Abstract
Tumor-originated and undefined extracellular matrices (ECMs) such as Matrigel™ have been widely used in three-dimensional (3D) cell and tissue culture, but their use is unacceptable in clinical cell therapies. In this study, we proposed a 3D cellulose nanofiber (CNF) hydrogel that has great potential as a defined tissue-engineering scaffold, especially for osteoblast culture. The CNF hydrogel showed attractive features as a cell scaffold material. It exhibited a ~ 1.4-fold higher diffusion coefficient (~ 2.98 × 10−7 cm2/s) of macromolecules such as bovine serum albumin than does Matrigel™ (< 2.2 × 10−7 cm2/s) due to the former’s higher porosity (> 95%) and pore size (~ 310.8 μm). Most pre-osteoblast cells that are encapsulated in the CNF hydrogel were immediately locked without sinking by instant hydrogen bond cross-linking between CNFs, whereas cells encapsulated in Matrigel™ sank to the bottom of the scaffold due to the slow sol–gel transition (> 20 min). The elastic modulus of the cell-encapsulated CNF hydrogel could be reinforced by further calcium-mediated cross-linking without cytotoxicity. As a result, the pre-osteoblast cells in the CNF hydrogels were homogeneously distributed in the 3D structure, proliferated for 3 weeks, and successfully differentiated. Overall, CNFs showed that it has potential to be used in tissue engineering as a defined ECM component.
Graphical abstract
Similar content being viewed by others
References
Abbott RD, Kaplan DL (2015) Strategies for improving the physiological relevance of human engineered tissues. Trends Biotechnol 33(7):401–407. https://doi.org/10.1016/j.tibtech.2015.04.003
Abe K, Iwamoto S, Yano H (2007) Obtaining cellulose nanofibers with a uniform width of 15 nm from wood. Biomacromol 8(10):3276–3278. https://doi.org/10.1021/bm700624p
Beniash E, Hartgerink JD, Storrie H, Stendahl JC, Stupp SI (2005) Self-assembling peptide amphiphile nanofiber matrices for cell entrapment. Acta Biomater 1(4):387–397. https://doi.org/10.1016/j.actbio.2005.04.002
Chatterjee K et al (2010) The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials 31(19):5051–5062. https://doi.org/10.1016/j.biomaterials.2010.03.024
Collins TJ (2007) ImageJ for microscopy. Biotechniques 43(1 Suppl):25–30. https://doi.org/10.2144/000112517
Dong H, Snyder JF, Williams KS, Andzelm JW (2013) Cation-induced hydrogels of cellulose nanofibrils with tunable moduli. Biomacromol 14(9):3338–3345. https://doi.org/10.1021/bm400993f
Dufresne A (2013) Nanocellulose: a new ageless bionanomaterial. Mater Today 16(6):220–227. https://doi.org/10.1016/j.mattod.2013.06.004
Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689. https://doi.org/10.1016/j.cell.2006.06.044
Evangelista MB et al (2007) Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix. Biomaterials 28(25):3644–3655. https://doi.org/10.1016/j.biomaterials.2007.04.028
Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy DC, Barlow SK, Langer R (1994) Biodegradable polymer scaffolds for tissue engineering. Nat Biotechnol 12(7):689–693. https://doi.org/10.1038/nbt0794-689
Fridman R et al (1991) Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel. J Natl Cancer Inst 83(11):769–774. https://doi.org/10.1093/jnci/83.11.769
Frith JE et al (2013) An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration. Biomaterials 34(37):9430–9440. https://doi.org/10.1016/j.biomaterials.2013.08.072
Grant DS, Kinsella JL, Kibbey MC, LaFlamme S, Burbelo PD, Goldstein AL, Kleinman HK (1995) Matrigel induces thymosin beta 4 gene in differentiating endothelial cells. J Cell Sci 108(12):3685–3694
