Antheraea pernyi silk fibroin maintains ... - Wiley Online Library

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Shen-Zhou Lu c. , Xue-Guang Zhang b a Department of Immunology, Binzhou Medical college, Shandong Province, Yantai 264003, People's Republic of China.

Cell Biology International 33 (2009) 1127e1134 www.elsevier.com/locate/cellbi

Antheraea pernyi silk fibroin maintains the immunosupressive properties of human bone marrow mesenchymal stem cells Xi-Ying Luan a,b,*, Guan-Hua Huo a, Ming-Zhong Li c, Shen-Zhou Lu c, Xue-Guang Zhang b b

a Department of Immunology, Binzhou Medical college, Shandong Province, Yantai 264003, People’s Republic of China Institute of Medical Biotechnology, Suzhou University; Jiangsu Province Key Laboratory of Stem Cell, Suzhou 215007, People’s Republic of China c School of Materials Engineering, Suzhou University, Suzhou 215006, People’s Republic of China

Received 29 October 2008; revised 3 May 2009; accepted 25 July 2009

Abstract We reported previously that regenerated Antheraea pernyi silk fibroin (A. pernyi SF) could support the attachment and growth of human bone marrow mesenchymal stem cells (hBMSCs). In this work, the immunosupressive effects of hBMSCs cultured on the A. pernyi SF films on T-cells were investigated in vitro. The production of IL-6, CD80, CD86 and HLA-DR by the hBMSCs was also observed. The study showed that hBMSCs cultured on the regenerated A. pernyi SF films still kept their immunosupression on T-cell proliferation and IL-2 secretion. Moreover, regenerated A. pernyi SF like regenerated Bombyx mori SF and collagen did not elicit T-cell proliferation but it could support the expression of IL-6 and surface antigen of hBMSCs. Regenerated A. pernyi SF can maintain the function of hBMSCs in immunomodulation and cytokines production, which has the potential utility of hBMSCs combined with A. pernyi SF in tissue replacement and repair. Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Human bone marrow mesenchymal stem cells; Immunomodulation; T-cells; Antheraea pernyi silk fibroin; Silk

1. Introduction Silk fibroin of the domestic silkworm Bombyx mori has been studied extensively, and has a long medical history as surgical sutures. Many studies revealed that B. mori SF could support the adhesion and proliferation of many kinds of cells, and offer versatility in matrix scaffold design for a number of tissue engineering needs (Chiarini et al., 2003; Inouye et al., 1998; Min et al., 2004; Sofia et al., 2001; Altman et al., 2002). The reported antigenic properties of silk are associated with sericins, the residual glue-like proteins forming a coat around silk fibers (Soong and Kenyon, 1984). Recent studies yielded improved protocols for silk purification by completely removing sericins, and enabled preparation of silk that has no

* Corresponding author. Department of Immunology, Binzhou Medical college, Shandong Province, Yantai 264003, People’s Republic of China. Tel.: þ86 535 6913454. E-mail address: [email protected] (X.-Y. Luan).

antigenic effects (Sofia et al., 2001). However, the study of Antheraea pernyi SF is at an early stage. A. pernyi SF is another kind of silk fibroin besides B. mori SF. Previously, we reported that A. pernyi SF supported the attachment and growth of hBMSCs (Luan et al., 2006). This result not only indicated the promising application of A. pernyi SF in the tissue engineering but also formed basis for the use of A. pernyi SF combined with hBMSCs in biomedical area. However, the immunological properties of A. pernyi SF are unclear. hBMSCs have the capacity to differentiate into multiple mesenchymal tissues, including bone, cartilage, muscle, and fat depending on specific growth supplements (Kopen et al., 1999; Pittenger et al., 1999). Moreover, many studies reveled that mesenchymal stem cells (MSCs) from various species can exert profound immunosupression by inhibiting T-cell response to various challenges such as mitogens, recall antigens and alloantigens (Morandi et al., 2008; Di Nicola et al., 2002; Krampera et al., 2003; Aggarwal and Pittenger, 2005; Maitra et al., 2004). The capacities of hBMSCs to differentiate into multiple mesenchymal tissues and immune modulation

