VIABILITY AND MORPHOLOGY OF HUMAN DENTAL PULP STEM CELLS IN THE PRESENCE OF CITRUS PECTIN

Abstract

Pectin is a galacturonic acid rich heteropolysaccharide which regulates plant cell metabolism. Plenty of fresh fruits and fruit pomaces from fruit juice production can be used as a raw material in commercial pectin production. Pectin occupies a large global market size especially in food industry and the utilization of waste materials for obtaining pectin molecules as a high value-added product makes it very favorite industrial material. Besides food industry, pectin is gaining attention in tissue engineering and drug development studies. In this study, the effects of citrus pectin on viability and morphology of human dental pulp stem cell (hDPSC) were investigated. The cells were cultured in the presence of pectin in culture medium (0.43, 0.85 and 1.7 mg/mL) for eight days. Resazurin application and MTT assay were applied on day one and eight for cellular viability. Cellular morphology was investigated by invert phase contrast microscope, live/dead cell staining and F-actin/nucleus immunofluorescence staining. MTT analysis results indicated that the viability of hDPSCs decreased significantly due to dissolved pectin in culture medium at applied concentrations. There was no significant morphological difference in the cells under invert phase contrast microscope and no significant staining difference in live/dead cell staining images. On the other hand, F-actin/nucleus staining showed that there were some condensed and crescent cell nuclei in the pectin applied groups when compared to the control groups which may be related to apoptosis. In conclusion, the viability of hDPSCs decreased and crescent cell nuclei formation was observed due to the presence of citrus pectin in the cell culture medium.

Keywords

• Pectin,human dental pulp,mesenchymal stem cell

References

1. [1] D. Mohnen, "Pectin structure and biosynthesis," Curr Opin Plant Biol, vol. 11, no. 3, pp. 266-77, Jun 2008, doi: 10.1016/j.pbi.2008.03.006.
2. [2] A. G. J. Voragen, G.-J. Coenen, R. P. Verhoef, and H. A. Schols, "Pectin, a versatile polysaccharide present in plant cell walls," Structural Chemistry, vol. 20, no. 2, p. 263, 2009/03/13 2009, doi: 10.1007/s11224-009-9442-z.
3. [3] F. Capel, T. Nicolai, D. Durand, P. Boulenguer, and V. Langendorff, "Calcium and acid induced gelation of (amidated) low methoxyl pectin," Food Hydrocolloids, vol. 20, no. 6, pp. 901-907, 2006, doi: 10.1016/j.foodhyd.2005.09.004.
4. [4] P. Sriamornsak, "Chemistry of pectin and its pharmaceutical uses: A review," Silpakorn University International Journal, vol. 3, pp. 206-228, 01/01 2003.
5. [5] W. G. T. Willats, J. P. Knox, and J. D. Mikkelsen, "Pectin: new insights into an old polymer are starting to gel," Trends in Food Science & Technology, vol. 17, no. 3, pp. 97-104, 2006, doi: 10.1016/j.tifs.2005.10.008.
6. [6] M. Pagliaro, R. Ciriminna, A. Fidalgo, R. Delisi, and L. Ilharco, "Pectin Production and Global Market," Agro Food Industry Hi Tech, vol. 27, 09/15 2016.
7. [7] L. Delphi and H. Sepehri, "Apple pectin: A natural source for cancer suppression in 4T1 breast cancer cells in vitro and express p53 in mouse bearing 4T1 cancer tumors, in vivo," Biomed Pharmacother, vol. 84, pp. 637-644, Dec 2016, doi: 10.1016/j.biopha.2016.09.080.
8. [8] M. Bergman, M. Djaldetti, H. Salman, and H. Bessler, "Effect of citrus pectin on malignant cell proliferation," Biomed Pharmacother, vol. 64, no. 1, pp. 44-7, Jan 2010, doi: 10.1016/j.biopha.2009.03.004.

