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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 18  |  Issue : 1  |  Page : 82

Evaluation and comparison number of gingival fibroblast and osteosarcoma cell (MG-63 cell line) adhesive to mocugraft, alloderm, and collagen membrane with or without advanced platelet-rich fibrin


Department of Periodontics, Faculty of Dentistry, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran

Date of Submission06-Apr-2020
Date of Acceptance19-Apr-2021
Date of Web Publication21-Oct-2021

Correspondence Address:
Dr. Shirin Zahra Farhad
Department of Periodontics, Faculty of Dentistry, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-3327.328750

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  Abstract 


Background: The tissue engineering has recently shown a significant progress in the fields of membranes and biosynthetic materials. Advanced platelet-rich fibrin (A-PRF) contains functional molecules that have newly shown great interest in regenerative therapies. The purpose of this study was to evaluate the effect of A-PRF on the adhesion of gingival fibroblast cells and osteosarcoma cells to different membranes.
Materials and Methods: In this experimental in vitro study, three collagen, alloderm, and mucograft membranes were studied, which were cut into four 5 mm × 5 mm pieces and placed in the bottom of a 24-well culture medium. One milliliter of A-PRF was added to two wells from each group and the other two wells remained without A-PRF. The gingival fibroblasts and osteosarcoma cells were individually added to each well. The cell adhesion was studied using an electron microscope after 24 h. The data were analyzed by independent t-test, one-way analysis of variance, and least significant difference test.
Results: In the presence of A-PRF, there was a significant higher osteoblast adhesion to collagen membrane compared to alloderm and mucograft membranes (P < 0.001). In the absence of A-PRF, adhesion of osteoblasts to collagen membrane was significantly higher than alloderm and mucograft (P = 0.019). Moreover, in the presence of A-PRF, fibroblast adhesion to collagen membrane was significantly higher than alloderm and mucograft membranes (P < 0.001). Furthermore, in the absence of A-PRF, no significant difference was found among the study groups (P = 0.830).
Conclusion: A-PRF was effective on fibroblast adhesion to the collagen membrane, which is similar to its absence. A-PRF was also found to be very effective on the adhesion of fibroblast cells to the collagen membrane, and in its absence, even less adhesion was observed compared to the other membranes. The presence or absence of A-PRF showed no significant differences in both cells' adhesion for alloderm and mucograft membranes.

Keywords: Advanced platelet-rich fibrin, alloderm, cell adhesion, collagen membrane, guided tissue regeneration, mocugraft


How to cite this article:
Reisie BH, Farhad SZ, Sadeh SA. Evaluation and comparison number of gingival fibroblast and osteosarcoma cell (MG-63 cell line) adhesive to mocugraft, alloderm, and collagen membrane with or without advanced platelet-rich fibrin. Dent Res J 2021;18:82

How to cite this URL:
Reisie BH, Farhad SZ, Sadeh SA. Evaluation and comparison number of gingival fibroblast and osteosarcoma cell (MG-63 cell line) adhesive to mocugraft, alloderm, and collagen membrane with or without advanced platelet-rich fibrin. Dent Res J [serial online] 2021 [cited 2021 Dec 4];18:82. Available from: https://www.drjjournal.net/text.asp?2021/18/1/82/328750




  Introduction Top


The final objective of periodontal treatments is to control the periodontal tissue inflammation and predictable regeneration periodontium, which are lost as the results of periodontal diseases. In order to improve the periodontium regeneration, it is important to direct those tissues capable of regenerating.[1],[2] Various clinical studies have shown a significant reduction in the pocket depth probe and clinical attachment level gain after performing regenerative treatments using different absorbable and nonabsorbable membranes.[3],[4]

Fibroblasts and osteoblasts play an important role in wound healing. These cells are rapidly present and proliferate at the site of injury and can also accelerate the healing process. They are also responsible for the development of various growth factors, which finally lead to extracellular matrix deposition and epithelial cell differentiation.[5],[6]