1065-6995/$ - see front matter Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2009.07.005

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make them attractive for cellular therapy and in tissue engineering. Therefore, further preclinical and clinical studies on the potential of mesenchymal stem cells are necessary for regenerative medicine and cellular immunotherapy (Wulf et al., 2006). On the other hand, future approaches using A. pernyi SF biomaterial for tissue engineering could involve hBMSCs. So before A. pernyi SF combined with hBMSCs can be broadly used for engineering of human autologous tissues, a more comprehensive understanding of the cellular basis of A. pernyi SF biocompatibility and the influence of A. pernyi SF on hBMSCs immunogenicity and immune modulation are needed. In experiments described here, we investigated whether regenerated A. pernyi SF can induce the proliferation of T-cells and maintain the low immunogenicity and immune modulation of hBMSCs. The regenerated B. mori SF collagen type I were used as control. The cytokine produced by hBMSCs cultured on the regenerated A. pernyi SF films was also observed. The results showed that A. pernyi SF did not activate T-cells alone, but could keep the low immunogenicity and immunoregulation properties of hBMSCs. It did not influence the cytokine secretion and immune phenotype expression from hBMSCs.

patient gave informed consent for the use of the sample for research, were isolated using previously described methods (Luan et al., 2006; Jiang et al., 2005). Briefly, the bone marrow aspirate was diluted with PBS. The cells were prepared by gradient centrifugation at 900 g for 30 min on FicollePlaque at a density of 1.077 g/ml (Sigma, St. Louis, MO). Then the cells were washed with PBS, and centrifuged. Cells were resuspended in hBMSCs medium consisting of Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG) (Gibco, Grand Island, N.Y. USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, N.Y. USA), 100 U/ml penicillin, and 100 mg/ml streptomycin, 2 mM glutamine and seeded in 25 cm2 flasks at a density of 1  106 cells/ml. The cultures were maintained at 37  C in an atmosphere of 5%CO2. Medium was replaced and the nonadherent cells were removed at 48 h of initial culture and 3 or 4 days thereafter. When 70e80% confluent, adherent cells were trypsinized (0.25% trypsin), harvested and split at 1:2 ratio. Cells were identified by morphology observed by light microscope and showed positive surface staining for CD29, CD105, CD166 and CD44 but negative for CD34, CD45 and CD14 surface expression. Cells after three passages were used in the experiment to ensure depletion of monocytes/ macrophages.

2. Materials and methods 2.3. T-cell isolation and culture 2.1. Films preparation Regenerated A. pernyi SF solution: the A. pernyi silk was treated three times in 0.25 wt% Na2CO3 solution at 98e 100  C for 30 min to remove sericin. After being air-dried the degummed silk was dissolved in solvent Ca(NO3)2 at 105  C through stirring for 5 h. Then the mixed solution was dialyzed and filtered to get A. pernyi SF solution with concentration of about 1.9 wt%, which was determined by weighing the remaining solid after drying. Regenerated B. mori SF solution: the B. mori domestic silk was treated three times in 0.05 wt% Na2CO3 solution at 98e 100  C for 30 min to remove sericin. After being air-dried the degummed silk was dissolved in ternary solvent CaCl2$CH3CH2OH$H2O (mole ratio ¼ 1:2:8) at 70  2  C through stirring. Then the mixed solution was dialyzed and filtered to get fibroin solution with concentration of about 3.0 wt%, which was determined by the method described above. Then, 6-, 24-, 96-well tissue culture plastic and flasks were coated with the solution of A. pernyi SF, B. mori SF and collagen (sigma, St. Louis, MO) respectively (300 ml/well for 6-well plate, 60 ml/ well for 24-well plate, 30 ml/well for 96-well plate and 600 ml for 25 cm2 flask). The plates were dried at 60  C, fixed in 75% ethanol (v./v.) overnight. Washed with phosphate-buffered saline (PBS) twice and dried at 60  C followed by irradiation treatment to sterilize. 2.2. Generation of hBMSCs The hBMSCs from normal bone marrow, which were provided by Suzhou University affiliated hospital and the

