Materials Science and Engineering C 44 (2014) 440–448
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Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor Zhongchun Su a,1, Huan Ma a,1, Zhengzheng Wu a,1, Huilan Zeng b, Zhizhong Li c, Yuechun Wang d, Gexiu Liu d, Bin Xu e, Yongliang Lin e, Peng Zhang e, Xing Wei a,⁎ a
Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Jinan University, Guangzhou 510632, China Department of Hematology, The First Affiliated Hospital, Jinan University, Guangzhou 510632, China Department of Bone, The First Affiliated Hospital, Jinan University, Guangzhou 510632, China d Department of Physiology, School of Medicine, Jinan University, Guangzhou 510632, China e Grandhope Biotech Co., Ltd., Building D, #408, Guangzhou International Business Incubator, Guangzhou Science Park, Guangzhou 510663, Guangdong, China b c
a r t i c l e
i n f o
Article history: Received 17 February 2014 Received in revised form 31 May 2014 Accepted 13 July 2014 Available online 19 July 2014 Keywords: Decellularized scaffolds Growth factors Skin repair
a b s t r a c t Current therapy for skin wound healing still relies on skin transplantation. Many studies were done to try to find out ways to replace skin transplantation, but there is still no effective alternative therapy. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments, and scaffolds loaded with hyaluronic acid (HA) and epidermal growth factor (EGF) were tested for their effect on wound healing. MTT assay showed that EGF increased NIH3T3 cell viability and confirmed that EGF used in this study was biologically active in vitro. Scanning electron microscope (SEM) showed that HA stably attached to scaffolds even after soaking in PBS for 48 h. ELISA assay showed that HA increased the adsorption of EGF to scaffolds and sustained the release of EGF from scaffolds. Animal study showed that the wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF scaffolds on days 10, 15 and 20 post-surgery. Hematoxylin–Eosin (HE) staining confirmed these results by showing that on days 10, 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing HA and EGF than scaffolds alone. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing EGF than scaffolds alone. Skin appendages were observed on day 20 only in the wound covered with scaffolds containing HA and EGF. These results demonstrate that the scaffolds containing HA and EGF can enhance wound healing. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Skin is the largest organ system in animals and provides the front defense barrier against different damages to animal bodies [1,2]. Injuries to skins caused by various physical factors or chemical agents can induce wound healing and skin regeneration [3,4]. Wound healing is the process in which cells in the body regenerate and repair to reduce size of damaged or necrotic area [5]. Wound healing involves a set of events that include inflammation surrounding a region of injury, wound cell migration and mitosis, angiogenesis and the development of granulation tissue, repair of the connective tissue, regeneration of extracellular matrix and remodeling that leads to a healed wound [6,7]. ⁎ Corresponding author. Tel./fax: +86 20 85222759. E-mail address: [email protected] (X. Wei). 1 The first three authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.msec.2014.07.039 0928-4931/© 2014 Elsevier B.V. All rights reserved.
The effects of growth factors on wound healing have been extensively studied and are found to be involved in these processes [8,9]. Almost all growth factors are peptides that bind to the target cell surface receptors. Commonly, the receptor kinase activity is essential for inducing the biological activities of various growth factors including epidermal growth factor (EGF), transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF), and different growth factors can affect the normal and pathological wound healing process [10]. EGF is known to be a potent stimulator of cellular proliferation. It has been tested for its effects on wound healing [11,12]. EGF is beneficial to wound healing because of its effects on keratinocytes, fibroblasts and vascular endothelial cells and promotes the formation of granulation tissue and re-epithelialization. The action of EGF on the cells including keratinocytes, fibroblasts and endothelial cells in wound area can be regulated by local production of EGF through autocrine and paracrine mechanisms [10]. EGF can stimulate the growth and
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differentiation of keratinocyte as well as the proliferation and migration of fibroblast and vascular endothelial cells [10,13]. Besides growth factors, other molecules also play important roles in wound healing. Hyaluronic acid (HA) is a major extracellular matrix component. It is important for wound healing because of its various biological activities [14]. Due to its unique physical properties, HA creates an excellent wound healing environment and has multifaceted roles in wound healing and scarring [15]. HA can be induced to be synthesized in wounds and plays an important role in scarless wound healing of fetal skin [16]. Wound dressing composed of HA and collagen sponge containing EGF has been investigated [17,18]. Decellularized tissues as biological scaffolds are tested in pre-clinical animal studies and clinical applications [19]. Decellularized scaffolds possess many good properties compared with artificial scaffolds [20]. Their exceptional cellular affinity and the ability to provide extracellular matrix resembling in vivo environment enable them to provide a proper environment for cellular growth, migration and differentiation and for protein expression, tissue deposition and angiogenesis [21]. Decellularized scaffolds are used for the skin engraftments and cosmetic surgery and are an ideal tissue source for tissue transplantation [22]. They are able to accelerate skin regeneration after they are applied to full-thickness skin defects [23]. They can promote re-epithelialization, formation of granulation tissue and other skin appendages as well as neovascularization in the early phase of implantation [24]. Decellularized scaffolds represent a promising biomaterial in future clinical applications. The effective wound healing strategies have been extensively studied for decades. Due to high complexity and poorly-known mechanisms of wound healing processes, many unavoidable obstacles and shortcomings exist with the current methods. These obstacles are divided into 3 groups: (1) Low secretion of growth factor in chronic wound. Previous study found that fluid from acute healing wounds contained approximately ten-fold higher level of EGF than from chronic wounds [25]. (2) Excessive degradation of growth factor in the wound because of high concentration of digestive enzymes. Previous study reported that an ointment containing EGF and a protease inhibitor could accelerate wound healing through stabilizing the topical EGF [26]. (3) The change of physiological response of skin cells to growth factor. For example, many studies indicate that aberrant transforming growth factor β (TGF-β) signaling plays a key role in the etiology of the pathophysiological mechanisms involved in hypertrophic scarring which often occurs after deep burn injury or trauma [27]. Many previous studies applying EGF in wound area failed to provide positive effects [28]. To address the under-secretion of the growth factors in the wound, artificial administration of growth factors through wound is explored, but the complexity has caused not satisfactory wound healing outcome in various wounds, including burns, diabetic, trauma and other chronic ulcers [29]. Thus, studies have been performed to find strategies to overcome those obstacles and increase EGF effect on wound healing, and for example, use of highly positive charged LMWP conjugated to the N-terminal of EGF significantly can increase the EGF permeability and decrease effective dose of EGF [12]. Studies of prolonged retention and sustained release of EGF are urgently required. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments. Decellularized scaffolds containing HA and EGF were compared with EGF alone for effect on the recovery of skin wounds. This study may provide effective method for the use of decellularized scaffolds in combination of EGF and HA for the promotion of wound healing. 2. Materials and methods 2.1. Materials DMEM cell culture medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Penicillin/streptomycin and dimethyl sulphoxide (DMSO) were purchased from Sigma (St Louis,
