BMTBiomaterials TranslationalTBA2096-112XBiomaterials Translational10.3877/cma.j.issn.2096-112X.2020.01.009Research ArticleHyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic ratshttps://artdesignp.com/journal/BMT/1/1/10.3877/cma.j.issn.2096-112X.2020.01.009YuanJishan,MaturavongsaditPanita,ZhouZhihui,LvBin,LinYuan,YangJia,Amie LuckanagulJittima2020112020-12-28Tobacco mosaic virus (TMV) has been studied as a multi-functional agent for bone tissue engineering. An osteo-inductive effect of wild-type TMV has been reported, as it can significantly enhance the bone differentiation potential of bone marrow stromal cells both on a two-dimensional substrate and in a three-dimensional (3D) hydrogel system. A TMV mutant (TMV-RGD1) was created which featured the adhesion peptide arginyl-glycyl-aspartic acid (RGD), the most common peptide motif responsible for cell adhesion to the extracellular matrix, on the surface of the virus particle to enhance the bio-functionality of the scaffold material. We hypothesised that the incorporation of either wild-type TMV or TMV-RGD1 in the 3D hydrogel scaffold would induce bone healing in critical size defects of the cranial segmental bone. We have previously tested the virus-functionalised scaffolds, in vitro, with a hyaluronic acid-based system as an in-situ hydrogel platform for 3D cell encapsulation, culture, and differentiation. The results of these experiments suggested the potential of the virus-functionalised hydrogel to promote in vitro stem cell differentiation. The hydrogel-forming system we employed was shown to be safe and biocompatible in vivo. Here, we further explored the physiological responses regarding bone regeneration of a calvarial defect in both normal and osteoporotic ovariectomized rat models. Our results, based on histological analysis in both animal models, suggested that both wild-type TMV and TMV-RGD1 functionalised hydrogels could accelerate bone regeneration, without systemic toxicity, evaluated by blood counts. New bone formation was intensified by the incorporation of the RGD-mutant viral particles. This finding increased the potential for use of the rod-shaped plant virus as a platform for the addition of powerful biofunctionality for tissue engineering applications. This study was approved by the Ethics Committee on Animal Use of the Zhenjiang Affiliated First People’s Hospital affiliated to Jiangsu University.bone regeneration, calvarial defect, hydrogel, osteoporosis, tobacco mosaic virus

1. Kaur, G.; Valarmathi, M. T.; Potts, J. D.; Jabbari, E.; Sabo-Attwood, T.; Wang, Q. Regulation of osteogenic differentiation of rat bone marrow stromal cells on 2D nanorod substrates. Biomaterials. 2010, 31, 1732-1741.2. Kaur, G.; Wang, C.; Sun, J.; Wang, Q. The synergistic effects of multivalent ligand display and nanotopography on osteogenic differentiation of rat bone marrow stem cells. Biomaterials. 2010, 31, 5813-5824.3. Maturavongsadit, P.; Luckanagul, J. A.; Metavarayuth, K.; Zhao, X.; Chen, L.; Lin, Y.; Wang, Q. Promotion of in vitro chondrogenesis of mesenchymal stem cells using in situ hyaluronic hydrogel functionalized with rod-like viral nanoparticles. Biomacromolecules. 2016, 17, 1930-1938.4. Metavarayuth, K.; Sitasuwan, P.; Luckanagul, J. A.; Feng, S.; Wang, Q. Virus nanoparticles mediated osteogenic differentiation of bone derived mesenchymal stem cells. Adv Sci (Weinh). 2015, 2, 1500026.5. Sitasuwan, P.; Lee, L. A.; Bo, P.; Davis, E. N.; Lin, Y.; Wang, Q. A plant virus substrate induces early upregulation of BMP2 for rapid bone formation. Integr Biol (Camb). 2012, 4, 651-660.6. Zhao, X.; Lin, Y.; Wang, Q. Virus-based scaffolds for tissue engineering applications. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2015, 7, 534-547.7. Nguyen, H. G.; Metavarayuth, K.; Wang, Q. Upregulation of osteogenesis of mesenchymal stem cells with virus-based thin films. Nanotheranostics. 2018, 2, 42-58.8. Griffith, L. G.; Naughton, G. Tissue engineering--current challenges and expanding opportunities. Science. 2002, 295, 1009-1014.9. Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Microscale technologies for tissue engineering and biology. Proc Natl Acad Sci U S A. 2006, 103, 2480-2487.10. Khademhosseini, A.; Vacanti, J. P.; Langer, R. Progress in tissue engineering. Sci Am. 2009, 300, 64-71.11. Khademhosseini, A.; Langer, R. Microengineered hydrogels for tissue engineering. Biomaterials. 2007, 28, 5087-5092.12. Zhu, J. Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering. Biomaterials. 