T-cells from peripheral blood provided by a healthy volunteer who gave informed consent for the use of the sample for research, were prepared by FicollePlaque density centrifugation for 20 min at 600 g. Then the cells were washed by PBS and purified with sheep red blood cells. The isolated T-cells were labeled with the fluorescein isothiocyanate (FITC)-CD3 antibody (invitrogen, USA) and were always more than 90% positive. All T-cells were cultured in RPMI 1640 supplemented with 5  105 M 2-mercaptoethanol (2-M), 10% fetal calf serum (FCS) (Gibco, Grand Island, N.Y., USA), 100 U/ml penicillin and 100 mg/ml streptomycin. 2.4. T-cell proliferation assay T-cell proliferation assay were performed in 96-well plates which were coated with the regenerated A. pernyi SF, regenerated B. mori SF and type I collagen solution, respectively, in total volume of 200 ml RPMI 1640. hBMSCs were plated in triplicates at 1  104 cells/well in 200 ml complete DMEM-LG medium and were allowed adhere to the plate for 2e3 h. Then the cells were irradiated (30 Gry) and the media were discarded. T-cells were resuspended at 1  106 cells/ml and added to above wells with or without hBMSCs in the presence of the mitogen Phytohemagglutinin (PHA) (10 mg/ml) (Sigma, St. Louis, MO), anti-CD3 monoclonal antibody (mAb) (invitrogen, USA) combining anti-CD28 mAb (produced by our laboratory) (Sun et al., 2005; Qiu et al., 2005), or the irradiated allogeneic peripheral blood mononuclear cell (PBMC). The ratio of hBMSCs to

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T-cells was 1:10, and the ratio of PBMC to T-cells was 1:1. T-cells alone were used as negative control. T-cells plus PHA, anti-CD3 mAb or irradiated allogeneic PBMC were used as positive control. The cells were cultured at 37  C in an atmosphere of 5%CO2. 3H-thymidine 1 mCi (1.85  104 Bq) was added to each well 18 h before the end of 72 h culture. Finally, T-cells were harvested and detected by standard 3 H-thymidine incorporation assay. T-cell proliferation was represented as the incorporated radioactivity in count per minute (cpm), or showed as stimulator index (SI) which was defined as follows: SI ¼ mean cpm of triplicate wells of experimental group/mean cpm of triplicate wells of negative control group.

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2.5. Flow cytometry analysis hBMSCs were stained with the following phycoerythrin (PE)-conjugated or FITC-conjugated mouse anti-human CD34, CD44, CD45, CD105, CD166, CD29, CD14, CD80, CD86 and HLA-DR (All fluorescein direct conjugated antibodies were purchased from invitrogen, USA) at 4  C for 30 min, washed with PBS and analyzed by flow cytometry (BeckmaneCoulter USA). 2.6. Cytokines detection The levels of IL-6 and IL-2 in media were detected by enzyme-linked immunosorbent assay (ELISA) after 72 h (for

Fig. 1. Morphology and phenotype of hBMSCs cultured in vitro. (I) cells isolated from human bone marrow were expanded easily. (a) Three days after initial plating the cells adhered to a plastic surface and formed single cell with spindle shape. (b) Cultured at two weeks after initial plating, cells became confluent and began to form colonies with long spindle-shaped fibroblastic cells. (c) Cultured at seven passages, original magnification 40. (II) After three passages cells used in this cultured system showed positive for CD29, CD166, CD105 and CD44 but negative for CD34, CD45 and CD14. The flow cytometry plot is the representative of one out of three experiments of identical design. All above results confirmed that the cells were hBMSCs but non-hematopoietic cells.