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MO, USA). Ethanol, phosphate buffered saline (PBS), SDS and sodium chloride (NaCl) were purchased from Chemical Regent (Guangzhou, China). Benzalkonium bromide solution was purchased from Baiyun Pharmaceutical (Nanchang, China). Polypropylene oxide was purchased from Hainan Petrochemical Works (Jiangsu, China). HA was purchased from Freda Biotechnology Company (Shandong, China). EGF was purchased from PeproTech (Rocky Hill, NJ, USA). Atropine sulfate was purchased from Jixing Pharmaceutical Company (Shandong, China). Pelltobarbitalum natricum was purchased from Chemical Plant (Beijing, China). 2.2. MTT assay To understand the optimum concentration of EGF for the best interaction with the cells so that it can be used in further stages of the research and confirm that EGF used in this study is biologically active, MTT assay was performed. NIH3T3 cells (mouse embryonic fibroblasts) were obtained from American Type Culture Collection (ATCC) and seeded at passage 3 to 5 in 24-well plate (Corning, Acton, MA, USA) at a density of 8 × 103 cells/well. The culture medium was DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. After cells were cultured for 24 h, the culture medium was replaced with DMEM containing 0.5% FBS, and cells were cultured for 12 h. Cells were cultured for 72 h in DMEM containing 0.5% FBS and different concentrations of EGF. Then, 100 μl MTT solution was added to each well and incubated for 4 h. The culture medium was replaced by 300 μl DMSO. Then, 100 μl liquid was transferred from each well to a 96-well plate (Corning, Acton, MA, USA) and measured at 570 nm by a microplate reader (Bio-Rad 680, Hercules, CA, USA). The group containing 0 ng/ml EGF was used as a negative control. 2.3. Preparation of decellularized scaffolds Decellularized scaffolds were prepared from fresh and healthy pig peritoneum. The peritoneum was soaked overnight at 4 °C in 0.1% benzalkonium bromide solution as an antiseptic to inhibit the microbial growth in the scaffolds. It was soaked for 1 h in 70% aqueous solution and overnight in 30% aqueous solution. It was repeatedly rinsed in water to remove residual ethanol and soaked in water for at least 1 h at 4 °C to make cells in the tissue expand. Cells in the tissue were removed by ultrasonic treatment as previously described [30]. Briefly, the decellularization system using sonication treatment consisted of an ultrasonic horn (Sonifier 250A, Branson Ultrasonics Co., Shanghai, China), a constant temperature water bath (Shanghai Anfamayaxi Bio Co., Shanghai, China) and a custom-made reactor. A solution (pH 5.6) containing 2% SDS and 0.3% NaCl was used as detergent solution to improve the efficiency of cell removal. Ultrasonic power was set to 30 W of continuous oscillation, and the frequency of ultrasound was 20 kHz. The ultrasonic treatment was carried out for 24 h. Then, 0.1 M polypropylene oxide was prepared and adjusted to pH 8.5–10.5 with NaOH. The peritoneum, following sonication treatment, was soaked in 0.1 M polypropylene oxide for one week at room temperature for fully crosslinking the tissue, and the solution was changed every 2 to 3 days. The peritoneum was thoroughly rinsed with PBS to remove residual polypropylene oxide. It was cut into different sizes, and the surface was coated with 1% sodium hyaluronate. It was sterilized in a plastic bag with 60Co irradiation. 2.4. Examination of the attachment of HA to scaffold by scanning electron microscope (SEM) and HA release experiment Decellularized scaffolds were placed at room temperature to dry before SEM, and cut into pieces of 0.5 × 0.5 cm. A layer of conductive adhesive was pasted to the dedicated SEM object stage, and samples were adhered to the stage, keeping observation surface up. Sample was sputtered with gold (Au) for 60 s using a Fine Coater
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(JFC-1200, JEOL). The ultrastructure of the adventitial and intimal surfaces and vertical sections of the samples was examined by SEM (Philips XL-30, Amsterdam, Netherlands). The attachment of HA to scaffolds was performed by the complete immersion of scaffolds in HA solution as previously described [31]. Decellularized scaffolds were cut into pieces of 2.5 × 2.5 cm in sterile condition. They were placed in a sterile 6-well plate (Corning), and 2.5 ml of 1% HA solution was added to each well. After scaffolds were incubated for 24 h at room temperature, they were washed with PBS with gentle shaking for 5 min each time for a total of 3 times. Scaffolds soaking in PBS containing no HA as controls. Then, scaffolds containing HA were soaked in PBS at 37 °C for 12, 24 and 72 h to release adsorbed HA from scaffolds. Soaking solution was replaced with fresh PBS after soaking for 12 and 24 h. Wash solutions and soaking solutions were analyzed for the release of HA by ELISA test (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instruction. Absorbance was read at 450 nm by a spectrophotometer (Bio-Rad 680). Scaffolds were examined by SEM. 2.5. The incubation of decellularized scaffolds with EGF solution Decellularized scaffolds containing HA were soaked for 12 h at room temperature in 2.5 ml of 1 μg/ml EGF solution in a sterile 6-well plate. Then, 50 μl of soaking solution was taken from each well after 3, 6, 9 or 12 h of incubation, and was examined by ELISA test (R&D Systems) to detect the amount of EGF remaining in each soaking solution. Absorbance was read at 450 nm by a spectrophotometer (Bio-Rad 680). Scaffolds containing no HA were used as controls.
days post-surgery and processed for HE staining (Beyotime Biotechnology Co., Shanghai, China). The thickness of dermis layer of each wound was determined.
2.8. The calculation of wound healing rate The lengths of upside, downside, left side and right side of each wound were measured after the excision of skin areas as original wound areas and on day 5, 10, 15 or 20 post-surgery. The sizes of wound areas were calculated as follows: wound area = (upside + downside) / 2 × (left side + right side) / 2. The wound healing rate was calculated as follows: wound healing rate = (original wound area − wound area on different days post-surgery) / original wound area.
2.9. Statistical analysis All experiments in this study were independently repeated at least 3 times with similar results. The relative percentage and values are presented as the mean and standard deviation (SD) of the mean. Student's t-test was used for Fig. 1B for the comparison between the averages of two independent groups, and one-way ANOVA followed by Fisher LSD was used for the other figures for multiple comparisons. In all statistical analyses, P b 0.05 was considered significant.
2.6. Release of EGF from decellularized scaffolds Decellularized scaffolds containing HA and EGF were washed with PBS with gentle shaking for 5 min each time for a total of 3 times. Decellularized scaffolds containing HA and EGF were soaked in PBS in a sterile 6-well plate to release adsorbed EGF from scaffolds. Soaking solution was replaced with fresh PBS after 0.5, 1, 2, or 3 days of incubation. Wash solutions and soaking solutions were examined for EGF concentrations by ELISA test, and absorbance was read by a spectrophotometer at 450 nm (Bio-Rad 680). Scaffolds containing no HA were used as controls. 2.7. Animal skin experiment The animals used in this study were from Medical Laboratory Animal Center of Guangdong (Guangzhou, China). All procedures involving experimental animals were conducted in accordance with the institutional guideline and approved by the Animal Care Committee of the Jinan University (protocol SCXK 2003-0002). Decellularized scaffolds were cut into 4 × 5 cm in the sterile condition. They were soaked in 8 ml of 1% HA solution for 24 h and washed for 3 times with PBS. Then, they were soaked in 8 ml of 1 μg/ml EGF solution for 12 h and used for the following animal experiment. Rabbits were anesthetized by the injection of 0.5 ml atropine sulfate and 0.15 ml of 3% pelltobarbitalum natricum per kg. The hair in 4 skin areas on the left and right sides of each rabbit spine was removed, and the 4 skin areas were disinfected. Four full-thickness rectangular wounds were created at sizes of 2.5 × 3 cm on each animal's skin. The wounds were covered by the vaseline oil gauzes or scaffolds, which were sewed to the wound edge by non-absorbable 0 suture (Jinhuan Medical Devices Factory, Yangzhou, China). One wound was covered with vaseline oil gauze, the second with a decellularized scaffold alone, the third with a decellularized scaffold containing 1 μg/ml EGF, and the fourth with a decellularized scaffold containing HA and 1 μg/ml EGF. Animals were sacrificed on day 5, 10, 15 or 20 post-surgery, and each group contained 5 animals. Vaseline oil gauzes and decellularized scaffolds were gently peeled off the wounds. A piece of skin was taken from each wound on different
Fig. 1. Promotion of NIH3T3 cell viability by EGF. (A) NIH3T3 cells were cultured in culture medium containing different concentrations of EGF and examined by MTT assay. The maximum cell viability was achieved at 25 ng/ml of EGF. (B) NIH3T3 cells were cultured for different periods of time in culture medium containing 25 ng/ml EGF. Cell viability increased during 96 h of cell culture. *P b 0.01 compared with the no EGF control (n = 5).