Periodontal wound healing requires a series of interactions among epithelial cells, gingival fibroblasts, periodontal ligament cells, and osteoblasts. The presence of growth factors and cytokines in platelets also play a key role in the inflammation and in wound-healing process.[7] Platelets also act as a matrix for connective tissue by secreting fibrin and fibronectin, and by these adhesive molecules, they facilitate cell migration.[8] This has led to the theory of using platelets as a therapeutic tool to improve the tissue repairing, especially healing of periodontal wounds.[9]

The use of collagen membranes is recommended to avoid second surgery (it was indicated that the use of absorbable membranes eliminates the need for the second stage surgery to remove the membrane compared to nonabsorbable membranes.); however, it has been shown that, early membrane breakdown and downward epithelial growth can affect the treatment outcomes.[10]

The membrane used for regenerative treatments should have a number of specific features. Accordingly, an important feature of the membrane is its ability of improving the cell adhesion, proliferation, and differentiation. Several membranes are designed to meet these requirements, but some factors such as membrane composition, surface tissue morphology, size of the pores, and duration of membrane function that may affect the results of regenerative therapies, have not been still well understood.[11]

The cell adhesion to the surface substrate consists of a four-stage sequence including absorption of glycoproteins on the substrate surface, cell contact, adhesion, and diffusion, respectively.[12] The cell migration and proliferation occur after these four stages. Since the cell adhesion to all the tested membranes occurred, the cell migration and proliferation would also occur at the surface, which indicate the biocompatibility and nontoxicity of the studied membranes.

Hakki et al.[13] in their research studied the gingival fibroblast cell adhesion and proliferation on three collagen, alloderm, and glycolic lactic copolymer membranes and observed that adhesion and proliferation on two collagen and alloderm membranes were similar and also higher than glycolic lactic copolymer membrane group. Vahabi et al.[14] by evaluating the binding of fibroblast and osteoblast cells to three TXT-200, human collagen, and animal collagen membranes found that the binding of each cell in platelet-rich plasma (PRP)-activated group was >5% fetal bovine serum. Locci et al.[15] suggested that collagen membranes have the best ability in binding and inducing osteoblast cell adhesion.

Considering that the use of different types of membranes is common for periodontal treatments, the higher the adhesion of fibroblasts and osteoblasts to the respective membranes, the lower the probability of membrane displacement and subsequent disruption of tissue regeneration. The number of gingival fibroblasts and osteosarcoma cells (MG-63 cell line) attached to the three mucogrel, alloderm, and collagen membranes was determined in the presence or in the absence of advanced platelet-rich fibrin (A-PRF).


  Materials and Methods Top


In this experimental in vitro study, gingival fibroblast cells (NCBI Codece C165) and human osteosarcoma cells (MG-63) were prepared from Pasteur Institute. The cells were incubated at 37°C under the moist conditions, exposed to 5% carbon dioxide, and then passaged five times in a 24-well culture medium to reach the sufficient numbers.

In this study, three collagen (Regen [Itb, Tehran, Iran]), alloderm (Regen [Itb, Tehran, Iran]) (Freeze derived, cell_ free human dermal matrix.), and mucograft (Botiss dental, Berlin, Germany) (Pure collagen type I and III matrix porcine origin without any future cross linkage) membranes were studied for adhesion of osteoblast and gingival fibroblast cells. Furthermore, four pieces of 5 mm × 5 mm were prepared from each membrane.

In order to prepare A-PRF, the patient's blood sample was placed in a 2 ml tube without anticoagulant and then centrifuged immediately at 1500 rpm for 14 min. After centrifugation, the product was composed of three layers as follows: The top layer consisting of cell-free plasma containing low platelet counts, A-PRF clot in the middle, and the red blood cells at the bottom of the test tube. By removing the supernatant and the remainder of the product from the centrifuge tube and then isolating the sediment (red blood cells); fibrin clot (A-PRF) remained. After isolating the A-PRF layer, it was placed on a heater to be used at the same temperature of 36°C.

In order to prepare the membranes in groups containing A-PRF, 1 ml of prepared A-PRF was added to the membranes, then incubated for 2 h at 4°C, and finally washed with 5 ml of PBS. After that, 104 fibroblast cells and 104 MG-63 cells were added to all groups and incubated for 24 h at 37°C under the moist conditions, and exposed to 1% carbon dioxide. By passing 24 h, the cell binding in the control group (without any membrane and A-PRF) was evaluated using light microscopy and cell adhesion was assured.