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IL-2), 4 days and 7 days (for IL-6), respectively. The measurement was performed according to the manufacturer’s protocols (R&D Systems, USA). 2.7. Statistical analysis Data were collected from triplicate samples and were expressed as the mean  standard deviation (SD). Statistical analysis was performed with SAS 6.0 program and a significance level of 0.05 was chosen. 3. Results 3.1. Morphology and phenotypic characteristics of hBMSCs hBMSCs were cultured as described in Section 2. The morphology of hBMSCs was showed in Fig. 1(I). After the initial 3 days of primary culture, hBMSCs adhered to a plastic surface and presented a small population of single cells with spindle shape. Two weeks after initial plating, the cells looked like long spindle-shaped fibroblastic cells, began to form colonies and became confluent. A homogenous cell population was obtained after three passages. The cell surface antigens detected by flow cytometry showed that hBMSCs were positive for CD29, CD166, CD105 and CD44 but negative for CD34, CD45 and CD14 (Fig. 1(II)), which was consistent with other reports (Luan et al., 2006; Ya~nez et al., 2006). 3.2. A. pernyi SF supports the secretion of IL-6 from hBMSCs The results of ELISA assay showed that the regenerated A. pernyi SF did not alter the secretion of IL-6 from hBMSCs. There were no significant difference between the regenerated A. pernyi SF group and other groups (Fig. 2), which were detected at day 4 and 7, respectively. This suggested that hBMSCs cultured on A. pernyi SF films could maintain their function of supporting expansion of hematopoietic cells in vitro.

Fig. 2. Level of IL-6 secreted from hBMSCs seeded on (a) regenerated B. mori SF films; (b) regenerated A. pernyi SF films; (c) collagen films and (d) tissue culture plastic analyzed by ELISA at day 4 and 7 respectively. Data from three independent experiments with triplicate wells in each condition.

tissue culture plastic. This result suggested that A. pernyi SF alone did not elicit T-cell proliferation and had low immunogenicity. 3.4. Immunomodulation of hBMSCs cultured on the A. pernyi SF films In order to confirm the hBMSCs could suppress activation and proliferation of T-cells, we challenged T-cells with different antigenic stimuli in the presence or absence of hBMSCs. As shown in Fig. 4(I), hBMSCs strongly inhibited the proliferation of T-cells activated by PHA, anti-CD3 mAb combining anti-CD28 mAb or allogeneic PBMC (P < 0.01). Furthermore, the suppression was in dose dependent manner (Fig. 4(II)). The similar results were obtained from the other stimulation ways (data not shown). To determine whether A. pernyi SF could maintain the immunosupression mediated by hBMSCs, T-cells were cultured on the A. pernyi SF films in the presence or absence

3.3. A. pernyi SF does not induce T-cell proliferation Previous researches indicated that B. mori SF has no antigenic. However, the immunogenic properties of A. pernyi SF remain to be clarified. The ability of regenerated A. pernyi SF stimulating on T-cells was assayed by the method of 3 H-TdR in this work. T-cells were cultured on the regenerated A. pernyi SF films, regenerated B. mori SF, collagen films and tissue culture plastic, respectively. PHA was used as positive control. T-cells were effectively activated by PHA (Fig. 3). Regenerated A. pernyi SF, similar to regenerated B. mori SF and collagen, failed to induce T-cell proliferation, which were significant difference compared with PHA group (P < 0.01). But there were no significant difference among the regenerated A. pernyi SF, regenerated B. mori SF, collagen and

Fig. 3. Regenerated A. pernyi SF did not elicit T-cell proliferation. T-cells were cultured on regenerated B. mori SF films (a); regenerated A. pernyi SF films (b); collagen films (c) and tissue culture plastic (d) without any stimulation. While T-cells activated by PHA was used as positive control (e). T-cell proliferation was assayed by 3H-TdR at 72 h. Data from three independent experiments with triplicate wells in each condition. Asterisks indicate significant differences compared with PHA stimulated group (*P < 0.01).