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3. Results 3.1. Promotion of NIH3T3 cell viability by EGF NIH3T3 cell viability increased as EGF concentrations increased until 25 ng/ml, at which the maximum cell viability was achieved (Fig. 1A). EGF increased cell viability during 96 h of cell culture (Fig. 1B). These results confirmed that EGF used in this study was biologically active in vitro. 3.2. Characterization of decellularized scaffolds and examination of the attachment of HA to decellularized scaffolds A piece of decellularized scaffold showed a smooth surface (Fig. 2A). Decellularized scaffolds were also examined by SEM. The examination of adventitial and intimal surfaces and longitudinal sections of decellularized scaffolds showed that they were composed of the compact and long fibers and contained no cells. The adventitia of the decellularized scaffolds had a relatively smooth surface. The intima of the decellarized scaffolds showed a relatively rough surface containing thick fibers in different directions. Vertical sections of the decellularized scaffolds showed a compact layer consisting of many fibers (Fig. 2B). After scaffolds were incubated with HA, they were washed 3 times with PBS, and soaked in PBS for 24 and 48 h to release attached HA from scaffolds. Scaffold sections were examined by SEM. Results showed that scaffolds were covered with HA before and after soaking in PBS (Fig. 2C). Wash solutions and soaking solutions were analyzed for the release of HA from scaffolds by ELISA test. HA concentrations in wash solutions decreased significantly after each wash, and there was no significant difference in HA concentrations in soaking solutions
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after soaking in PBS for 24 and 48 h (Fig. 2D). These results suggest that HA stably attached to scaffolds. HA of known concentration was purchased from the company and used to validate the method for quantification of HA by ELISA. HA solution of known concentration 500 μg/ml was determined as 496 μg/ml by ELISA. 3.3. Measurement of EGF adsorbed to decellularized scaffolds by ELISA The amount of EGF remaining in the soaking solution decreased gradually along with the increase of immersion time (Fig. 3A), suggesting that the amount of EGF adsorbed to scaffolds increased gradually along with the increase of immersion time. In addition, there was less EGF remaining in the soaking solution in case of scaffolds containing HA than no HA, suggesting that HA increased EGF adsorption to scaffolds. EGF of known concentration was purchased from the company and used to validate the method for quantification of EGF by ELISA. EGF solution of known concentration 1.00 μg/ml was determined as 0.98 μg/ml by ELISA. 3.4. Measurement of EGF released from decellularized scaffolds by ELISA EGF concentrations in each of the 3 wash solutions were measured by ELISA. The results showed that less amount of free EGF was washed out of scaffolds containing HA than no HA (Fig. 3B). After 3 washes, scaffolds were soaked in PBS, and 50 μl PBS was taken at different incubation times and measured by ELISA to detect EGF concentrations. The amount of EGF released from scaffolds decreased along with the increase of immersion time (Fig. 3C). In addition, more EGF was released from scaffolds containing HA than no HA, confirming that HA increased the amount of EGF attached to scaffolds.
Fig. 2. Examination of the scaffold and attachment of HA to decellularized scaffold by scanning electron microscope (SEM). (A) A piece of decellularized scaffold showed a smooth surface. (B) Decellularized scaffolds were examined by SEM. The adventitia of the scaffolds had a relatively smooth surface. The intima of the scaffolds showed a relatively rough surface containing thick fibers in different directions. The vertical sections of the decellularized scaffolds showed a compact layer consisting of many fibers. (C) After scaffold was soaked in HA solution, it was washed 3 times with PBS. Scaffold containing HA was soaked in PBS for 24 and 48 h to release adsorbed HA. Scaffold sections containing HA were examined by SEM. Scaffold soaking in PBS containing no HA was used as a control. Results showed that scaffolds were covered by HA before and after soaking in PBS. (D) Wash solution and soaking solution were analyzed for HA concentrations by ELISA test. Results showed that HA concentrations decreased after each wash, and there was no significant difference in HA concentrations after soaking in PBS for 24 and 48 h. *P b 0.01 compared with the release of HA for 48 h (n = 5).
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Fig. 2 (continued).
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the third with a scaffold soaked in 1 μg/ml EGF solution, and the fourth with a scaffold soaked in 1% HA solution and then, in 1 μg/ml EGF solution. Vaseline gauze and scaffolds were removed from the wounds on different days post-surgery, and the wounds were examined for their recovery (Fig. 4A, B and Table 1). There was no significant difference in wound healing rates on day 5 post-surgery. The wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 postsurgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF on days 10, 15 and 20 post-surgery. 3.6. Observation of skin tissue regeneration after HE staining A small piece of skin tissue was removed from each wound, and frozen sections of skin tissue were examined after HE staining (Fig. 5A, B). On day 5 post-surgery, there was no obvious difference in the recovery of 4 wounds. Epidermis and dermis layers were removed during surgery and were not yet regenerated, and only the hypodermis layer was observed. On day 10 post-surgery, the thicker dermis layer was observed in the 2 wounds covered with scaffolds containing EGF than the other 2 wounds containing no EGF. On days 15 and 20 postsurgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing EGF than the other 2 wounds containing no EGF and in addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing HA and EGF than EGF alone. Skin appendages were observed on day 20 only in the wound covered with scaffold containing HA and EGF. 4. Discussion
Fig. 3. Detection of EGF adsorbed to decellularized scaffold by ELISA test. (A) Scaffold was soaked in 1 μg/ml EGF. EGF remaining in the soaking solution was detected by ELISA to estimate amount of EGF adsorbed into scaffold. EGF concentrations in soaking solution decreased along with the increase of immersion time, suggesting that EGF adsorbed into scaffold increased along with the increase of immersion time. (B) Decellularized scaffold containing EGF was washed 3 times to release the free EGF. EGF concentrations decreased after each wash. (C) Scaffold following 3 washes was soaked in PBS to release EGF. EGF concentrations released from scaffold decreased along with the increase of incubation time. Effect of HA on EGF adsorption into scaffold was also compared with no HA control. * or #P b 0.01 compared with the respective first sample (n = 5).
3.5. Observation of the wound healing on different days post-surgery Four skin wounds were generated in the dorsal area of each rabbit and used as skin wound animal model. The first wound was covered with vaseline gauze; the second with a decellularized scaffold only;
Wound healing is a complex process and includes three major phases: inflammation response, new tissue formation and tissue remodeling [3]. Each phase has its distinctive mechanism and is also interconnected with the other phases. Extracellular matrix (ECM) is composed of collagens, proteoglycans, structural proteins and basement membrane and plays a critical role in providing mechanical support and regulating cell activities [32]. Decellularized scaffolds, which are most close to natural ECM, were thus used in this study. EGF and some other growth factors including VEGF, FGF and HGF have been studied for their effects on wound healing. EGF is effective and crucial components in wound healing microenvironment and has effects on the proliferation of endothelial cells and fibroblasts and the promotion of wound skin regeneration by regulating relevant gene expression through binding to its cell surface receptor and modulating relevant receptor kinase signaling pathway [33,34]. HA, together with fibronectin, glycosaminoglycans, proteoglycans and collagen, is one component in ECM, which provides scaffolds for cell adhesion, growth, movement and differentiation mainly in the stage of granulation tissue formation during wound healing [35,36]. HA is shown to play an important role in wound healing process [37]. In this study, therefore, decellularized scaffolds loaded with HA and EGF were studied for their effect on wound healing. The same method used in this study for coating the scaffolds with HA was used in previous studies, and the effectiveness and validation of this method were also defined [31]. Although the complete mechanism of coating HA to the decellularized scaffolds remains unclear, some mechanisms could be predicted from previous studies [38–40]. Due to the self-association ability of HA [38], HA coating was successfully formed on the surface of the scaffolds in this study (Fig. 2C). Previous studies showed that the mechanism of self-assembly could be applied by HA and other ECM components, for example collagen and proteoglycan, to form robust and elastic membrane complex [39,40], and the
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Fig. 4. Examination of wound healing on different days post-surgery. (A) Four skin wounds were generated in each rabbit back skin area. The first wound was covered with vaseline oil gauze (1), the second with scaffold alone (2), the third with scaffold containing 1 μg/ml EGF (3), and the fourth with scaffold containing HA and 1 μg/ml EGF (4). Vaseline oil gauzes or scaffolds were gently peeled off the wounds on days 5, 10, 15 and 20 post-surgery. (B) Calculation of wound healing rate. Each wound area was measured before and after transplantation. Size of wound area and wound healing rate were calculated for each wound. Results showed that on days 10, 15 and 20 post-surgery, the wounds covered with the scaffolds containing EGF were smaller than no EGF. In addition, the wounds covered with the scaffolds containing HA and EGF were smaller than EGF alone. * P b 0.01 compared with the respective vaseline oil gauzes (n = 5).
electrostatic complexation play a significant factor in the self-assembly process of HA, collagen or proteoglycan [39]. Because of limited transdermal permeability, low sustainability and insufficient release rate of EGF in vivo, occasional undesirable wound healing outcomes were observed when EGF was applied [28,41,42]. In this study, in vitro study showed that HA increased the adsorption
Table 1 Wound healing rates on different days post-surgery. Days post-surgery
Wound healing rate (%) Vaseline oil gauze
Scaffold
5 10 15 20
12.76 19.19 38.37 59.10
14.03 29.26 42.80 70.14
± ± ± ±
3.20 4.40 5.90 7.50
Each group contained 5 animals.
