The osteogenesis of bacterial cellulose scaffold loaded with fisetin

Document Type: Original Article


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Research Center for Animal Development and Applied Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

3 Biotechnology Research Center, Institute of Pharmaceutical Technology, Mashhad University of Medical Sciences, Mashhad, Iran

4 School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Bacterial cellulose (BC) has applications in medical science, it is easily synthesized, economic and purer compared to plant cellulose. The present study aimed to evaluate BC, a biocompatible natural polymer, as a scaffold for the bone marrow mesenchymal stem cells (BMSCs) loaded with fisetin, a phytoestrogen.
Materials and Methods: BC hydrogel scaffold was prepared from Gluconaceter xylinus and characterized through scanning electron microscopy (SEM). Biocompatibility of BC was measured by MTT assay, BMSCs were obtained from femur of rat and the osteogenic potential of the BC scaffold cultured with BMSCs and loaded with fisetin, was investigated by measuring the alkaline phosphatase (ALP) activity, alizarin red staining (ARS) and real-time PCR in terms of osteoblast-specific marker, osteocalcin (OCN) and osteopontin (OPN).
Results: Biocompatibility results did not show any toxic effects of BC scaffold on BMSCs, while it increased cell viability. The data showed that BC loaded fisetin differentiated BMSCs into osteoblasts as demonstrated by ALP activity assays and ARS in vitro. Moreover, results from gene expression assay showed the expression of OCN and OPN genes was increased in cells that were seeded on the BC scaffold loaded with fisetin.
Conclusion: According to the results of the present study, BC loaded with fisetin is an effective strategy to promote osteogenic differentiation and a proper localized delivery system, which could be a potential candidate in bone tissue engineering.


