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Novel 3D-printed methacrylated chitosan-laponite nanosilicate composite scaffolds enhance cell growth and biomineral formation in MC3T3 pre-osteoblasts

  • Tugba Cebe (a1), Neelam Ahuja (a2), Felipe Monte (a1), Kamal Awad (a3), Kimaya Vyavhare (a1), Pranesh Aswath (a1), Jian Huang (a2), Marco Brotto (a2) and Venu Varanasi (a4)...


This study compared the effect of gelatin- and chitosan-based scaffolds on osteoblast biomineralization. These scaffolds have been modified using methacrylate and laponite nanosilicates to improve their mechanical strength and support osteoblast function. Scaffold materials were prepared to have the same compressive strength (14–15 MPa) such that differences in cell response would be isolated to differences in biopolymer chemistry. The materials were tested for rheological properties to optimize the bio-ink for successful 3D printing using a robocast-assisted deposition system. Osteoblasts were cultured on the surface of 3D-printed methacrylated chitosan-laponite (MAC-Lp), methacrylated gelatin-laponite (MAG-Lp), MAC, and MAG scaffolds. MAC-Lp scaffolds showed increased cell viability, cell growth, and biomineral formation as compared to MAG-Lp scaffolds. FTIR results showed the presence of higher biomineral phosphate and extracellular matrix (ECM) collagen-like amide formation on MAC-Lp scaffolds as compared to MAG-Lp scaffolds. MAC-Lp scaffolds showed increased density of ECM-like tissue from SEM analysis, stained mineral nodules from Alizarin staining, and the existence of Ca–P species evident by X-ray absorbance near edge structure analysis. In conclusion, MAC-Lp scaffolds enhanced osteoblast growth and biomineral formation as compared to MAG-Lp scaffolds.


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1.Saxena, S., Ray, A., Kapil, A., Pavon Djavid, G., Letourneur, D., Gupta, B., and Meddahi-Pellé, A.: Development of a new polypropylene-based suture: Plasma grafting, surface treatment, characterization, and biocompatibility studies. Macromolecular bioscience. 11, 373 (2011).
2.Chiarello, E., Cadossi, M., Tedesco, G., Capra, P., Calamelli, C., Shehu, A., and Giannini, S.: Autograft, allograft and bone substitutes in reconstructive orthopedic surgery. Aging: Clin. Exp. Res. 25, 101 (2013).
3.Grove, J.R.: Autograft, allograft and xenograft options in the treatment of neglected Achilles tendon ruptures: A historical review with illustration of surgical repair. Surgeon. 15, 47 (2008).
4.Peppas, N.A. and Langer, R.: New challenges in biomaterials. Science 263, 1715 (1994).
5.Censi, R., Schuurman, W., Malda, J., di Dato, G., Burgisser, P.E., Dhert, W.J.A., van Nostrum, C.F., di Martino, P., Vermonden, T., and Hennink, W.E.: A printable photopolymerizable thermosensitive p(HPMAm-lactate)-PEG hydrogel for tissue engineering. Adv. Funct. Mater. 21, 1833 (2011).
6.Wangtueai, S. and Noomhorm, A.: Processing optimization and characterization of gelatin from lizardfish (Saurida spp.) scales. LWT–Food Sci. Technol. 42, 825 (2009).
7.Schwenke, K.D.: The Science and Technology of Gelatin. Herausgegeben von A. G. Ward u. A. Courts, XVI und 564 Seiten mit zahlreichen Abb. u. Tab., Academic Press London, New York, San Francisco 1977. Preis: 18,00 £; 39,50 $. Food/Nahrung. 22, 444 (1978).
8.Maurer, P.H.: II. Antigenicity of gelatin in rabbits and other species. J. Exp. Med. 100, 515 (1954).
9.Zhu, J. and Marchant, R.: Design properties of hydrogel tissue-engineering scaffolds. Expert Rev. Med. Devices 8, 607 (2011).
10.Haraguchi, K. and Li, H-J.: Mechanical properties and structure of polymer−clay nanocomposite gels with high clay content. Macromolecules 39, 1898 (2006).