± ± ± ±
5.00 4.00 6.00 6.70
Scaffold + 1 μg/ml EGF
Scaffold + HA + 1 μg/ml EGF
16.16 40.34 56.24 83.00
15.19 49.86 70.94 87.41
± ± ± ±
4.00 5.80 7.30 8.40
± ± ± ±
3.00 7.30 9.20 11.00
of EGF to scaffolds and sustained the release of EGF from scaffolds (Fig. 3), and the possible reasons are as follows. The electrostatic interactions between EGF and the unreacted carboxylic groups in HA may increase EGF adsorption and decrease EGF release [43]. In addition, due to the highly hygroscopic property of HA [44], HA coated on scaffolds can keep scaffolds wet, and due to the hydrophilicity of EGF [12], hydrophilic EGF is easy to attach to the wet scaffolds. So, more EGF and sustained release of EGF from scaffolds can improve skin wound healing. EGF can bind to EGF receptors on cell surface and activate the related kinase pathway to promote wound healing [45]. A good wound healing environment is also created by HA and can help wound healing process [15]. Previous study showed that full-thickness wounds created on rats and covered with cotton gauze had only 40% healing rate and no epidermal layer formation after two weeks post-surgery [46], whereas in this study, wounds covered with decellularized scaffolds containing HA and EGF had wound healing rate of 70.94% and obvious epidermal formation after 15 days post-surgery (Fig. 4, Table 1).
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Fig. 5. Examination of skin tissue after HE staining. (A) After rabbits were sacrificed, a piece of skin was taken from each wound and processed for HE staining. (B) The thickness of dermis layer of each wound was determined. Results showed that on day 5 post-surgery, there was no obvious difference in the recovery of the 4 wounds. On day 10 post-surgery, the thicker dermis layer was observed in the wounds covered with scaffolds containing EGF than no EGF. On days 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing EGF than no EGF. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing HA and EGF than EGF alone. *P b 0.01 compared with the respective vaseline oil gauzes (n = 5).
Previous study also found that full thickness surgical wounds created on dorsal midline area of domestic pigs had a wound healing rate of 49.9% and no skin appendage formation after 21 days post-surgery [47], whereas in this study, wounds covered with scaffolds containing HA and EGF had a wound healing rate of 87.41% and skin appendage formation after 20 days post-surgery (Fig. 4, Table 1). In this study, the wounds covered with scaffolds containing HA and EGF had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to vaseline oil gauze with wound healing rates of 19.19%, 38.37% and 59.10% (Fig. 4, Table 1). These results clearly demonstrate that the decellularized scaffolds loaded with EGF and HA significantly promote the wound healing compared to natural healing rate. Previous studies also examined the effects of different scaffolds containing EGF on wound healing [29,48]. For example, self-assembling peptide (SAP) nanofiber scaffold by
mimicking the structure and porosity of ECM showed the ability to slow the release of EGF, which enabled a prolonged release of EGF to wound area and accelerated the wound closure [48]. These studies together with this work demonstrated that sufficient delivery and sustainable release of bioactive agents such as EGF are important considerations for wound healing. Even though many studies have been done in deciphering the molecular and genetic pathways of wound healing process, the complex mechanisms still remain largely unknown [3,33,34]. Many individual components are identified to be involved in promoting wound healing. But, wound healing in vivo is a complex process and requires many individual components working together to enhance wound healing process. Single-agent therapy, for example, the administration of a growth factor, sometimes has only moderate effects on wound healing, especially for the wound resulting from diabetes or radiation exposure.
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Therefore, therapy with multiple agents together may give more effective results. In this study, decellularized scaffolds in combination of HA and EGF were investigated, and combination of both EGF and HA showed more effective effects on wound healing than EGF alone. We clearly show the synergistic effects of HA and EGF, two important components of ECM, as well as decellularized scaffolds on wound healing through the increased adsorption of EGF to scaffolds and sustained release of EGF from scaffolds by HA. In addition to HA and EGF, many other components including other growth factors, other ECM proteins and their receptors, various enzymes and proteases are found to be involved in re-epithelialization and granulation tissue formation which are crucial events during skin tissue regeneration. Thus, it is possible to speculate that the combination of some of these other components may enhance the therapeutic effect on wound healing. 5. Conclusion Our results demonstrated that HA increases the adsorption of EGF into decellularized scaffolds and sustains the release of EGF from scaffolds. Scaffolds loaded with HA and EGF significantly promote the recovery of skin wound and may be possibly tested in the future in clinical study for the treatment of human skin wounds. Acknowledgments This work was supported by National Natural Science Foundation of China (grant no. 30870650 and grant no. 31171304 to Xing Wei), Research Foundation for Doctoral Discipline of Higher Education (grant no. 20114401110007 to Xing Wei), and the Fundamental Research Funds for the Central Universities (grant no. 21612107 to Xing Wei). References [1] R. Richardson, K. Slanchev, C. Kraus, P. Knyphausen, S. Eming, M. Hammerschmidt, Adult zebrafish as a model system for cutaneous wound-healing research, J. Invest. Dermatol. 133 (2013) 1655–1665. [2] A.W. Seifert, S.G. Kiama, M.G. Seifert, J.R. Goheen, T.M. Palmer, M. Maden, Skin shedding and tissue regeneration in African spiny mice (Acomys), Nature 489 (2012) 561–565. [3] G.C. Gurtner, S. Werner, Y. Barrandon, M.T. Longaker, Wound repair and regeneration, Nature 453 (2008) 314–321. [4] H. Liu, L. Mu, J. Tang, C. Shen, C. Gao, M. Rong, et al., A potential wound healingpromoting peptide from frog skin, Int. J. Biochem. Cell Biol. 49 (2014) 32–41. [5] R.F. Pereira, C.C. Barrias, P.L. Granja, P.J. Bartolo, Advanced biofabrication strategies for skin regeneration and repair, Nanomedicine 8 (2013) 603–621. [6] V.W. Wong, G.C. Gurtner, Tissue engineering for the management of chronic wounds: current concepts and future perspectives, Exp. Dermatol. 21 (2012) 729–734. [7] K. Akhundov, G. Pietramaggiori, S.D. Waselle, S. Guerid, C. Scaletta, N. Hirt-Burri, et al., Development of a cost-effective method for platelet-rich plasma (PRP) preparation for topical wound healing, Ann. Burns Fire Disasters 25 (2012) 207–213. [8] S. Malerich, D. Berson, Next generation cosmeceuticals: the latest in peptides, growth factors, cytokines, and stem cells, Dermatol. Clin. 32 (2014) 13–21. [9] G. Bellavia, P. Fasanaro, R. Melchionna, M.C. Capogrossi, M. Napolitano, Transcriptional control of skin reepithelialization, J. Dermatol. Sci. 73 (2014) 3–9. [10] T.N. Demidova-Rice, M.R. Hamblin, I.M. Herman, Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 2: role of growth factors in normal and pathological wound healing: therapeutic potential and methods of delivery, Adv. Skin Wound Care 25 (2012) 349–370. [11] M.T. Rose, Effect of growth factors on the migration of equine oral and limb fibroblasts using an in vitro scratch assay, Vet. J. 193 (2012) 539–544. [12] J.K. Choi, J.-H. Jang, W.-H. Jang, J. Kim, I.-H. Bae, J. Bae, et al., The effect of epidermal growth factor (EGF) conjugated with low-molecular-weight protamine (LMWP) on wound healing of the skin, Biomaterials 33 (2012) 8579–8590. [13] E.K. Tiaka, N. Papanas, A.C. Manolakis, G.S. Georgiadis, Epidermal growth factor in the treatment of diabetic foot ulcers an update, Perspect. Vasc. Surg. Endovasc. Ther. 24 (2012) 37–44. [14] A. Mineo, R. Suzuki, Y. Kuroyanagi, Development of an artificial dermis composed of hyaluronic acid and collagen, J. Biomater. Sci. Polym. Ed. 24 (2013) 726–740. [15] S. Reitinger, G. Lepperdinger, Hyaluronan, a ready choice to fuel regeneration: a mini-review, Gerontology 59 (2012) 71–76. [16] M. Prosdocimi, C. Bevilacqua, Exogenous hyaluronic acid and wound healing: an updated vision, Panminerva Med. 54 (2012) 129–135.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor Zhongchun Su a,1, Huan Ma a,1, Zhengzheng Wu a,1, Huilan Zeng b, Zhizhong Li c, Yuechun Wang d, Gexiu Liu d, Bin Xu e, Yongliang Lin e, Peng Zhang e, Xing Wei a,⁎ a
Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Jinan University, Guangzhou 510632, China Department of Hematology, The First Affiliated Hospital, Jinan University, Guangzhou 510632, China Department of Bone, The First Affiliated Hospital, Jinan University, Guangzhou 510632, China d Department of Physiology, School of Medicine, Jinan University, Guangzhou 510632, China e Grandhope Biotech Co., Ltd., Building D, #408, Guangzhou International Business Incubator, Guangzhou Science Park, Guangzhou 510663, Guangdong, China b c
a r t i c l e
i n f o
Article history: Received 17 February 2014 Received in revised form 31 May 2014 Accepted 13 July 2014 Available online 19 July 2014 Keywords: Decellularized scaffolds Growth factors Skin repair
a b s t r a c t Current therapy for skin wound healing still relies on skin transplantation. Many studies were done to try to find out ways to replace skin transplantation, but there is still no effective alternative therapy. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments, and scaffolds loaded with hyaluronic acid (HA) and epidermal growth factor (EGF) were tested for their effect on wound healing. MTT assay showed that EGF increased NIH3T3 cell viability and confirmed that EGF used in this study was biologically active in vitro. Scanning electron microscope (SEM) showed that HA stably attached to scaffolds even after soaking in PBS for 48 h. ELISA assay showed that HA increased the adsorption of EGF to scaffolds and sustained the release of EGF from scaffolds. Animal study showed that the wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF scaffolds on days 10, 15 and 20 post-surgery. Hematoxylin–Eosin (HE) staining confirmed these results by showing that on days 10, 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing HA and EGF than scaffolds alone. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing EGF than scaffolds alone. Skin appendages were observed on day 20 only in the wound covered with scaffolds containing HA and EGF. These results demonstrate that the scaffolds containing HA and EGF can enhance wound healing. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Skin is the largest organ system in animals and provides the front defense barrier against different damages to animal bodies [1,2]. Injuries to skins caused by various physical factors or chemical agents can induce wound healing and skin regeneration [3,4]. Wound healing is the process in which cells in the body regenerate and repair to reduce size of damaged or necrotic area [5]. Wound healing involves a set of events that include inflammation surrounding a region of injury, wound cell migration and mitosis, angiogenesis and the development of granulation tissue, repair of the connective tissue, regeneration of extracellular matrix and remodeling that leads to a healed wound [6,7]. ⁎ Corresponding author. Tel./fax: +86 20 85222759. E-mail address: [email protected] (X. Wei). 1 The first three authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.msec.2014.07.039 0928-4931/© 2014 Elsevier B.V. All rights reserved.
The effects of growth factors on wound healing have been extensively studied and are found to be involved in these processes [8,9]. Almost all growth factors are peptides that bind to the target cell surface receptors. Commonly, the receptor kinase activity is essential for inducing the biological activities of various growth factors including epidermal growth factor (EGF), transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF), and different growth factors can affect the normal and pathological wound healing process [10]. EGF is known to be a potent stimulator of cellular proliferation. It has been tested for its effects on wound healing [11,12]. EGF is beneficial to wound healing because of its effects on keratinocytes, fibroblasts and vascular endothelial cells and promotes the formation of granulation tissue and re-epithelialization. The action of EGF on the cells including keratinocytes, fibroblasts and endothelial cells in wound area can be regulated by local production of EGF through autocrine and paracrine mechanisms [10]. EGF can stimulate the growth and
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differentiation of keratinocyte as well as the proliferation and migration of fibroblast and vascular endothelial cells [10,13]. Besides growth factors, other molecules also play important roles in wound healing. Hyaluronic acid (HA) is a major extracellular matrix component. It is important for wound healing because of its various biological activities [14]. Due to its unique physical properties, HA creates an excellent wound healing environment and has multifaceted roles in wound healing and scarring [15]. HA can be induced to be synthesized in wounds and plays an important role in scarless wound healing of fetal skin [16]. Wound dressing composed of HA and collagen sponge containing EGF has been investigated [17,18]. Decellularized tissues as biological scaffolds are tested in pre-clinical animal studies and clinical applications [19]. Decellularized scaffolds possess many good properties compared with artificial scaffolds [20]. Their exceptional cellular affinity and the ability to provide extracellular matrix resembling in vivo environment enable them to provide a proper environment for cellular growth, migration and differentiation and for protein expression, tissue deposition and angiogenesis [21]. Decellularized scaffolds are used for the skin engraftments and cosmetic surgery and are an ideal tissue source for tissue transplantation [22]. They are able to accelerate skin regeneration after they are applied to full-thickness skin defects [23]. They can promote re-epithelialization, formation of granulation tissue and other skin appendages as well as neovascularization in the early phase of implantation [24]. Decellularized scaffolds represent a promising biomaterial in future clinical applications. The effective wound healing strategies have been extensively studied for decades. Due to high complexity and poorly-known mechanisms of wound healing processes, many unavoidable obstacles and shortcomings exist with the current methods. These obstacles are divided into 3 groups: (1) Low secretion of growth factor in chronic wound. Previous study found that fluid from acute healing wounds contained approximately ten-fold higher level of EGF than from chronic wounds [25]. (2) Excessive degradation of growth factor in the wound because of high concentration of digestive enzymes. Previous study reported that an ointment containing EGF and a protease inhibitor could accelerate wound healing through stabilizing the topical EGF [26]. (3) The change of physiological response of skin cells to growth factor. For example, many studies indicate that aberrant transforming growth factor β (TGF-β) signaling plays a key role in the etiology of the pathophysiological mechanisms involved in hypertrophic scarring which often occurs after deep burn injury or trauma [27]. Many previous studies applying EGF in wound area failed to provide positive effects [28]. To address the under-secretion of the growth factors in the wound, artificial administration of growth factors through wound is explored, but the complexity has caused not satisfactory wound healing outcome in various wounds, including burns, diabetic, trauma and other chronic ulcers [29]. Thus, studies have been performed to find strategies to overcome those obstacles and increase EGF effect on wound healing, and for example, use of highly positive charged LMWP conjugated to the N-terminal of EGF significantly can increase the EGF permeability and decrease effective dose of EGF [12]. Studies of prolonged retention and sustained release of EGF are urgently required. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments. Decellularized scaffolds containing HA and EGF were compared with EGF alone for effect on the recovery of skin wounds. This study may provide effective method for the use of decellularized scaffolds in combination of EGF and HA for the promotion of wound healing. 2. Materials and methods 2.1. Materials DMEM cell culture medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Penicillin/streptomycin and dimethyl sulphoxide (DMSO) were purchased from Sigma (St Louis,