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Fig. 4. Regenerated A. pernyi SF maintained the immunosuppressive effects mediated by hBMSCs on T-cell proliferation. (I) hBMSCs suppressed the proliferation of T-cells induced by PHA, anti-CD3 mAb or by allogeneic PBMC. (II) The immunosupression of hBMSCs on the proliferation of T-cells induced by PHA was in dose dependant manner. (III) Regenerated A. pernyi SF kept the inhibitory effects of hBMSCs on the PHA induced T-cell proliferation. (IV) Regenerated A. pernyi SF did not enhance hBMSCs to elicit T-cell proliferation. T-cells were cultured on the (a) regenerated B. mori SF films; (b) regenerated A. pernyi SF films; (c) collagen films and (d) tissue culture plastic, in the presence (-) (hBMSCs/T-cells ration ¼ 1:10) or absence (,) of hBMSCs. T-cell proliferation was detected by 3 H-TdR. Data from three independent experiments with triplicate wells in each condition. Asterisks indicate significant differences compared with the absence of hBMSCs group (*P < 0.01).

of hBMSCs under the stimulating by PHA. As shown in Fig. 4(III), hBMSCs cultured on the A. pernyi SF, B. mori SF, collagen films and tissue plastic obviously inhibited T-cell proliferation induced by PHA compared with the absence of hBMSCs in each group (P < 0.01). However, the inhibitory effects were similar among the groups of A. pernyi SF, B. mori SF, collagen films and tissue culture plastic. The result indicated that A. pernyi SF did not influence the immunosupressive effect of hBMSCs on T-cells response to PHA. The similar results were obtained from T-cells stimulated by anti-CD3 mAb (data not shown). In addition, regenerated A. pernyi SF, regenerated B. mori SF and collagen films did not enhance the stimulation abilities of hBMSCs on T-cells. As shown in Fig. 4(IV), T-cells and the irradiated hBMSCs were seeded on the biomaterial films, 3HTdR analysis showed that there were no obvious difference between the groups of tissue culture plastic and A. pernyi SF, B. mori SF or collagen. Furthermore, the level of IL-2 from T-cells was detected by ELISA to investigate the inhibitory effects of hBMSCs cultured on A. pernyi SF films. The hBMSCs strongly

suppressed the production of IL-2 from T-cells stimulated by PHA compared with the absence of hBMSCs in each group (P < 0.01). Moreover, the inhibitory effect of hBMSCs on the production of IL-2 from T-cells activated by PHA on the A. pernyi SF films was almost same as that on B. mori SF, collagen films and tissue culture plastic (Fig. 5). 3.5. Immunophenotype of hBMSCs cultured on the A. pernyi SF films Previous study suggested that hBMSCs did not express the costimulator molecules such as CD80, CD86 and HLA-DR (Jiang et al., 2002). The expression of CD80, CD86 and HLADR on hBMSCs seeded on each biomaterial films was detected by flow cytometry to evaluate whether the regenerated A. pernyi SF could support the expression of immunophenotype on hBMSCs. As Fig. 6 showed that there was no obvious change of the immunophenotype expression between the experimental groups and the control group. Taken together, the results demonstrated that regenerated A. pernyi SF did not alter the low immunogenicity of hBMSCs.

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4. Discussion

Fig. 5. hBMSCs cultured on A. pernyi SF films still inhibited the secretion of IL-2 from T-cells activated by PHA. in the presence (-) (hBMSCs/T-cells ration ¼ 1:10) or absence (,) of hBMSCs. (a) Regenerated B. mori SF films; (b) regenerated A. pernyi SF films; (c) collagen films and (d) tissue culture plastic detected by ELISA at day 3. Data from three independent experiments with triplicate wells in each condition. Asterisks indicate significant differences compared with the absence of hBMSCs group (*P < 0.01).

In previous study, we reported the compliant films of regenerated A. pernyi SF by chemical crosslinking, and then in later research we showed that regenerated A. pernyi SF could support attachment and growth of hBMSCs (Luan et al., 2006; Li et al., 2003). In this work we demonstrated that the levels of IL-6 secreted by hBMSCs seeded on A. pernyi SF, B. mori SF, collagen films and tissue plastic were similar. The utilities of hBMSCs for supporting the expansion of hematopoietic cells ex vivo were reported (Maitra et al., 2004; Angelopoulou et al., 2003). The expanded mesenchymal stromal cells in cultures may produce many growth factors associated with hematopoietic support, such as IL-6, IL-7, IL-8, LIF, SCF, M-CSF and Flt-3 ligand, among them IL-6 is known as an important immunoregulatory factor. Our study showed that A. pernyi SF did not influence the secretion of IL-6 from hBMSCs, which suggested that it did not alter the support hematopoietic function of hBMSCs.