9. [9] S. Hosseini, K. Parastouei, and F. Khodaiyan, "Simultaneous extraction optimization and characterization of pectin and phenolics from sour cherry pomace," Int J Biol Macromol, vol. 158, pp. 911-921, Apr 30 2020, doi: 10.1016/j.ijbiomac.2020.04.241.
10. [10] W. Zhang, F. Xie, X. Lan, S. Gong, and Z. Wang, "Characteristics of pectin from black cherry tomato waste modified by dynamic high-pressure microfluidization," Journal of Food Engineering, vol. 216, pp. 90-97, 2018, doi: 10.1016/j.jfoodeng.2017.07.032.
11. [11] S. M. Hosseini, S. H. Hoseinifar, M. Mazandarani, H. Paknejad, H. Van Doan, and E. R. El-Haroun, "The potential benefits of orange peels derived pectin on serum and skin mucus immune parameters, antioxidant defence and growth performance in common carp (Cyprinus carpio)," Fish Shellfish Immunol, vol. 103, pp. 17-22, Apr 20 2020, doi: 10.1016/j.fsi.2020.04.019.
12. [12] Y. Jin and N. Yang, "Array-induced voltages assisted extraction of pectin from grapefruit (Citrus paradisi Macf.) peel and its characterization," Int J Biol Macromol, vol. 152, pp. 1205-1212, Jun 1 2020, doi: 10.1016/j.ijbiomac.2019.10.215.
13. [13] E. H. Cho, H. T. Jung, B. H. Lee, H. S. Kim, J. K. Rhee, and S. H. Yoo, "Green process development for apple-peel pectin production by organic acid extraction," Carbohydr Polym, vol. 204, pp. 97-103, Jan 15 2019, doi: 10.1016/j.carbpol.2018.09.086.
14. [14] D. L. Su et al., "Efficient extraction and characterization of pectin from orange peel by a combined surfactant and microwave assisted process," Food Chem, vol. 286, pp. 1-7, Jul 15 2019, doi: 10.1016/j.foodchem.2019.01.200.
15. [15] Y. Guan et al., "Components of heat-treated Helianthus annuus L. pectin inhibit tumor growth and promote immunity in a mouse CT26 tumor model," Journal of Functional Foods, vol. 48, pp. 190-199, 2018, doi: 10.1016/j.jff.2018.07.001.
16. [16] R. G. Zhu, Y. D. Sun, Y. T. Hou, J. G. Fan, G. Chen, and T. P. Li, "Pectin penta-oligogalacturonide reduces cholesterol accumulation by promoting bile acid biosynthesis and excretion in high-cholesterol-fed mice," Chem Biol Interact, vol. 272, pp. 153-159, Jun 25 2017, doi: 10.1016/j.cbi.2017.05.018.
17. [17] C. Ramachandran, B. J. Wilk, A. Hotchkiss, H. Chau, I. Eliaz, and S. J. Melnick, "Activation of human T-helper/inducer cell, T-cytotoxic cell, B-cell, and natural killer (NK)-cells and induction of natural killer cell activity against K562 chronic myeloid leukemia cells with modified citrus pectin," BMC Complement Altern Med, vol. 11, p. 59, Aug 4 2011, doi: 10.1186/1472-6882-11-59.
18. [18] I. Eliaz and A. Raz, "Pleiotropic Effects of Modified Citrus Pectin," Nutrients, vol. 11, no. 11, Nov 1 2019, doi: 10.3390/nu11112619.
19. [19] J. C. Amorim, L. C. Vriesmann, C. L. Petkowicz, G. R. Martinez, and G. R. Noleto, "Modified pectin from Theobroma cacao induces potent pro-inflammatory activity in murine peritoneal macrophage," Int J Biol Macromol, vol. 92, pp. 1040-1048, Nov 2016, doi: 10.1016/j.ijbiomac.2016.08.015.
20. [20] S. M. Sureban et al., "Dietary Pectin Increases Intestinal Crypt Stem Cell Survival following Radiation Injury," PLoS One, vol. 10, no. 8, p. e0135561, 2015, doi: 10.1371/journal.pone.0135561.