11.Tian, W.M., Hou, S.P., Ma, J., Zhang, C.L., Xu, Q.Y., Lee, I.S., Li, H.D., Spector, M., and Cui, F.Z.: Hyaluronic acid–poly-D-lysine-based three-dimensional hydrogel for traumatic brain injury. Tissue Eng. 11, 513 (2005).
12.Clark, A.H., Richardson, R.K., Ross-Murphy, S.B., and Stubbs, J.M.: Structural and mechanical properties of agar/gelatin co-gels. Small-deformation studies. Macromolecules 16, 1367 (1983).
13.Yasuda, K., Ping Gong, J., Katsuyama, Y., Nakayama, A., Tanabe, Y., Kondo, E., Ueno, M., and Osada, Y.: Biomechanical properties of high-toughness double network hydrogels. Biomaterials 26, 4468 (2005).
14.Iyer, P., Walker, K., and Madihally, S.: Increased matrix synthesis by fibroblasts with decreased proliferation on synthetic chitosan-gelatin porous structures. Biotechnol. Bioeng. 109, 1314 (2012).
15.Shigemasa, Y., Saito, K., Sashiwa, H., and Saimoto, H.: Enzymatic degradation of chitins and partially deacetylated chitins. Int. J. Biol. Macromol. 16, 43 (1994).
16.Khor, E. and Lim, L.: Implantable applications of chitin and chitosan. Biomaterials 24, 2339 (2003).
17.Madihally, S.V. and Matthew, H.W.T.: Porous chitosan scaffolds for tissue engineering. Biomaterials 20, 1133 (1999).
18.Suh, J.K. and Matthew, H.W.: Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: A review. Biomaterials 21, 2589 (2000).
19.Mi, F-L., Tan, Y-C., Liang, H-F., and Sung, H-W.: In vivo biocompatibility and degradability of a novel injectable-chitosan-based implant. Biomaterials 23, 181 (2002).
20.Aiba, S., Minoura, N., Taguchi, K., and Fujiwara, Y.: Covalent immobilization of chitosan derivatives onto polymeric film surfaces with the use of a photosensitive hetero-bifunctional crosslinking reagent. Biomaterials 8, 481 (1987).
21.Cai, K., Yao, K., Cui, Y., Lin, S., Yang, Z., Li, X., Xie, H., Qing, T., and Luo, J.: Surface modification of poly(D,L-lactic acid) with chitosan and its effects on the culture of osteoblasts in vitro. J. Biomed. Mater. Res. 60, 398 (2002).
22.Lahiji, A., Sohrabi, A., Hungerford, D.S., and Frondoza, C.G.: Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. J. Biomed. Mater. Res. 51, 586 (2000).
23.Arpornmaeklong, P., Pripatnanont, P., and Suwatwirote, N.: Properties of chitosan-collagen sponges and osteogenic differentiation of rat-bone-marrow stromal cells. Int. J. Oral Maxillofac. Surg. 37, 357 (2008).
24.Li, B., Wang, Y., Jia, D., and Zhou, Y.: Gradient structural bone-like apatite induced by chitosan hydrogel via ion assembly. J. Biomater. Sci., Polym. Ed. 22, 505 (2011).
25.Jiang, T., Abdel Fattah, W., and Laurencin, C.: In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering. Biomaterials 27, 4894 (2006).
26.Kim, I-Y., Seo, S-J., Moon, H-S., Yoo, M-K., Park, I-Y., Kim, B-C., and Cho, C-S.: Chitosan and its derivatives for tissue engineering applications. Biotechnol. Adv. 26, 1 (2008).
27.Harward, M.E.: An introduction to clay colloid chemistry. For clay technologists, geologists, and soil scientists. H. van Olphen, ed. Interscience (Wiley), New York, 1963. xvi + 301 pp. Illus. $10. Science 143, 1023 (1964).
28.Wu, C-J., Gaharwar, A.K., Schexnailder, P.J., and Schmidt, G.: Development of biomedical polymer-silicate nanocomposites: A materials science perspective. Materials 3, 2986 (2010).