Fig. 6. Surface antigen expression of hBMSCs seeded on the (a) regenerated B. mori SF films; (b) regenerated A. pernyi SF films; (c) collagen films and (d) tissue culture plastic analyzed by flow cytometry at day 7. The flow cytometry plot is representative of one out of three experiments of identical design.

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Silks from B. mori SF used for tissue replacement and repair, in particular as a substitute biomaterial for ligaments and sutures were reported by many researchers (Lu et al., 2007; Hino et al., 2006). It was reported that the inflammatory reaction of BMSCs cultured on silk, silk-RGD and collagen films was similar, supporting the suitability of silk-based biomaterials as scaffolds for stem cell-based engineering approaches of autologous tissues (Meinel et al., 2005). However, the immunogenicity of A. pernyi SF combining with hBMSCs remains to be understood. The one of aims of this study was to assay the response of T-cells to A. pernyi SF films in order to observe the immunogenicity of A. pernyi SF and to provide evidence for the feasibility of its usage in tissue engineering. Due to minimal tissue reaction after implantation and low immunogenicity, the protein-based biomaterial collagen and the regenerated B. mori SF were used widely. (Meinel et al., 2005; Riesle et al., 1998; Adelmann et al., 1972; Adelmann, 1972; Panilaitis et al., 2003; Zhang et al., 2006). In our study, T-cells were seeded on A. pernyi SF, B. mori SF, collagen films and tissue plastic to observe the stimulation on the proliferation of T-cells by each biomaterial. A. pernyi SF similar to B. mori SF, collagen and tissue culture plastic failed to induce the proliferation of T-cells, which indicating its lower immunogenicty. Recently, increasing evidence indicated that hBMSCs had low immunogenicty and inhibited T-cell proliferation, which is beneficial for using it in tissue engineering (Morandi et al., 2008; Aggarwal and Pittenger, 2005; Maitra et al., 2004; Schuleri et al., 2008). In this research we investigated whether hBMSCs seeded on regenerated A. pernyi SF films could keep their immunoregulation. We found that hBMSCs seeded on the A. pernyi SF, B. mori SF and collagen films did not obviously induce T-cell proliferation, compared with tissue plastic group. Likewise, hBMSCs cultured on every biomaterial still mediated the un-response of T-cells to PHA and strongly inhibited T-cell proliferation. Furthermore, the ELISA detection showed that hBMSCs seeded on A. pernyi SF films still strongly suppressed production of IL-2 by activated T-cells. CD80, CD86 and HLA-DR are important costimulator molecules that play key roles in activation and proliferation of T-cells. It had been reported that hBMSCs did not express CD80, CD86 and HLA-DR (Jiang et al., 2002). However, whether A. pernyi SF, B. mori SF and collagen could alter the expression of CD80, CD86 and HLA-DR on hBMSCs is unclear now. The flow cytometry analysis results indicated that the expression of CD80, CD86 and HLA-DR on hBMSCs cultured on regenerated A. pernyi SF films had no obvious changes compared with other groups. All these results revealed that hBMSCs seeded on the A. pernyi SF did not alter their low immunogenicty and immunomodulation properties. 5. Conclusions In conclusion, we have shown in vitro that A. pernyi SF failed to induce the proliferation of T-cells, and kept the immunosupressive effects of hBMSCs on T-cell proliferation and cytokine secretion. Moreover, A. pernyi SF could maintain

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the production of IL-6 and immunophenotype of hBMSCs. All these results indicated that A. pernyi SF not only has low immunogenicty but also can keep the immunosupressive effects of hBMSCs. Therefore, the suitability of hBMSCs combined with A. pernyi SF in tissue engineering area was supported by our study.

Acknowledgements This work was supported by China National ‘‘973’’ Program (No.2005CB623906), Shangdong Province Natural Science Foundation of China (No. Y2006C02), and National Defence Foundation of China (No. 2003-44).

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