Main Subjects

1. Yang L, Tsang KY, Tang HC, Chan D, Cheah KS. Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation. Proc Natl Acad Sci USA 2014; 111: 97-121.
2.  Mollazadeh S,  Fazly Bazzaz  BS, Kerachian MA. Role of apoptosis in pathogenesis and treatment of bone-related diseases. J Orthop Surg Res 2015; 10:1-7.
3. Gareta E, Coathup MJ, Blunn GW. Osteoinduction of bone grafting materials for bone repair and regeneration. Bone 2015; 81:112-123.
4. Li X, Wang L, Fan Y, Feng Q, Cui FZ, Watari F. Nanostructured scaffolds for bone tissue engineering. J Biomed Mater Res A 2013; 101: 2424-2435.
5. Huang Y, Zhu C, Yang J, Nie Y, Chen C, Sun D. Recent advances in bacterial cellulose.  Cellulose 2014; 21: 1-30.
6. Khan S,  Ul-Islam M,  Ullah MW,  Ikram M, Subhan F, Kim Y. Engineered regenerated bacterial cellulose scaffolds for application in in vitro tissue regeneration. RSC Adv 2015; 5: 84565–84573.
7. Keshk SM. Bacterial Cellulose Production and its Industrial Applications.  J Bioproces Biotechniq 2014; 4: 150-160.
8. Zaborowska M, Bodin A, Bäckdahl H, Popp J, Goldstein A, Gatenholm P. Microporous bacterial cellulose as a potential scaffold for bone regeneration. Acta Biomater 2010; 6: 2540-2547.
9. Granero-Molto F, Weis JA, Longobardi L, Spagnoli A. Role of mesenchymal stem cells in regenerative medicine: application to bone and cartilage repair. Expert Opin Biol Ther 2008; 8: 255-268.
10. Saeed H, Ahsan M, Saleem Z, Iqtedar M, Islam M, Danish Z, et al. Mesenchymal stem cells (MSCs) as skeletal therapeutics - an update. J Biomed Sci 2016; 16:23-41.
11. Jensen E, Gopalakrishnan R, Westendorf J. Regulation of Gene Expression in Osteoblasts. Biofactors 2010; 36: 25–32.
12. Cintron D, Rodriguez-Gutierrez R, Serrano V, Latortue-Albino P, Erwin PJ, Murad MH. Effect of estrogen replacement therapy on bone and cardiovascular outcomes in women with turner syndrome: a systematic review and meta-analysis. Endocrine 2017; 55: 366-375.
13. Jia M, Dahlman-Wright K, Gustafsson J. Estrogen receptor alpha and beta in health and disease. Best Pract Res Clin Endocrinol Metab 2015; 29: 557–568.
14. Moreira AC, Silva AM, Santos MS, Sardao VA. Phytoestrogens as alternative hormone replacement therapy in menopause: What is real, what is unknown. J Steroid Biochem Mol Biol 2014; 143: 61-71.
15. Lagari VS, Levis S. Phytoestrogens in the Prevention of Postmenopausal Bone Loss. J Clin Densitom 2013; 16: 445-449.
16. Chen X, Uzuner U, Li M, Shi W, Yuan JS, Dai SY. Phytoestrogens and mycoestrogens induce signature structure dynamics changes on estrogen receptor α. Int J Environ Res Public Health 2016; 13: 869-883.
17. Pal HC, Diamond AC,  Strickland LR, Kappes JC, Katiyar SK, Elmets CA, Athar M. Fisetin, a dietary flavonoid, augments the anti-invasive and anti-metastatic potential of sorafenib in melanoma. Oncotarget 2016; 7: 1227-1241.
18.  Kazemi F, Azin M, Ashori A. Production of bacterial cellulose using different carbon sources and culture media. Carbohydr Polym 2015; 117: 518–523.
19. Rahmanifar F, Tamadon A, Mehrabani D, Zare S, Abasi S, Keshavarz S. Histomorphometric evaluation of treatment of rat azoosper-mic seminiferous tubules by allotransplantation of bone marrow-derived mesenchymal stem cells. Iran J Basic Med Sci 2016; 19: 653-661.
20. Mohammadian M, Boskabady M, Ragerdi I, Jahromi P, Omidi A, Nejad A. Effect of bone marrow derived mesenchymal stem cells on lung pathology and inflammation in ovalbumin‐induced asthma in mouse.Iran J Basic Med Sci 2016; 19:  55‐63.
21. Vidyasekar P, Shyamsunder P,  Sahoo S, Verma R. Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells. In Vitro Cell Dev Biol Anim. 2016; 52: 204-217.
22. Khalaj Z, Sahebghadam Lotfi A, Kabir-Salmani M. Laminin matrix promotes hepatogenic terminal differentiation of human bone marrow mesenchymal stem cells. Iran J Basic Med Sci 2016; 19: 34-42.
23. Yaghoobi M, Hashemi-Najafabadi S, Soleimani M, Vasheghani-Farahani E, Mousavi SM. Osteogenic Differentiation and Mineralization on Compact Multilayer nHA-PCL Electrospun Scaffolds in a Perfusion Bioreactor. Iran J Biotech 2016; 14: 41-49.
24.Faia-Torres, A, Guimond-Lischer S, Rottmar M, Charnley M, Goren T, Maniura-Weber K, Neves N M. Differential regulation of osteogenic differentiation of stem cells on surface roughness gradients. Biomaterials 2014; 35: 9023–9032.