Under the electron microscope, the surface of each membrane was divided into four parts. In each quadrant, a point image with a ×300 magnification was taken, in which, 16 images were obtained from each one of the membranes. Accordingly, eight images showed the number of fibroblasts attached to the membrane without the presence of A-PRF, and the other eight images showed cells attached to the surface of the membrane in the presence of A-PRF.

The obtained data were analyzed by independent t-test, one-way analysis of variance (ANOVA), least significant difference, and software SPSS 22 (IBM, Armonk, USA), and also the statistical level was considered as 0.05.

To evaluate the adhesion of osteoblast cells in each membrane alone, based on independent t-test.


  Results Top


According to two-way ANOVA, the membrane type was effective on the cell adhesion (P < 0.01).

To evaluate the adhesion of osteoblast cells in each membrane separately, using independent t-test, it was shown that the adhesion of osteoblast cells to alloderm (P = 0.11) and mucograft (P = 0.643) membranes was slightly higher in the absence of A-PRF, and this difference was not statistically significant. The cell adhesion to collagen membrane was higher in the presence of A-PRF, which was not statistically significant (P = 0.736) [Table 1].
Table 1: Mean of osteoblast cell adhesion in each membrane

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The results of comparison between the studied membranes based on the one-way ANOVA showed that, in the presence of A-PRF, osteoblast adhesion to collagen membrane was higher than alloderm and mucograft membranes and this difference was statistically significant (P < 0.001). Furthermore, the adhesion of osteoblasts to collagen membrane was significantly higher than alloderm and mucograft (P = 0.019) when A-PRF was absent in the medium.

According to paired comparison of the groups, in the presence of A-PRF, the adhesion of the osteoblasts to collagen membrane was significantly higher than those of alloderm (P < 0.001) and mucograft (P < 0.001). The adhesion of osteoblasts to collagen membrane was significantly higher compared to alloderm (P = 0.024) and mucograft (P = 0.009), and in the presence of A-PRF, osteoblast adhesion to mucograft membrane was higher than alloderm; however, this difference was not statistically significant. The cell adhesion to alloderm was higher than mucograft in the absence of A-PRF, but again, this difference was not statistically significant (P = 0.655) [Figure 1].
Figure 1: Mean of osteoblast cell adhesion in each membrane.

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Considering the adhesion of fibroblast cells individually in each membrane, independent t-test showed that, adhesion of fibroblast cells to alloderm (P = 0.745) and mucograft (P = 0.500) membranes was slightly higher in the absence of A-PRF, and this difference was not statistically significant. The cell adhesion to collagen membrane was higher in the presence of A-PRF, which was statistically significant (P < 0.001) [Table 2].
Table 2: Mean of adhesion of fibroblast cells in each membrane

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Comparison of the studied membranes by the one-way ANOVA showed that, in the presence of A-PRF, fibroblast adhesion to collagen membrane was higher than the other two membranes of alloderm and mucograft, and this difference was statistically significant (P < 0.001). When A-PRF was absent in the medium, adhesion of fibroblasts to mucograft membrane was higher than alloderm and collagen, which was not statistically significant (P = 0.830).

Furthermore, according to paired comparison of the groups, fibroblast adhesion to collagen membrane was significantly higher than alloderm (P < 0.001) and mucograft (P < 0.001). Moreover, in the presence of A-PRF, fibroblast adhesion to mucograft membrane was more than alloderm; however, this difference was not statistically significant (P = 0.607) [Figure 2].
Figure 2: Mean of adhesion of fibroblast cells in each membrane.

Click here to view



  Discussion Top


Separately studying each membrane showed that, adhesion of osteoblast cells to alloderm and mucograft membranes was slightly higher when A-PRF was absent; however, this difference was not statistically significant. For collagen membrane, adhesion of osteoblast cells was higher in the presence of A-PRF, but the difference was not significant.