Hachet E, Van den Berghe H, Bayma E, Block MR, Auzély-Velty R (2012) Design of biomimetic cell-interactive substrates using hyaluronic acid hydrogels with tunable mechanical properties. Biomacromol 13(6):1818–1827. https://doi.org/10.1021/bm300324m
Hsiong SX, Boontheekul T, Huebsch N, Mooney DJ (2008a) Cyclic arginine-glycine-aspartate peptides enhance three-dimensional stem cell osteogenic differentiation. Tissue Eng Part A 15(2):263–272. https://doi.org/10.1089/ten.tea.2007.0411
Hsiong SX, Carampin P, Kong HJ, Lee KY, Mooney DJ (2008b) Differentiation stage alters matrix control of stem cells. J Biomed Mater Res Part A 85(1):145–156. https://doi.org/10.1002/jbm.a.31521
Huang H, Ding Y, Sun XS, Nguyen TA (2013) Peptide hydrogelation and cell encapsulation for 3D culture of MCF-7 breast cancer cells. PLoS One 8(3):e59482. https://doi.org/10.1371/journal.pone.0059482
Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21:2529–2543
Jing J, Fournier A, Szarpak-Jankowska A, Block MR, Auzély-Velty R (2015) Type, density, and presentation of grafted adhesion peptides on polysaccharide-based hydrogels control preosteoblast behavior and differentiation. Biomacromol 16(3):715–722. https://doi.org/10.1021/bm501613u
Kang B-J, Ryu HH, Park S-S, Kim Y, Woo H-H, Kim WH, Kweon O-K (2012) Effect of matrigel on the osteogenic potential of canine adipose tissue-derived mesenchymal stem cells. J Vet Med Sci 74(7):827–836. https://doi.org/10.1292/jvms.11-0484
Karageorgiou V, Kaplan D (2005) Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26(27):5474–5491. https://doi.org/10.1016/j.biomaterials.2005.02.002
Kim HD, Valentini RF (2002) Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro. J Biomed Mater Res Part A 59(3):573–584. https://doi.org/10.1002/jbm.10011
Kim BJ, Kim S, Oh DX, Masic A, Cha HJ, Hwang DS (2015) Mussel-inspired adhesive protein-based electrospun nanofibers reinforced by Fe(III)–DOPA complexation. J Mater Chem B 3(1):112–118. https://doi.org/10.1039/C4TB01496K
Kinsella JL, Grant DS, Weeks BS, Kleinman HK (1992) Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel. Exp Cell Res 199(1):56–62. https://doi.org/10.1016/0014-4827(92)90461-G
Koutsopoulos S, Unsworth LD, Nagai Y, Zhang S (2009) Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold. Proc Natl Acad Sci 106(12):4623–4628. https://doi.org/10.1073/pnas.0807506106
Krontiras P, Gatenholm P, Hägg DA (2015) Adipogenic differentiation of stem cells in three-dimensional porous bacterial nanocellulose scaffolds. J Biomed Mater Res Part B 103(1):195–203. https://doi.org/10.1002/jbm.b.33198
Lian JB, Stein GS (2003) Runx2/Cbfa1: a multifunctional regulator of bone formation. Curr Pharm Des 9(32):2677–2685. https://doi.org/10.2174/1381612033453659
Lian J et al (1989) Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression. Proc Natl Acad Sci 86(4):1143–1147. https://doi.org/10.1073/pnas.86.4.1143
Malinen MM, Kanninen LK, Corlu A, Isoniemi HM, Lou Y-R, Yliperttula ML, Urtti AO (2014) Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels. Biomaterials 35(19):5110–5121. https://doi.org/10.1016/j.biomaterials.2014.03.020
Mehrotra M, Krane SM, Walters K, Pilbeam C (2004) Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells. J Cell Biochem 93(4):741–752. https://doi.org/10.1002/jcb.20138
Merriman HL et al (1995) The tissue-specific nuclear matrix protein, NMP-2, is a member of the AML/PEBP2/runt domain transcription factor family: interactions with the osteocalcin gene promoter. Biochemistry 34(40):13125–13132. https://doi.org/10.1021/bi00040a025