21. [21] D. Govindaraj, M. Rajan, A. A. Hatamleh, and M. A. Munusamy, "From waste to high-value product: Jackfruit peel derived pectin/apatite bionanocomposites for bone healing applications," Int J Biol Macromol, vol. 106, pp. 293-301, Jan 2018, doi: 10.1016/j.ijbiomac.2017.08.017.
22. [22] J. Li et al., "Modulation of mesenchymal stem cells behaviors by chitosan/gelatin/pectin network films," J Biomed Mater Res B Appl Biomater, vol. 95, no. 2, pp. 308-19, Nov 2010, doi: 10.1002/jbm.b.31715.
23. [23] F. Chen et al., "Self-crosslinking and injectable hyaluronic acid/RGD-functionalized pectin hydrogel for cartilage tissue engineering," Carbohydr Polym, vol. 166, pp. 31-44, Jun 15 2017, doi: 10.1016/j.carbpol.2017.02.059.
24. [24] M. Dziadek, A. Zima, E. Cichoń, J. Czechowska, and A. Ślósarczyk, "Biomicroconcretes based on the hybrid HAp/CTS granules, α-TCP and pectins as a novel injectable bone substitutes," Materials Letters, vol. 265, 2020, doi: 10.1016/j.matlet.2020.127457.
25. [25] M. Ghorbani, L. Roshangar, and J. Soleimani Rad, "Development of reinforced chitosan/pectin scaffold by using the cellulose nanocrystals as nanofillers: An injectable hydrogel for tissue engineering," European Polymer Journal, vol. 130, 2020, doi: 10.1016/j.eurpolymj.2020.109697.
26. [26] N. Li et al., "Fabrication and Characterization of Pectin Hydrogel Nanofiber Scaffolds for Differentiation of Mesenchymal Stem Cells into Vascular Cells," ACS Biomaterials Science & Engineering, vol. 5, no. 12, pp. 6511-6519, 2019, doi: 10.1021/acsbiomaterials.9b01178.
27. [27] D. Platt and A. Raz, "Modulation of the lung colonization of B16-F1 melanoma cells by citrus pectin," (in eng), J Natl Cancer Inst, vol. 84, no. 6, pp. 438-42, Mar 18 1992, doi: 10.1093/jnci/84.6.438.
28. [28] G. Päth, N. Perakakis, C. S. Mantzoros, and J. Seufert, "Stem cells in the treatment of diabetes mellitus — Focus on mesenchymal stem cells," Metabolism, vol. 90, pp. 1-15, 2019, doi: 10.1016/j.metabol.2018.10.005.
29. [29] D. T. Chu et al., "Adipose Tissue Stem Cells for Therapy: An Update on the Progress of Isolation, Culture, Storage, and Clinical Application," J Clin Med, vol. 8, no. 7, Jun 26 2019, doi: 10.3390/jcm8070917.
30. [30] S. Morikawa et al., "Applications of Mesenchymal Stem Cells and Neural Crest Cells in Craniofacial Skeletal Research," Stem Cells Int, vol. 2016, p. 2849879, 2016, doi: 10.1155/2016/2849879.
31. [31] S. C. Neves et al., "Biofunctionalized pectin hydrogels as 3D cellular microenvironments," J Mater Chem B, vol. 3, no. 10, pp. 2096-2108, Mar 14 2015, doi: 10.1039/c4tb00885e.
32. [32] A. Polymeri, W. V. Giannobile, and D. Kaigler, "Bone Marrow Stromal Stem Cells in Tissue Engineering and Regenerative Medicine," Horm Metab Res, vol. 48, no. 11, pp. 700-713, Nov 2016, doi: 10.1055/s-0042-118458.
33. [33] A. R. Silini et al., "Immunological and Differentiation Properties of Amniotic Cells Are Retained After Immobilization in Pectin Gel," Cell Transplant, vol. 27, no. 1, pp. 70-76, Jan 2018, doi: 10.1177/0963689717738786.
34. [34] J. G. Martins, S. E. A. Camargo, T. T. Bishop, K. C. Popat, M. J. Kipper, and A. F. Martins, "Pectin-chitosan membrane scaffold imparts controlled stem cell adhesion and proliferation," Carbohydr Polym, vol. 197, pp. 47-56, Oct 1 2018, doi: 10.1016/j.carbpol.2018.05.062.