However, the results of comparison between the studied membranes showed that, the adhesion of osteoblast cells to collagen membrane was significantly higher than that of mucograft and alloderm in the absence of A-PRF, which is consistent with the results of the studies by Wang et al.[16] and Nagahara et al.[17]

Locci et al. in their study suggested that, collagen membrane had the best abilities in binding and inducing osteoblast cell adhesion.[15]

According to the results of the present study, in the presence of A-PRF, the adhesion of osteoblast cells to collagen membrane was significantly higher than mucograft and alloderm membranes, which is consistent with the results of studies by Vahabi et al.[14] and Simon et al.[18]

In a comparative study performed on PRF and collagen membrane, Gassling et al.[19] showed that the levels of proliferation and metabolic activities of osteoblast cells at PRF level was higher than that of collagen membrane. Furthermore, in a study by Wu et al.,[20] human-derived PRF was studied. Accordingly, it was found that binding and proliferation of osteoblast cells increased in the presence of PRF, and by evaluating the effective mechanisms using western blot test, it was found that a significant increase in collagen-dependent proteins (Heat shock protein 47), HSP47 (Lysyl oxidase), and LOX leads to the effect of PRF on bone regeneration. The study of the effect of TGF B-1, as a collagen gene inducer, that is normally secreted by PRF, on HSP47 that was produced from osteoblast cells showed that TGF B-1 could produce 3-fold mRNA HSP47 compared to its absence. This dose-dependent increase was observed in both HSP47 mRNA and collagen alpha 1 mRNA.[21]

However, the results of a study by Döri et al.[22] showed that, clinical parameters for the treatment of intracerebral guided tissue regeneration (GTR) with PRP, collagen membrane and autogenous mineralized bone were not different from those that were treated with similar materials, but without the use of PRP. The reason for this difference may be the use of PRP rather than PRF, and the study was an in vivo study, not an in vitro study.

Separately studying of each membrane revealed that, the adhesion of fibroblast cells to alloderm and mucograft membranes was slightly higher when A-PRF was absent; however, this difference was not statistically significant. For collagen membrane, the adhesion of fibroblast cells was higher in the presence of A-PRF, and the difference was statistically significant.

The adhesion of fibroblast cells was not significantly different among the three membranes studied in the absence of A-PRF; however it can be said that, the most important effect of A-PRF on fibroblast cell absorption is on collagen membrane, as in its absence fibroblast binding is lower than other membranes, which is consistent with the results of studies by Hakki et al.,[13] Alpar et al.,[23] and Wang et al.[24]

Rodrigues et al.[5] examined the binding and aggregation of fibroblasts on alloderm membrane and finally concluded that, on the 7th day, the cells were distributed as a discontinuous layer on the membrane, which continued until the day 14, but from day 14 to day 21, it reduced a little. In a study by Ojeh et al.,[25] the structure of dans alloderm reduced the cell migration into it. But Hillmann et al.[26] stated that, denser the matrix fibers carrying alloderm cell, the lower the cell migration, and low porosity and small pores in alloderm reduced the nutrient distribution and consequently caused disruption of migration and cell binding, which is inconsistent with the result of the present study, because it requires a longer study period that was less in the present study. Also, perhaps fibroblasts were more likely to be adsorbed to other membranes than alloderm if the study duration was longer.

In a study by Chang et al.,[27] the level of fibroblast cell absorption on the three types of membranes of Gore-Tex, Gore-Resolut XT (nonabsorbable membranes), and INION GTR (absorbable membranes) was higher in the presence of PRP compared to the absence of PRP. The highest cell adhesion was for INION membrane.

According to the study results, the adhesion to the absorbable membrane is better than the nonabsorbable membrane. Kasaj et al.[1] in their study showed that, the materials in the used membranes can improve cellular uptake and proliferation in the periodontal or bone tissue regeneration process, and also the absorbable membranes can stimulate the cell proliferation better than the nonabsorbable membranes.

Therefore, it seems that collagen receptor substrate could promote the growth and differentiation of many cells in the culture medium to a greater extent compared to those with glass and plastic substrates.[28],[29]

According to the results, although the effect of A-PRF was solely on the adhesion of fibroblasts to collagen membrane and it was not very effective on the cell adhesion to membranes generally, due to its important role in the cell differentiation, its usage in adjacent membrane should not be undamaged.