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MO, USA). Ethanol, phosphate buffered saline (PBS), SDS and sodium chloride (NaCl) were purchased from Chemical Regent (Guangzhou, China). Benzalkonium bromide solution was purchased from Baiyun Pharmaceutical (Nanchang, China). Polypropylene oxide was purchased from Hainan Petrochemical Works (Jiangsu, China). HA was purchased from Freda Biotechnology Company (Shandong, China). EGF was purchased from PeproTech (Rocky Hill, NJ, USA). Atropine sulfate was purchased from Jixing Pharmaceutical Company (Shandong, China). Pelltobarbitalum natricum was purchased from Chemical Plant (Beijing, China). 2.2. MTT assay To understand the optimum concentration of EGF for the best interaction with the cells so that it can be used in further stages of the research and confirm that EGF used in this study is biologically active, MTT assay was performed. NIH3T3 cells (mouse embryonic fibroblasts) were obtained from American Type Culture Collection (ATCC) and seeded at passage 3 to 5 in 24-well plate (Corning, Acton, MA, USA) at a density of 8 × 103 cells/well. The culture medium was DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. After cells were cultured for 24 h, the culture medium was replaced with DMEM containing 0.5% FBS, and cells were cultured for 12 h. Cells were cultured for 72 h in DMEM containing 0.5% FBS and different concentrations of EGF. Then, 100 μl MTT solution was added to each well and incubated for 4 h. The culture medium was replaced by 300 μl DMSO. Then, 100 μl liquid was transferred from each well to a 96-well plate (Corning, Acton, MA, USA) and measured at 570 nm by a microplate reader (Bio-Rad 680, Hercules, CA, USA). The group containing 0 ng/ml EGF was used as a negative control. 2.3. Preparation of decellularized scaffolds Decellularized scaffolds were prepared from fresh and healthy pig peritoneum. The peritoneum was soaked overnight at 4 °C in 0.1% benzalkonium bromide solution as an antiseptic to inhibit the microbial growth in the scaffolds. It was soaked for 1 h in 70% aqueous solution and overnight in 30% aqueous solution. It was repeatedly rinsed in water to remove residual ethanol and soaked in water for at least 1 h at 4 °C to make cells in the tissue expand. Cells in the tissue were removed by ultrasonic treatment as previously described [30]. Briefly, the decellularization system using sonication treatment consisted of an ultrasonic horn (Sonifier 250A, Branson Ultrasonics Co., Shanghai, China), a constant temperature water bath (Shanghai Anfamayaxi Bio Co., Shanghai, China) and a custom-made reactor. A solution (pH 5.6) containing 2% SDS and 0.3% NaCl was used as detergent solution to improve the efficiency of cell removal. Ultrasonic power was set to 30 W of continuous oscillation, and the frequency of ultrasound was 20 kHz. The ultrasonic treatment was carried out for 24 h. Then, 0.1 M polypropylene oxide was prepared and adjusted to pH 8.5–10.5 with NaOH. The peritoneum, following sonication treatment, was soaked in 0.1 M polypropylene oxide for one week at room temperature for fully crosslinking the tissue, and the solution was changed every 2 to 3 days. The peritoneum was thoroughly rinsed with PBS to remove residual polypropylene oxide. It was cut into different sizes, and the surface was coated with 1% sodium hyaluronate. It was sterilized in a plastic bag with 60Co irradiation. 2.4. Examination of the attachment of HA to scaffold by scanning electron microscope (SEM) and HA release experiment Decellularized scaffolds were placed at room temperature to dry before SEM, and cut into pieces of 0.5 × 0.5 cm. A layer of conductive adhesive was pasted to the dedicated SEM object stage, and samples were adhered to the stage, keeping observation surface up. Sample was sputtered with gold (Au) for 60 s using a Fine Coater
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(JFC-1200, JEOL). The ultrastructure of the adventitial and intimal surfaces and vertical sections of the samples was examined by SEM (Philips XL-30, Amsterdam, Netherlands). The attachment of HA to scaffolds was performed by the complete immersion of scaffolds in HA solution as previously described [31]. Decellularized scaffolds were cut into pieces of 2.5 × 2.5 cm in sterile condition. They were placed in a sterile 6-well plate (Corning), and 2.5 ml of 1% HA solution was added to each well. After scaffolds were incubated for 24 h at room temperature, they were washed with PBS with gentle shaking for 5 min each time for a total of 3 times. Scaffolds soaking in PBS containing no HA as controls. Then, scaffolds containing HA were soaked in PBS at 37 °C for 12, 24 and 72 h to release adsorbed HA from scaffolds. Soaking solution was replaced with fresh PBS after soaking for 12 and 24 h. Wash solutions and soaking solutions were analyzed for the release of HA by ELISA test (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instruction. Absorbance was read at 450 nm by a spectrophotometer (Bio-Rad 680). Scaffolds were examined by SEM. 2.5. The incubation of decellularized scaffolds with EGF solution Decellularized scaffolds containing HA were soaked for 12 h at room temperature in 2.5 ml of 1 μg/ml EGF solution in a sterile 6-well plate. Then, 50 μl of soaking solution was taken from each well after 3, 6, 9 or 12 h of incubation, and was examined by ELISA test (R&D Systems) to detect the amount of EGF remaining in each soaking solution. Absorbance was read at 450 nm by a spectrophotometer (Bio-Rad 680). Scaffolds containing no HA were used as controls.
days post-surgery and processed for HE staining (Beyotime Biotechnology Co., Shanghai, China). The thickness of dermis layer of each wound was determined.
2.8. The calculation of wound healing rate The lengths of upside, downside, left side and right side of each wound were measured after the excision of skin areas as original wound areas and on day 5, 10, 15 or 20 post-surgery. The sizes of wound areas were calculated as follows: wound area = (upside + downside) / 2 × (left side + right side) / 2. The wound healing rate was calculated as follows: wound healing rate = (original wound area − wound area on different days post-surgery) / original wound area.
2.9. Statistical analysis All experiments in this study were independently repeated at least 3 times with similar results. The relative percentage and values are presented as the mean and standard deviation (SD) of the mean. Student's t-test was used for Fig. 1B for the comparison between the averages of two independent groups, and one-way ANOVA followed by Fisher LSD was used for the other figures for multiple comparisons. In all statistical analyses, P b 0.05 was considered significant.
2.6. Release of EGF from decellularized scaffolds Decellularized scaffolds containing HA and EGF were washed with PBS with gentle shaking for 5 min each time for a total of 3 times. Decellularized scaffolds containing HA and EGF were soaked in PBS in a sterile 6-well plate to release adsorbed EGF from scaffolds. Soaking solution was replaced with fresh PBS after 0.5, 1, 2, or 3 days of incubation. Wash solutions and soaking solutions were examined for EGF concentrations by ELISA test, and absorbance was read by a spectrophotometer at 450 nm (Bio-Rad 680). Scaffolds containing no HA were used as controls. 2.7. Animal skin experiment The animals used in this study were from Medical Laboratory Animal Center of Guangdong (Guangzhou, China). All procedures involving experimental animals were conducted in accordance with the institutional guideline and approved by the Animal Care Committee of the Jinan University (protocol SCXK 2003-0002). Decellularized scaffolds were cut into 4 × 5 cm in the sterile condition. They were soaked in 8 ml of 1% HA solution for 24 h and washed for 3 times with PBS. Then, they were soaked in 8 ml of 1 μg/ml EGF solution for 12 h and used for the following animal experiment. Rabbits were anesthetized by the injection of 0.5 ml atropine sulfate and 0.15 ml of 3% pelltobarbitalum natricum per kg. The hair in 4 skin areas on the left and right sides of each rabbit spine was removed, and the 4 skin areas were disinfected. Four full-thickness rectangular wounds were created at sizes of 2.5 × 3 cm on each animal's skin. The wounds were covered by the vaseline oil gauzes or scaffolds, which were sewed to the wound edge by non-absorbable 0 suture (Jinhuan Medical Devices Factory, Yangzhou, China). One wound was covered with vaseline oil gauze, the second with a decellularized scaffold alone, the third with a decellularized scaffold containing 1 μg/ml EGF, and the fourth with a decellularized scaffold containing HA and 1 μg/ml EGF. Animals were sacrificed on day 5, 10, 15 or 20 post-surgery, and each group contained 5 animals. Vaseline oil gauzes and decellularized scaffolds were gently peeled off the wounds. A piece of skin was taken from each wound on different
Fig. 1. Promotion of NIH3T3 cell viability by EGF. (A) NIH3T3 cells were cultured in culture medium containing different concentrations of EGF and examined by MTT assay. The maximum cell viability was achieved at 25 ng/ml of EGF. (B) NIH3T3 cells were cultured for different periods of time in culture medium containing 25 ng/ml EGF. Cell viability increased during 96 h of cell culture. *P b 0.01 compared with the no EGF control (n = 5).