Mouw JK, Ou G, Weaver VM (2014) Extracellular matrix assembly: a multiscale deconstruction. Nat Rev Mol Cell Biol 15(12):771–785. https://doi.org/10.1038/nrm3902
Nam J, Johnson J, Lannutti JJ, Agarwal S (2011) Modulation of embryonic mesenchymal progenitor cell differentiation via control over pure mechanical modulus in electrospun nanofibers. Acta Biomater 7(4):1516–1524. https://doi.org/10.1016/j.actbio.2010.11.022
Nazarov R, Jin H-J, Kaplan DL (2004) Porous 3-D scaffolds from regenerated silk fibroin. Biomacromol 5(3):718–726. https://doi.org/10.1021/bm034327e
Nguyen HL, Jo YK, Cha M, Cha YJ, Yoon DK, Sanandiya ND, Prajatelistia E, Oh DX, Hwang DS (2016) Mussel-inspired anisotropic nanocellulose and silver nanoparticle composite with improved mechanical properties, electrical conductivity and antibacterial activity. Polymers 8(3):102. https://doi.org/10.3390/polym8030102
Nguyen HL, Hanif J, Park SA, Choi BG, Tran TH, Hwang DS, Park JY, Hwang SY, Oh DX (2018) Sustainable boron nitride nanosheet-reinforced cellulose nanofiber composite film with oxygen barrier without the cost of color and cytotoxicity. Polymers 10(5):501. https://doi.org/10.3390/polym10050501
O’Brien FJ, Harley B, Yannas IV, Gibson LJ (2005) The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 26(4):433–441. https://doi.org/10.1016/j.biomaterials.2004.02.052
Oh SH, Park IK, Kim JM, Lee JH (2007) In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method. Biomaterials 28(9):1664–1671. https://doi.org/10.1016/j.biomaterials.2006.11.024
Oh DX, Kim S, Lee D, Hwang DS (2015) Tunicate-mimetic nanofibrous hydrogel adhesive with improved wet adhesion. Acta Biomater 20:104–112. https://doi.org/10.1016/j.actbio.2015.03.031
Park SN, Park JC, Kim HO, Song MJ, Suh H (2002) Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking. Biomaterials 23(4):1205–1212. https://doi.org/10.1016/S0142-9612(01)00235-6
Pek YS, Wan AC, Shekaran A, Zhuo L, Ying JY (2008) A thixotropic nanocomposite gel for three-dimensional cell culture. Nat Nanotechnol 3(11):671–675. https://doi.org/10.1038/nnano.2008.270
Pietrucha K (2005) Changes in denaturation and rheological properties of collagen–hyaluronic acid scaffolds as a result of temperature dependencies. Int J Biol Macromol 36(5):299–304. https://doi.org/10.1016/j.ijbiomac.2005.07.004
Pluen A et al (2001) Role of tumor–host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc Natl Acad Sci 98(8):4628–4633. https://doi.org/10.1073/pnas.081626898
Ramanujan S, Pluen A, McKee TD, Brown EB, Boucher Y, Jain RK (2002) Diffusion and convection in collagen gels: implications for transport in the tumor interstitium. Biophys J 83(3):1650–1660
Riccio M, Resca E, Maraldi T, Pisciotta A, Ferrari A, Bruzzesi G, De Pol A (2010) Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures. Eur J Histochem 54(4):e46. https://doi.org/10.4081/ejh.2010.e46
Rocha LB, Goissis G, Rossi MA (2002) Biocompatibility of anionic collagen matrix as scaffold for bone healing. Biomaterials 23(2):449–456. https://doi.org/10.1016/S0142-9612(01)00126-0
Ruel-Gariepy E, Leroux J-C (2004) In situ-forming hydrogels—review of temperature-sensitive systems. Eur J Pharm Biopharm 58(2):409–426. https://doi.org/10.1016/j.ejpb.2004.03.019
Rungby J, Kassem M, Eriksen EF, Danscher G (1993) The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background. Histochem J 25(6):446–451. https://doi.org/10.1007/BF00157809
Saito T, Isogai A (2004) TEMPO-mediated oxidation of native cellulose. The effect of oxidation conditions on chemical and crystal structures of the water-insoluble fractions. Biomacromol 5(5):1983–1989. https://doi.org/10.1021/bm0497769