25. Birmingham E, Niebur GL, McHugh PE, Shaw G, Barry FP, McNamara LM. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche. Eur Cells Mater 2012; 12: 13-27.
26. Wang X, Ding B , Li B. Biomimetic electrospun nanofibrous structures for tissue engineering. Mater Today 2013; 16(6): 229-241.
27. Thibault RA, Mikos AG, Kasper FK. Scaffold/Extracellular Matrix Hybrid Constructs for Bone-Tissue Engineering. Adv Healthc Mater 2013; 2: 13-24.
28. Rajwade JM, Paknikar KM, Kumbhar JV. Applications of bacterial cellulose and its composites in biomedicine. Appl Microbiol Biotechnol 2015; 99: 2491-2511.
29. Castilho M, Rodrigues G, Pires I, Gouveia B, Pereira M, Moseke C. Fabrication of individual alginate-TCP scaffolds for bone tissue engineering by means of powder printing. Biofabrication 2015; 7: 1351-1362.
30. Geisel N, Clasohm J, Shi X, Lamboni L, Yang J, Mattern K, Yang G . Microstructured multilevel bacterial cellulose allows the guided growth of neural stem cells Small 2016; 12: 5407-5413.
31. Holzwarth JM, Ma PX. Biomimetic nanofibrous scaffolds for bone tissue engineering. Biomaterials. 2011; 32: 9622-9629.
32. Ullah H, Santos HA, Khan, T. Applications of bacterial cellulose in food, cosmetics and drug delivery. Cellulose 2016; 23: 2291–2314.
33. Granero-Molto F, Weis J.A, Longobardi L, Spagnoli A. Role of mesenchymal stem cells in regenerative medicine: application to bone and cartilage repair. Expert Opin Biol Ther 2008; 8: 255-268.
34. Lall RK, Adhami VM, Mukhtar H. Dietary flavonoid fisetin for cancer prevention and treatment. Mol Nutr Food Res 2016; 60: 1396-1405.
35. Schilling T, Ebert R, Raaijmakers N, Schütze N, Jakob F. Effects of phytoestrogens and other plant-derived compounds on mesenchymal stem cells, bone maintenance and regeneration. Steroid Biochem Mol Biol 2014; 139: 252-261.
36. Casarin RC, Casati MZ, Pimentel SP, Cirano FR, Algayer M, Pires PR, et al. Resveratrol improves bone repair by modulation of bone morphogenetic proteins and osteopontin gene expression in rats. Int J Oral Maxillofac Surg 2014; 43: 900-906.
37. Mollazadeh S, Neshati V, Fazly Bazzaz BS, Iranshahi M, Mojarrad M, Naderi-Meshkin H, Kerachian MA. Standardized Sophora pachycarpa root extract enhances osteogenic differentiation in adipose-derived human mesenchymal stem cells. Phytother Res 2017; 31: 792-800.
38. Golub EE, Boesze-Battaglia K. The role of alkaline phosphatase in mineralization. Curr. Opin. Orthop 2007; 18: 444–448.
39. Gregory C, Gunn W, Peister A, Prockop D. An Alizarin red-based assay of mineralization by adherent cells in culture: Comparison with cetylpyridinium chloride extraction. Anal. Biochem 2004; 329: 77–84.
40.Wang C, Meng M, Tang X, Chen K, Zhang L, Liu , et al. The proliferation, differentiation, and mineralization effects of puerarin on osteoblasts in vitro. CJNM 2014; 12: 436-442.
41. Beederman M, Lamplot J, Nan G, Wang J, Liu X, Yin L. BMP signaling in mesenchymal stem cell differentiation and bone formation. J Biomed Sci Eng 2013; 6: 32–52.
42. Tazi N, Zhang Z, Messaddeq Y, Lopes  L, Zanardi L, Levinson D, Rouabhia M. Hydroxyapatite bioactivated bacterial cellulose promotes osteoblast growth and the formation of bone nodule. AMB Express 2012; 2: 61-71.
43. Favi P, Benson R, Neilsen N, Hammonds R, Bates C, Stephens C. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds. Mater Sci Eng C Mater Biol Appl 2013; 33: 1935-1944.
44. Qin L, Yao D, Zheng L, Liu W, Liu Z, Lei M. Phytomolecule icaritin incorporated PLGA/TCP scaffold for steroidassociated osteonecrosis: Proof-of-concept for prevention of hip joint collapse in bipedal emus and mechanistic study in quadrupedal rabbits. Biomaterials 2015; 59: 125-143.
45. Kamath M, Ahmed S, Dhanasekaran M, santosh S. Polycaprolactone scaffold engineered for sustained release of resveratrol: therapeutic enhancement in bone tissue engineering. Int J Nanomedicine 2014; 9: 183–195.
46. Dosier C, Erdman C, Park J, Schwartz Z, Boyana B, Guldberg R. Resveratrol effect on osteogenic differentiation of rat and human adipose derived stem cells in a 3-D culture environment. J Mech Behav Biomed Mater 2011; 11: 112-122.