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3. Results 3.1. Promotion of NIH3T3 cell viability by EGF NIH3T3 cell viability increased as EGF concentrations increased until 25 ng/ml, at which the maximum cell viability was achieved (Fig. 1A). EGF increased cell viability during 96 h of cell culture (Fig. 1B). These results confirmed that EGF used in this study was biologically active in vitro. 3.2. Characterization of decellularized scaffolds and examination of the attachment of HA to decellularized scaffolds A piece of decellularized scaffold showed a smooth surface (Fig. 2A). Decellularized scaffolds were also examined by SEM. The examination of adventitial and intimal surfaces and longitudinal sections of decellularized scaffolds showed that they were composed of the compact and long fibers and contained no cells. The adventitia of the decellularized scaffolds had a relatively smooth surface. The intima of the decellarized scaffolds showed a relatively rough surface containing thick fibers in different directions. Vertical sections of the decellularized scaffolds showed a compact layer consisting of many fibers (Fig. 2B). After scaffolds were incubated with HA, they were washed 3 times with PBS, and soaked in PBS for 24 and 48 h to release attached HA from scaffolds. Scaffold sections were examined by SEM. Results showed that scaffolds were covered with HA before and after soaking in PBS (Fig. 2C). Wash solutions and soaking solutions were analyzed for the release of HA from scaffolds by ELISA test. HA concentrations in wash solutions decreased significantly after each wash, and there was no significant difference in HA concentrations in soaking solutions
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after soaking in PBS for 24 and 48 h (Fig. 2D). These results suggest that HA stably attached to scaffolds. HA of known concentration was purchased from the company and used to validate the method for quantification of HA by ELISA. HA solution of known concentration 500 μg/ml was determined as 496 μg/ml by ELISA. 3.3. Measurement of EGF adsorbed to decellularized scaffolds by ELISA The amount of EGF remaining in the soaking solution decreased gradually along with the increase of immersion time (Fig. 3A), suggesting that the amount of EGF adsorbed to scaffolds increased gradually along with the increase of immersion time. In addition, there was less EGF remaining in the soaking solution in case of scaffolds containing HA than no HA, suggesting that HA increased EGF adsorption to scaffolds. EGF of known concentration was purchased from the company and used to validate the method for quantification of EGF by ELISA. EGF solution of known concentration 1.00 μg/ml was determined as 0.98 μg/ml by ELISA. 3.4. Measurement of EGF released from decellularized scaffolds by ELISA EGF concentrations in each of the 3 wash solutions were measured by ELISA. The results showed that less amount of free EGF was washed out of scaffolds containing HA than no HA (Fig. 3B). After 3 washes, scaffolds were soaked in PBS, and 50 μl PBS was taken at different incubation times and measured by ELISA to detect EGF concentrations. The amount of EGF released from scaffolds decreased along with the increase of immersion time (Fig. 3C). In addition, more EGF was released from scaffolds containing HA than no HA, confirming that HA increased the amount of EGF attached to scaffolds.
Fig. 2. Examination of the scaffold and attachment of HA to decellularized scaffold by scanning electron microscope (SEM). (A) A piece of decellularized scaffold showed a smooth surface. (B) Decellularized scaffolds were examined by SEM. The adventitia of the scaffolds had a relatively smooth surface. The intima of the scaffolds showed a relatively rough surface containing thick fibers in different directions. The vertical sections of the decellularized scaffolds showed a compact layer consisting of many fibers. (C) After scaffold was soaked in HA solution, it was washed 3 times with PBS. Scaffold containing HA was soaked in PBS for 24 and 48 h to release adsorbed HA. Scaffold sections containing HA were examined by SEM. Scaffold soaking in PBS containing no HA was used as a control. Results showed that scaffolds were covered by HA before and after soaking in PBS. (D) Wash solution and soaking solution were analyzed for HA concentrations by ELISA test. Results showed that HA concentrations decreased after each wash, and there was no significant difference in HA concentrations after soaking in PBS for 24 and 48 h. *P b 0.01 compared with the release of HA for 48 h (n = 5).
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Fig. 2 (continued).
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the third with a scaffold soaked in 1 μg/ml EGF solution, and the fourth with a scaffold soaked in 1% HA solution and then, in 1 μg/ml EGF solution. Vaseline gauze and scaffolds were removed from the wounds on different days post-surgery, and the wounds were examined for their recovery (Fig. 4A, B and Table 1). There was no significant difference in wound healing rates on day 5 post-surgery. The wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 postsurgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF on days 10, 15 and 20 post-surgery. 3.6. Observation of skin tissue regeneration after HE staining A small piece of skin tissue was removed from each wound, and frozen sections of skin tissue were examined after HE staining (Fig. 5A, B). On day 5 post-surgery, there was no obvious difference in the recovery of 4 wounds. Epidermis and dermis layers were removed during surgery and were not yet regenerated, and only the hypodermis layer was observed. On day 10 post-surgery, the thicker dermis layer was observed in the 2 wounds covered with scaffolds containing EGF than the other 2 wounds containing no EGF. On days 15 and 20 postsurgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing EGF than the other 2 wounds containing no EGF and in addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing HA and EGF than EGF alone. Skin appendages were observed on day 20 only in the wound covered with scaffold containing HA and EGF. 4. Discussion
Fig. 3. Detection of EGF adsorbed to decellularized scaffold by ELISA test. (A) Scaffold was soaked in 1 μg/ml EGF. EGF remaining in the soaking solution was detected by ELISA to estimate amount of EGF adsorbed into scaffold. EGF concentrations in soaking solution decreased along with the increase of immersion time, suggesting that EGF adsorbed into scaffold increased along with the increase of immersion time. (B) Decellularized scaffold containing EGF was washed 3 times to release the free EGF. EGF concentrations decreased after each wash. (C) Scaffold following 3 washes was soaked in PBS to release EGF. EGF concentrations released from scaffold decreased along with the increase of incubation time. Effect of HA on EGF adsorption into scaffold was also compared with no HA control. * or #P b 0.01 compared with the respective first sample (n = 5).
3.5. Observation of the wound healing on different days post-surgery Four skin wounds were generated in the dorsal area of each rabbit and used as skin wound animal model. The first wound was covered with vaseline gauze; the second with a decellularized scaffold only;
Wound healing is a complex process and includes three major phases: inflammation response, new tissue formation and tissue remodeling [3]. Each phase has its distinctive mechanism and is also interconnected with the other phases. Extracellular matrix (ECM) is composed of collagens, proteoglycans, structural proteins and basement membrane and plays a critical role in providing mechanical support and regulating cell activities [32]. Decellularized scaffolds, which are most close to natural ECM, were thus used in this study. EGF and some other growth factors including VEGF, FGF and HGF have been studied for their effects on wound healing. EGF is effective and crucial components in wound healing microenvironment and has effects on the proliferation of endothelial cells and fibroblasts and the promotion of wound skin regeneration by regulating relevant gene expression through binding to its cell surface receptor and modulating relevant receptor kinase signaling pathway [33,34]. HA, together with fibronectin, glycosaminoglycans, proteoglycans and collagen, is one component in ECM, which provides scaffolds for cell adhesion, growth, movement and differentiation mainly in the stage of granulation tissue formation during wound healing [35,36]. HA is shown to play an important role in wound healing process [37]. In this study, therefore, decellularized scaffolds loaded with HA and EGF were studied for their effect on wound healing. The same method used in this study for coating the scaffolds with HA was used in previous studies, and the effectiveness and validation of this method were also defined [31]. Although the complete mechanism of coating HA to the decellularized scaffolds remains unclear, some mechanisms could be predicted from previous studies [38–40]. Due to the self-association ability of HA [38], HA coating was successfully formed on the surface of the scaffolds in this study (Fig. 2C). Previous studies showed that the mechanism of self-assembly could be applied by HA and other ECM components, for example collagen and proteoglycan, to form robust and elastic membrane complex [39,40], and the
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Fig. 4. Examination of wound healing on different days post-surgery. (A) Four skin wounds were generated in each rabbit back skin area. The first wound was covered with vaseline oil gauze (1), the second with scaffold alone (2), the third with scaffold containing 1 μg/ml EGF (3), and the fourth with scaffold containing HA and 1 μg/ml EGF (4). Vaseline oil gauzes or scaffolds were gently peeled off the wounds on days 5, 10, 15 and 20 post-surgery. (B) Calculation of wound healing rate. Each wound area was measured before and after transplantation. Size of wound area and wound healing rate were calculated for each wound. Results showed that on days 10, 15 and 20 post-surgery, the wounds covered with the scaffolds containing EGF were smaller than no EGF. In addition, the wounds covered with the scaffolds containing HA and EGF were smaller than EGF alone. * P b 0.01 compared with the respective vaseline oil gauzes (n = 5).
electrostatic complexation play a significant factor in the self-assembly process of HA, collagen or proteoglycan [39]. Because of limited transdermal permeability, low sustainability and insufficient release rate of EGF in vivo, occasional undesirable wound healing outcomes were observed when EGF was applied [28,41,42]. In this study, in vitro study showed that HA increased the adsorption
Table 1 Wound healing rates on different days post-surgery. Days post-surgery
Wound healing rate (%) Vaseline oil gauze
Scaffold
5 10 15 20
12.76 19.19 38.37 59.10
14.03 29.26 42.80 70.14
± ± ± ±
3.20 4.40 5.90 7.50
Each group contained 5 animals.