  Conclusion Top


The adhesion of both osteoblast and fibroblast cells to alloderm and mucograft membranes were slightly higher when A-PRF was absent; however, these differences were not statistically significant in both of these cell types.

For collagen membrane, osteoblast cell adhesion was higher in the presence of A-PRF, but again this difference was not statistically significant. However, the most significant effect of A-PRF was on the adhesion of fibroblast cells to the collagen membrane, and the cell adhesion to the membrane was higher in the presence of A-PRF, and this difference was statistically significant.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.



 
  References Top

1.
Kasaj A, Reichert C, Götz H, Röhrig B, Smeets R, Willershausen B. In vitro evaluation of various bioabsorbable and nonresorbable barrier membranes for guided tissue regeneration. Head Face Med 2008;4:22.  Back to cited text no. 1
    
2.
Tatakis DN, Promsudthi A, Wikesjö UM. Devices for periodontal regeneration. Periodontol 2000 1999;19:59-73.  Back to cited text no. 2
    
3.
Cortellini P, Pini Prato G, Tonetti MS. Periodontal regeneration of human intrabony defects with bioresorbable membranes. A controlled clinical trial. J Periodontol 1996;67:217-23.  Back to cited text no. 3
    
4.
Machtei EE, Grossi SG, Dunford R, Zambon JJ, Genco RJ. Long-term stability of Class II furcation defects treated with barrier membranes. J Periodontol 1996;67:523-7.  Back to cited text no. 4
    
5.
Rodrigues AZ, Oliveira PT, Novaes AB Jr., Maia LP, Souza SL, Palioto DB. Evaluation of in vitro human gingival fibroblast seeding on acellular dermal matrix. Braz Dent J 2010;21:179-89.  Back to cited text no. 5
    
6.
Erdag G, Sheridan RL. Fibroblasts improve performance of cultured composite skin substitutes on athymic mice. Burns 2004;30:322-8.  Back to cited text no. 6
    
7.
Lindhe J, Westfelt E, Nyman S, Socransky SS, Haffajee AD. Long-term effect of surgical/non-surgical treatment of periodontal disease. J Clin Periodontol 1984;11:448-58.  Back to cited text no. 7
    
8.
Christan C, Dietrich T, Hägewald S, Kage A, Bernimoulin JP. White blood cell count in generalized aggressive periodontitis after non-surgical therapy. J Clin Periodontol 2002;29:201-6.  Back to cited text no. 8
    
9.
Purucker P, Mertes H, Goodson JM, Bernimoulin JP. Local versus systemic adjunctive antibiotic therapy in 28 patients with generalized aggressive periodontitis. J Periodontol 2001;72:1241-5.  Back to cited text no. 9
    
10.
Lindhe J, Lang NP, Berglundh T, Giannobile WV, Sanz M. Clinical Periodontology and Implant Dentistry. 6th ed.. Chicheste: Wiley; 2015. p. 916.  Back to cited text no. 10
    
11.
Takata T, Wang HL, Miyauchi M. Attachment, proliferation and differentiation of periodontal ligament cells on various guided tissue regeneration membranes. J Periodontal Res 2001;36:322-7.  Back to cited text no. 11
    
12.
Burridge K, Molony L, Kelly T. Adhesion plaques: sites of transmembrane interaction between the extracellular matrix and the actin cytoskeleton. J Cell Sci Suppl 1987;8:211-29.  Back to cited text no. 12
    
13.
Hakki SS, Korkusuz P, Purali N, Bozkurt B, Kus M, Duran I. Attachment, proliferation and collagen type I mRNA expression of human gingival fibroblasts on different biodegradable membranes. Connect Tissue Res 2013;54:260-6.  Back to cited text no. 13
    
14.
Vahabi S, Yadegary Z, Karamshahi M. Evaluating the adhesion of human gingival fibroblasts and MG-63 osteoblast-like cells to activated PRP-coated membranes. Cell Tissue Bank 2019;20:339-49.  Back to cited text no. 14
    