35. [35] F. Ahadi, S. Khorshidi, and A. Karkhaneh, "A hydrogel/fiber scaffold based on silk fibroin/oxidized pectin with sustainable release of vancomycin hydrochloride," European Polymer Journal, vol. 118, pp. 265-274, 2019, doi: 10.1016/j.eurpolymj.2019.06.001.
36. [36] Y. Komada et al., "Origins and properties of dental, thymic, and bone marrow mesenchymal cells and their stem cells," PLoS One, vol. 7, no. 11, p. e46436, 2012, doi: 10.1371/journal.pone.0046436.
37. [37] E. Anitua, M. Troya, and M. Zalduendo, "Progress in the use of dental pulp stem cells in regenerative medicine," Cytotherapy, vol. 20, no. 4, pp. 479-498, Apr 2018, doi: 10.1016/j.jcyt.2017.12.011.
38. [38] M. La Noce et al., "Dental pulp stem cells: state of the art and suggestions for a true translation of research into therapy," J Dent, vol. 42, no. 7, pp. 761-8, Jul 2014, doi: 10.1016/j.jdent.2014.02.018.
39. [39] M. C. Yurtsever, A. Kiremitci, and M. Gumusderelioglu, "Dopaminergic induction of human dental pulp stem cells by photobiomodulation: comparison of 660nm laser light and polychromatic light in the nir," J Photochem Photobiol B, vol. 204, p. 111742, Mar 2020, doi: 10.1016/j.jphotobiol.2019.111742.
40. [40] E. M. Czekanska, "Assessment of cell proliferation with resazurin-based fluorescent dye," (in eng), Methods Mol Biol, vol. 740, pp. 27-32, 2011, doi: 10.1007/978-1-61779-108-6_5.
41. [41] F. Asghari Sana, M. Capkin Yurtsever, G. Kaynak Bayrak, E. O. Tuncay, A. S. Kiremitci, and M. Gumusderelioglu, "Spreading, proliferation and differentiation of human dental pulp stem cells on chitosan scaffolds immobilized with RGD or fibronectin," Cytotechnology, vol. 69, no. 4, pp. 617-630, Aug 2017, doi: 10.1007/s10616-017-0072-9.
42. [42] L. Leclere et al., "Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells," PLoS One, vol. 10, no. 3, p. e0115831, 2015, doi: 10.1371/journal.pone.0115831.
43. [43] V. Hawach, Abdel-Massih, R. M., Boujaoude, M., "The Cytotoxic and Anti-proliferative Activity of High Molecular Weight Pectin and Modified Citrus Pectin," Functional Foods in Health and Disease, vol. 6, pp. 587-601, 2016.
44. [44] M. Kressel and P. Groscurth, "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA," (in eng), Cell Tissue Res, vol. 278, no. 3, pp. 549-56, Dec 1994, doi: 10.1007/bf00331373.
45. [45] H. Inohara and A. Raz, "Effects of natural complex carbohydrate (citrus pectin) on murine melanoma cell properties related to galectin-3 functions," Glycoconj J, vol. 11, no. 6, pp. 527-32, Dec 1994, doi: 10.1007/BF00731303.
46. [46] C. L. Jackson et al., "Pectin induces apoptosis in human prostate cancer cells: correlation of apoptotic function with pectin structure," Glycobiology, vol. 17, no. 8, pp. 805-19, Aug 2007, doi: 10.1093/glycob/cwm054.
47. [47] F. O. Ogutu, T. H. Mu, H. Sun, and M. Zhang, "Ultrasonic Modified Sweet Potato Pectin Induces Apoptosis like Cell Death in Colon Cancer (HT-29) Cell Line," Nutr Cancer, vol. 70, no. 1, pp. 136-145, Jan 2018, doi: 10.1080/01635581.2018.1406123.
48. [48] L. Leclere, P. V. Cutsem, and C. Michiels, "Anti-cancer activities of pH- or heat-modified pectin," Front Pharmacol, vol. 4, p. 128, Oct 8 2013, doi: 10.3389/fphar.2013.00128.
49. [49] W. Zhang, P. Xu, and H. Zhang, "Pectin in cancer therapy: A review," Trends in Food Science & Technology, vol. 44, no. 2, pp. 258-271, 2015, doi: 10.1016/j.tifs.2015.04.001.