29.Bordes, P., Pollet, E., and Avérous, L.: Nano-biocomposites: Biodegradable polyester/nanoclay systems. Prog. Polym. Sci. 34, 125 (2009).
30.Xavier, J., Thakur, T., Desai, P., Jaiswal, M., Sears, N., Cosgriff Hernandez, E., Kaunas, R., and Gaharwar, A.: Bioactive nanoengineered hydrogels for bone tissue engineering: A growth-factor-free approach. ACS Nano 9, 3109 (2015).
31.Gaharwar, A., Mihaila, S., Swami, A., Patel, A., Sant, S., Reis, R., Marques, A., Gomes, M., and Khademhosseini, A.: Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells. Adv. Mater. 25, 3329 (2013).
32.Cesarano III, J. and Calvert, P. D.: Freeforming objects with low-binder slurry. U.S. Patent 6, 326 (2000).
33.Lewis, J.A., Smay, J.E., Stuecker, J., and Cesarano, J.: Direct ink writing of three-dimensional ceramic structures. J. Am. Ceram. Soc. 89, 3599 (2006).
34.Nichol, J., Koshy, S., Bae, H., Hwang, C., Yamanlar, S., and Khademhosseini, A.: Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 31, 5536 (2010).
35.Lu, S., Song, X., Cao, D., Chen, Y., and Yao, K.: Preparation of water-soluble chitosan. J. Appl. Polym. Sci. 91, 3497 (2004).
36.Varanasi, V.G., Saiz, E., Loomer, P.M., Ancheta, B., Uritani, N., Ho, S.P., Tomsia, A.P., Marshall, S.J., and Marshall, G.W.: Enhanced osteocalcin expression by osteoblast-like cells (MC3T3-E1) exposed to bioactive coating glass (SiO2–CaO–P2O5–MgO–K2O–Na2O system) ions. Acta Biomater. 5, 3536 (2009).
37.Demirkiran, H., Hu, Y., Zuin, L., Appathurai, N., and Aswath, P.B.: XANES analysis of calcium and sodium phosphates and silicates and hydroxyapatite–Bioglass®45S5 co-sintered bioceramics. Mater. Sci. Eng., C 31, 134 (2011).
38.Rajendran, J., Gialanella, S., and Aswath, P.B.: XANES analysis of dried and calcined bones. Mater. Sci. Eng., C 33, 3968 (2013).
39.Aruwajoye, O.O., Kim, H.K., and Aswath, P.B.: Bone apatite composition of necrotic trabecular bone in the femoral head of immature piglets. Calcif. Tissue Int. 96, 324 (2015).
40.Kruse, J., Leinweber, P., Eckhardt, K-U., Godlinski, F., Hu, Y., and Zuin, L.: Phosphorus L2, 3-edge XANES: Overview of reference compounds. J. Synchrotron Radiat. 16, 247 (2009).
41.Mano, N., Mao, F., and Heller, A.: Characteristics of a miniature compartment-less glucose-O2 biofuel cell and its operation in a living plant. J. Am. Chem. Soc. 125, 6588 (2003).
42.Ravenelle, F., and Rahmouni, M.: Contramid®: High-amylose starch for controlled drug delivery. Polysaccharides for drug delivery and pharmaceutical applications 934, 79104 (2006).
43.Darder, A., Baltodano, M., and Torres, R.D.: Critical pedagogy: An introduction. In: The Critical Pedagogy Reader (Routledge, Abingdon, United Kingdom, 2003), pp. 121.
44.Saraiva, S.M., Miguel, S.P., Ribeiro, M.P., Coutinho, P., and Correia, I.J.: Synthesis and characterization of a photocrosslinkable chitosan-gelatin hydrogel aimed for tissue regeneration. RSC Adv. 5, 63478 (2015).
45.Han, J., Lei, T., and Wu, Q.: High-water-content mouldable polyvinyl alcohol-borax hydrogels reinforced by well-dispersed cellulose nanoparticles: Dynamic rheological properties and hydrogel formation mechanism. Carbohydr. Polym. 102, 306 (2014).