15.
Locci P, Calvitti M, Belcastro S, Pugliese M, Guerra M, Marinucci L, et al. Phenotype expression of gingival fibroblasts cultured on membranes used in guided tissue regeneration. J Periodontol 1997;68:857-63.  Back to cited text no. 15
    
16.
Wang HL, Miyauchi M, Takata T. Initial attachment of osteoblasts to various guided bone regeneration membranes: An in vitro study. J Periodontal Res 2002;37:340-4.  Back to cited text no. 16
    
17.
Nagahara K, Mouri K, Kanematsu N, Shrestha P, Meenaghan MA. Stimulation of in vivo calcification using collagen membranes cultured with osteoblastic cells in vitro: A preliminary report. Int J Oral Maxillofac Implants 1995;10:109-13.  Back to cited text no. 17
    
18.
Simon BI, Zatcoff AL, Kong JJW, O'Connell SM. Clinical and histological comparison of extraction socket healing following the use of autologous platelet-rich fibrin matrix (PRFM) to ridge preservation procedures employing demineralized freeze dried bone allograft material and membrane. Pen Dent J 2009;3:92-9.  Back to cited text no. 18
    
19.
Gassling V, Hedderich J, Açil Y, Purcz N, Wiltfang J, Douglas T. Comparison of platelet rich fibrin and collagen as osteoblast-seeded scaffolds for bone tissue engineering applications. Clin Oral Implants Res 2013;24:320-8.  Back to cited text no. 19
    
20.
Wu CL, Lee SS, Tsai CH, Lu KH, Zhao JH, Chang YC. Platelet-rich fibrin increases cell attachment, proliferation and collagen-related protein expression of human osteoblasts. Aust Dent J 2012;57:207-12.  Back to cited text no. 20
    
21.
Yamamura I, Hirata H, Hosokawa N, Nagata K. Transcriptional activation of the mouse HSP47 gene in mouse osteoblast MC3T3-E1 cells by TGF-beta 1. Biochem Biophys Res Commun 1998;244:68-74.  Back to cited text no. 21
    
22.
Döri F, Huszár T, Nikolidakis D, Arweiler NB, Gera I, Sculean A. Effect of platelet-rich plasma on the healing of intra-bony defects treated with a natural bone mineral and a collagen membrane. J Clin Periodontol 2007;34:254-61.  Back to cited text no. 22
    
23.
Alpar B, Leyhausen G, Günay H, Geurtsen W. Compatibility of resorbable and nonresorbable guided tissue regeneration membranes in cultures of primary human periodontal ligament fibroblasts and human osteoblast-like cells. Clin Oral Investig 2000;4:219-25.  Back to cited text no. 23
    
24.
Wang HJ, Chou TD, Tsou TL, Chen TM, Chen SL, Chen SG, et al. The application of new biosynthetic artificial skin for long-term temporary wound coverage. Burns 2005;31:991-7.  Back to cited text no. 24
    
25.
Ojeh NO, Frame JD, Navsaria HA. In vitro characterization of an artificial dermal scaffold. Tissue Eng 2001;7:457-72.  Back to cited text no. 25
    
26.
Hillmann G, Steinkamp-Zucht A, Geurtsen W, Gross G, Hoffmann A. Culture of primary human gingival fibroblasts on biodegradable membranes. Biomaterials 2002;23:1461-9.  Back to cited text no. 26
    
27.
Chang T, Liu Q, Marino V, Bartold PM. Attachment of periodontal fibroblasts to barrier membranes coated with platelet-rich plasma. Aust Dent J 2007;52:227-33.  Back to cited text no. 27
    
28.
Newsome DA. In vitro stimulation of cartilage in embryonic chick neural crest cells by products of rentinal pigmented epithelium. Dev Biol 1976;49:496-507.  Back to cited text no. 28
    
29.
Gospodarowicz D, Greenburg G, Birdwell CR. Determinatio n ofcellular shape by the extracellular matrix and its correlation with the control of cellular growth. Cancer Res 1978;38:4155-71.  Back to cited text no. 29
    


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