migration and expression of ECM regulatory factors in vitro. In addition to conditioned medium or direct stem cell injection, cell-free extract of ATSC can be the potential source for augmentation of wound healing. Acknowledgments
Fig. 4. ATSC-Ex modulates ECM expression in HDF. HDS was treated with vehicle or ATSC-Ex for 48 h and representative ECM proteins, COL-1 and MMP-1, in the conditioned medium is analyzed using western blot (A). Relative COL-1 and MMP-1 level from three independent experiments was calculated and represented as bar graph (B), *p < 0.01.
migration and ECM expressions of HDF in vitro by ATSC-Ex. Tissue-derived stem cells have promising implications in tissue engineering and regenerative medicine filed and ATSC is most abundant and easily obtainable source among adult stem cells. ATSC can be differentiated into endothelial cells [23,24], myocyte [25e27] and neuronal cells [28] under appropriate culture conditions. In addition, ATSC isolated from fat tissues have been considered as proper source of paracrine effect-based therapy to ameliorate pathologic conditions. Secretome of ATSC seems to have anti-apoptotic, angiogenic, anti-inflammatory, anti-wrinkle and wound healing effect [18e22]. Our results showed that extract of ATSC also has wound healing enhancing effects. After wounding of adult mammalian skin, intricate and complex healing processes are initiated to rebuild damaged skin [11,12]. Wound healing involves inflammation and formation and remodeling of skin tissue. Healing process such as proliferation, migration and ECM synthesize at the wound edge are examined in the beginning of skin restoration. HDFs proliferate to expand wounded area and are important in the repair of the damaged dermis by
This work was supported by the National Research Foundation of Korea (NRF) (2014R1A2A1A11051520), Korea Health 21 R&D Project (HI14C2348) by the Ministry of Health & Welfare, Republic of Korea, the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2016M3C7A1914002), and National Research Foundation of Korea (2015R1D1A1A01060056). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.01.103. References [1] C. Griffiths, J. Barker, T. Bleiker, R. Chalmers, D. Creamer, in: Rook's Textbook of Dermatology, ninth ed., John Wiley & Sons Inc., Chichester, West Sussex; Hoboken, NJ, 2016. [2] P.M. Elias, L.F. Eichenfield, J.F. Fowler Jr., P. Horowitz, R.P. McLeod, Update on the structure and function of the skin barrier: atopic dermatitis as an exemplar of clinical implications, Semin. Cutan. Med. Surg. 32 (2013) S21eS24. [3] M. Furuse, S. Tsukita, Claudins in occluding junctions of humans and flies, Trends Cell Biol. 16 (2006) 181e188. [4] A. Kubo, K. Nagao, M. Amagai, Epidermal barrier dysfunction and cutaneous sensitization in atopic diseases, J. Clin. Investig. 122 (2012) 440e447. [5] D.J. Prockop, K.I. Kivirikko, L. Tuderman, N.A. Guzman, The biosynthesis of collagen and its disorders (first of two parts), N. Engl. J. Med. 301 (1979) 13e23. [6] A. Gosain, L.A. DiPietro, Aging and wound healing, World J. Surg. 28 (2004) 321e326.
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