± ± ± ±
5.00 4.00 6.00 6.70
Scaffold + 1 μg/ml EGF
Scaffold + HA + 1 μg/ml EGF
16.16 40.34 56.24 83.00
15.19 49.86 70.94 87.41
± ± ± ±
4.00 5.80 7.30 8.40
± ± ± ±
3.00 7.30 9.20 11.00
of EGF to scaffolds and sustained the release of EGF from scaffolds (Fig. 3), and the possible reasons are as follows. The electrostatic interactions between EGF and the unreacted carboxylic groups in HA may increase EGF adsorption and decrease EGF release [43]. In addition, due to the highly hygroscopic property of HA [44], HA coated on scaffolds can keep scaffolds wet, and due to the hydrophilicity of EGF [12], hydrophilic EGF is easy to attach to the wet scaffolds. So, more EGF and sustained release of EGF from scaffolds can improve skin wound healing. EGF can bind to EGF receptors on cell surface and activate the related kinase pathway to promote wound healing [45]. A good wound healing environment is also created by HA and can help wound healing process [15]. Previous study showed that full-thickness wounds created on rats and covered with cotton gauze had only 40% healing rate and no epidermal layer formation after two weeks post-surgery [46], whereas in this study, wounds covered with decellularized scaffolds containing HA and EGF had wound healing rate of 70.94% and obvious epidermal formation after 15 days post-surgery (Fig. 4, Table 1).
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Fig. 5. Examination of skin tissue after HE staining. (A) After rabbits were sacrificed, a piece of skin was taken from each wound and processed for HE staining. (B) The thickness of dermis layer of each wound was determined. Results showed that on day 5 post-surgery, there was no obvious difference in the recovery of the 4 wounds. On day 10 post-surgery, the thicker dermis layer was observed in the wounds covered with scaffolds containing EGF than no EGF. On days 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing EGF than no EGF. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing HA and EGF than EGF alone. *P b 0.01 compared with the respective vaseline oil gauzes (n = 5).
Previous study also found that full thickness surgical wounds created on dorsal midline area of domestic pigs had a wound healing rate of 49.9% and no skin appendage formation after 21 days post-surgery [47], whereas in this study, wounds covered with scaffolds containing HA and EGF had a wound healing rate of 87.41% and skin appendage formation after 20 days post-surgery (Fig. 4, Table 1). In this study, the wounds covered with scaffolds containing HA and EGF had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to vaseline oil gauze with wound healing rates of 19.19%, 38.37% and 59.10% (Fig. 4, Table 1). These results clearly demonstrate that the decellularized scaffolds loaded with EGF and HA significantly promote the wound healing compared to natural healing rate. Previous studies also examined the effects of different scaffolds containing EGF on wound healing [29,48]. For example, self-assembling peptide (SAP) nanofiber scaffold by
mimicking the structure and porosity of ECM showed the ability to slow the release of EGF, which enabled a prolonged release of EGF to wound area and accelerated the wound closure [48]. These studies together with this work demonstrated that sufficient delivery and sustainable release of bioactive agents such as EGF are important considerations for wound healing. Even though many studies have been done in deciphering the molecular and genetic pathways of wound healing process, the complex mechanisms still remain largely unknown [3,33,34]. Many individual components are identified to be involved in promoting wound healing. But, wound healing in vivo is a complex process and requires many individual components working together to enhance wound healing process. Single-agent therapy, for example, the administration of a growth factor, sometimes has only moderate effects on wound healing, especially for the wound resulting from diabetes or radiation exposure.
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Therefore, therapy with multiple agents together may give more effective results. In this study, decellularized scaffolds in combination of HA and EGF were investigated, and combination of both EGF and HA showed more effective effects on wound healing than EGF alone. We clearly show the synergistic effects of HA and EGF, two important components of ECM, as well as decellularized scaffolds on wound healing through the increased adsorption of EGF to scaffolds and sustained release of EGF from scaffolds by HA. In addition to HA and EGF, many other components including other growth factors, other ECM proteins and their receptors, various enzymes and proteases are found to be involved in re-epithelialization and granulation tissue formation which are crucial events during skin tissue regeneration. Thus, it is possible to speculate that the combination of some of these other components may enhance the therapeutic effect on wound healing. 5. Conclusion Our results demonstrated that HA increases the adsorption of EGF into decellularized scaffolds and sustains the release of EGF from scaffolds. Scaffolds loaded with HA and EGF significantly promote the recovery of skin wound and may be possibly tested in the future in clinical study for the treatment of human skin wounds. Acknowledgments This work was supported by National Natural Science Foundation of China (grant no. 30870650 and grant no. 31171304 to Xing Wei), Research Foundation for Doctoral Discipline of Higher Education (grant no. 20114401110007 to Xing Wei), and the Fundamental Research Funds for the Central Universities (grant no. 21612107 to Xing Wei). References [1] R. Richardson, K. Slanchev, C. Kraus, P. Knyphausen, S. Eming, M. Hammerschmidt, Adult zebrafish as a model system for cutaneous wound-healing research, J. Invest. Dermatol. 133 (2013) 1655–1665. [2] A.W. Seifert, S.G. Kiama, M.G. Seifert, J.R. Goheen, T.M. Palmer, M. Maden, Skin shedding and tissue regeneration in African spiny mice (Acomys), Nature 489 (2012) 561–565. [3] G.C. Gurtner, S. Werner, Y. Barrandon, M.T. Longaker, Wound repair and regeneration, Nature 453 (2008) 314–321. [4] H. Liu, L. Mu, J. Tang, C. Shen, C. Gao, M. Rong, et al., A potential wound healingpromoting peptide from frog skin, Int. J. Biochem. Cell Biol. 49 (2014) 32–41. [5] R.F. Pereira, C.C. Barrias, P.L. Granja, P.J. Bartolo, Advanced biofabrication strategies for skin regeneration and repair, Nanomedicine 8 (2013) 603–621. [6] V.W. Wong, G.C. Gurtner, Tissue engineering for the management of chronic wounds: current concepts and future perspectives, Exp. Dermatol. 21 (2012) 729–734. [7] K. Akhundov, G. Pietramaggiori, S.D. Waselle, S. Guerid, C. Scaletta, N. Hirt-Burri, et al., Development of a cost-effective method for platelet-rich plasma (PRP) preparation for topical wound healing, Ann. Burns Fire Disasters 25 (2012) 207–213. [8] S. Malerich, D. Berson, Next generation cosmeceuticals: the latest in peptides, growth factors, cytokines, and stem cells, Dermatol. Clin. 32 (2014) 13–21. [9] G. Bellavia, P. Fasanaro, R. Melchionna, M.C. Capogrossi, M. Napolitano, Transcriptional control of skin reepithelialization, J. Dermatol. Sci. 73 (2014) 3–9. [10] T.N. Demidova-Rice, M.R. Hamblin, I.M. Herman, Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 2: role of growth factors in normal and pathological wound healing: therapeutic potential and methods of delivery, Adv. Skin Wound Care 25 (2012) 349–370. [11] M.T. Rose, Effect of growth factors on the migration of equine oral and limb fibroblasts using an in vitro scratch assay, Vet. J. 193 (2012) 539–544. [12] J.K. Choi, J.-H. Jang, W.-H. Jang, J. Kim, I.-H. Bae, J. Bae, et al., The effect of epidermal growth factor (EGF) conjugated with low-molecular-weight protamine (LMWP) on wound healing of the skin, Biomaterials 33 (2012) 8579–8590. [13] E.K. Tiaka, N. Papanas, A.C. Manolakis, G.S. Georgiadis, Epidermal growth factor in the treatment of diabetic foot ulcers an update, Perspect. Vasc. Surg. Endovasc. Ther. 24 (2012) 37–44. [14] A. Mineo, R. Suzuki, Y. Kuroyanagi, Development of an artificial dermis composed of hyaluronic acid and collagen, J. Biomater. Sci. Polym. Ed. 24 (2013) 726–740. [15] S. Reitinger, G. Lepperdinger, Hyaluronan, a ready choice to fuel regeneration: a mini-review, Gerontology 59 (2012) 71–76. [16] M. Prosdocimi, C. Bevilacqua, Exogenous hyaluronic acid and wound healing: an updated vision, Panminerva Med. 54 (2012) 129–135.
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