2010, 31, 4639-4656.13. Bichara, D. A.; Zhao, X.; Bodugoz-Senturk, H.; Ballyns, F. P.; Oral, E.; Randolph, M. A.; Bonassar, L. J.; Gill, T. J.; Muratoglu, O. K. Porous poly(vinyl alcohol)-hydrogel matrix-engineered biosynthetic cartilage. Tissue Eng Part A. 2011, 17, 301-309.14. Elisseeff, J. Injectable cartilage tissue engineering. Expert Opin Biol Ther. 2004, 4, 1849-1859.15. Aldaye, F. A.; Senapedis, W. T.; Silver, P. A.; Way, J. C. A structurally tunable DNA-based extracellular matrix. J Am Chem Soc. 2010, 132, 14727-14729.16. Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science. 2009, 324, 59-63.17. Luckanagul, J.; Lee, L. A.; Nguyen, Q. L.; Sitasuwan, P.; Yang, X.; Shazly, T.; Wang, Q. Porous alginate hydrogel functionalized with virus as three-dimensional scaffolds for bone differentiation. Biomacromolecules. 2012, 13, 3949-3958.18. Luckanagul, J. A.; Lee, L. A.; You, S.; Yang, X.; Wang, Q. Plant virus incorporated hydrogels as scaffolds for tissue engineering possess low immunogenicity in vivo. J Biomed Mater Res A. 2015, 103, 887-895.19. Luckanagul, J. A.; Metavarayuth, K.; Feng, S.; Maneesaay, P.; Clark, A. Y.; Yang, X.; García, A. J.; Wang, Q. Tobacco mosaic virus functionalized alginate hydrogel scaffolds for bone regeneration in rats with cranial defect. ACS Biomater Sci Eng. 2016, 2, 606-615.20. Mahajan, H. S.; Gattani, S. In situ gels of Metoclopramide Hydrochloride for intranasal delivery: in vitro evaluation and in vivo pharmacokinetic study in rabbits. Drug Deliv. 2010, 17, 19-27.21. Burdick, J. A.; Prestwich, G. D. Hyaluronic acid hydrogels for biomedical applications. Adv Mater. 2011, 23, H41-56.22. Maturavongsadit, P.; Bi, X.; Metavarayuth, K.; Luckanagul, J. A.; Wang, Q. Influence of cross-linkers on the in vitro chondrogenesis of mesenchymal stem cells in hyaluronic acid hydrogels. ACS Appl Mater Interfaces. 2017, 9, 3318-3329.23. Ananthanarayanan, B.; Kim, Y.; Kumar, S. Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform. Biomaterials. 2011, 32, 7913-7923.24. Jakob, F.; Ebert, R.; Ignatius, A.; Matsushita, T.; Watanabe, Y.; Groll, J.; Walles, H. Bone tissue engineering in osteoporosis. Maturitas. 2013, 75, 118-124.25. Lelovas, P. P.; Xanthos, T. T.; Thoma, S. E.; Lyritis, G. P.; Dontas, I. A. The laboratory rat as an animal model for osteoporosis research. Comp Med. 2008, 58, 424-430.26. Wronski, T. J.; Cintrón, M.; Dann, L. M. Temporal relationship between bone loss and increased bone turnover in ovariectomized rats. Calcif Tissue Int. 1988, 43, 179-183.27. Wronski, T. J.; Dann, L. M.; Scott, K. S.; Cintrón, M. Long-term effects of ovariectomy and aging on the rat skeleton. Calcif Tissue Int. 1989, 45, 360-366.28. Xin, Z.; Jin, C.; Chao, L.; Zheng, Z.; Liehu, C.; Panpan, P.; Weizong, W.; Xiao, Z.; Qingjie, Z.; Honggang, H.; Longjuan, Q.; Xiao, C.; Jiacan, S. A matrine derivative M54 suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss by targeting ribosomal protein S5. Front Pharmacol. 2018, 9, 22.29. Zhou, L.; Liu, Q.; Yang, M.; Wang, T.; Yao, J.; Cheng, J.; Yuan, J.; Lin, X.; Zhao, J.; Tickner, J.; Xu, J. Dihydroartemisinin, an anti-malaria drug, suppresses estrogen deficiency-induced osteoporosis, osteoclast formation, and RANKL-induced signaling pathways. J Bone Miner Res. 2016, 31, 964-974.30. Mardas, N.; Stavropoulos, A.; Karring, T. Calvarial bone regeneration by a combination of natural anorganic bovine-derived hydroxyapatite matrix coupled with a synthetic cell-binding peptide (PepGen): an experimental study in rats. Clin Oral Implants Res. 2008, 19, 1010-1015.31. Durão, S. F.; Gomes, P. S.; Colaço, B. J.; Silva, J. C.; Fonseca, H. M.; Duarte, J. R.; Felino, A. C.; Fernandes, M. H. The biomaterial-mediated healing of critical size bone defects in the ovariectomized rat. Osteoporos Int. 2014, 25, 1535-1545.32. Namkung-Matthai, H.; Appleyard, R.; Jansen, J.; Hao Lin, J.; Maastricht, S.; Swain, M.; Mason, R. S.; Murrell, G. A.; Diwan, A. D.; Diamond, T. Osteoporosis influences the early period of fracture healing in a rat osteoporotic model. Bone. 2001, 28, 80-86.33. Hao, Y. J.; Zhang, G.; Wang, Y. S.; Qin, L.; Hung, W. Y.; Leung, K.; Pei, F. X. Changes of microstructure and mineralized tissue in the middle and late phase of osteoporotic fracture healing in rats. Bone. 2007, 41, 631-638.34. Oberg, S.; Johansson, C.; Rosenquist, J. B. Bone formation after implantation of autolysed antigen extracted allogeneic bone in ovariectomized rabbits. Int J Oral Maxillofac Surg. 2003, 32, 628-632.