46.Shen, M., Li, L., Sun, Y., Xu, J., Guo, X., and Prud’homme, R.K.: Rheology and adhesion of poly(acrylic acid)/laponite nanocomposite hydrogels as biocompatible adhesives. Langmuir 30, 1636 (2014).
47.Gaharwar, A., Kishore, V., Rivera, C., Bullock, W., Wu, C-J., Akkus, O., and Schmidt, G.: Physically crosslinked nanocomposites from silicate-crosslinked PEO: Mechanical properties and osteogenic differentiation of human mesenchymal stem cells. Macromolecular bioscience. 12, 779 (2012).
48.Peter, M., Ganesh, N., Selvamurugan, N., Nair, S.V., Furuike, T., Tamura, H., and Jayakumar, R.: Preparation and characterization of chitosan–gelatin/nanohydroxyapatite composite scaffolds for tissue engineering applications. Carbohydr. Polym. 80, 687 (2010).
49.Pleshko, N., Boskey, A., and Mendelsohn, R.: Novel infrared spectroscopic method for the determination of crystallinity of hydroxyapatite minerals. Biophys. J. 60, 786 (1991).
50.Chen, P-Y. and McKittrick, J.: Compressive mechanical properties of demineralized and deproteinized cancellous bone. J. Mech. Behav. Biomed. Mater. 4, 961 (2011).
51.Xiaoyu, Z., Payal, B., Melissa, O., and Zanello, L.: 1α,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival. J. Steroid Biochem. Mol. Biol. 103, 457 (2007).
52.Gaharwar, A., Schexnailder, P., Jin, Q., Wu, C-J., and Schmidt, G.: Addition of chitosan to silicate cross-linked PEO for tuning osteoblast cell adhesion and mineralization. ACS Appl. Mater. Interfaces 2, 3119 (2010).
53.Reffitt, D.M., Ogston, N., Jugdaohsingh, R., Cheung, H.F.J., Evans, B.A.J., Thompson, R.P.H., Powell, J.J., and Hampson, G.N.: Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro. Bone 32, 127 (2003).
54.Varanasi, V., Owyoung, J., Saiz, E., Marshall, S., Marshall, G., and Loomer, P.: The ionic products of bioactive glass particle dissolution enhance periodontal ligament fibroblast osteocalcin expression and enhance early mineralized tissue development. J. Biomed. Mater. Res., Part A 98, 177 (2011).
55.Hoppe, A., Güldal, N.S., and Boccaccini, A.: A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 32, 2757 (2011).
56.Maehira, F., Iinuma, Y., Eguchi, Y., Miyagi, I., and Teruya, S.: Effects of soluble silicon compound and deep-sea water on biochemical and mechanical properties of bone and the related gene expression in mice. J. Bone Miner. Metab. 26, 446 (2008).
57.Izu, A., Kumai, T., Tohno, Y., Tohno, S., Minami, T., Yamada, G., and Yamada, M-O.: Silicon intake to vertebral columns of mice after dietary supply. Biol. Trace Elem. Res. 113, 297 (2006).
58.Yang Huixia, H.S., Wenbo, W., and Aiqin, W.: Composite hydrogel beads based on chitosan and laponite: Preparation, swelling, and drug release behaviour. Iran. Polym. J. 20, 479–490 (2011).
59.Murphy, C.M., Haugh, M.G., and O’Brien, F.J.: The effect of mean pore size on cell attachment, proliferation and migration in collagen–glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials 31, 461 (2010).
60.Raja, I.S. and Fathima, N.N.: Porosity and dielectric properties as tools to predict drug release trends from hydrogels. SpringerPlus 3, 393 (2014).


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Novel 3D-printed methacrylated chitosan-laponite nanosilicate composite scaffolds enhance cell growth and biomineral formation in MC3T3 pre-osteoblasts

  • Tugba Cebe (a1), Neelam Ahuja (a2), Felipe Monte (a1), Kamal Awad (a3), Kimaya Vyavhare (a1), Pranesh Aswath (a1), Jian Huang (a2), Marco Brotto (a2) and Venu Varanasi (a4)...


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