Saito T, Kimura S, Nishiyama Y, Isogai A (2007) Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose. Biomacromol 8(8):2485–2491. https://doi.org/10.1021/bm0703970
Schneider A et al (2006) Polyelectrolyte multilayers with a tunable Young’s modulus: influence of film stiffness on cell adhesion. Langmuir 22(3):1193–1200. https://doi.org/10.1021/la0521802
Shenoy V, Rosenblatt J (1995) Diffusion of macromolecules in collagen and hyaluronic acid, rigid-rod-flexible polymer, composite matrixes. Macromolecules 28(26):8751–8758. https://doi.org/10.1021/ma00130a007
Song J, Takeda M, Morimoto RI (2001) Bag1–Hsp70 mediates a physiological stress signalling pathway that regulates Raf-1/ERK and cell growth. Nat Cell Biol 3(3):276–282. https://doi.org/10.1038/35060068
Story BJ, Wagner WR, Gaisser DM, Cook SD, Rust-Dawicki AM (1998) In vivo performance of a modified CSTi dental implant coating. Int J Oral Maxillofacc Implants 13(6):749–757
Tibbitt MW, Anseth KS (2009) Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng 103(4):655–663. https://doi.org/10.1002/bit.22361
Torres-Rendon JG et al (2015) Bioactive gyroid scaffolds formed by sacrificial templating of nanocellulose and nanochitin hydrogels as instructive platforms for biomimetic tissue engineering. Adv Mater 27(19):2989–2995. https://doi.org/10.1002/adma.201405873
Traianedes K, Ng KW, Martin TJ, Findlay DM (1993) Cell substratum modulates responses of preosteoblasts to retinoic acid. J Cell Physiol 157(2):246–252. https://doi.org/10.1002/jcp.1041570206
Weaver VM, Petersen OW, Wang F, Larabell C, Briand P, Damsky C, Bissell MJ (1997) Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol 137(1):231–245. https://doi.org/10.1083/jcb
Wu H, Lozano G (1994) NF-kappa B activation of p53. A potential mechanism for suppressing cell growth in response to stress. J Biol Chem 269(31):20067–20074. https://doi.org/10.1074/jbc.269.20067
Yan LP, Oliveira JM, Oliveira AL, Caridade SG, Mano JF, Reis RL (2012) Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications. Acta Biomater 8(1):289–301. https://doi.org/10.1016/j.actbio.2011.09.037
Yeung T et al (2005) Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cytoskeleton 60(1):24–34. https://doi.org/10.1002/cm.20041
Yin N, Stilwell MD, Santos TM, Wang H, Weibel DB (2015) Agarose particle-templated porous bacterial cellulose and its application in cartilage growth in vitro. Acta Biomater 12:129–138. https://doi.org/10.1016/j.actbio.2014.10.019
Yoshikawa C, Hoshiba T, Sakakibara K, Tsujii Y (2018) Flocculation of cells by cellulose nanofibers modified with concentrated polymer brushes. ACS Appl Nano Mater 1(4):1450–1455. https://doi.org/10.1021/acsanm.8b00172
Zander NE, Dong H, Steele J, Grant JT (2014) Metal cation cross-linked nanocellulose hydrogels as tissue engineering substrates. ACS Appl Mater Interfaces 6(21):18502–18510. https://doi.org/10.1021/am506007z
Acknowledgments
We would like to acknowledge the financial support from the Grant the Marine Biotechnology program (Marine BioMaterials Research Center) funded by the Ministry of Oceans and Fisheries of Korea (D11013214H480000110). This work was also supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2016M1A5A1027592 and NRF-2017R1A2B3006354). DXO acknowledges KRICT SI-1809.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflicts of interest
All authors declare that there is no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kim, H.J., Oh, D.X., Choy, S. et al. 3D cellulose nanofiber scaffold with homogeneous cell population and long-term proliferation. Cellulose 25, 7299–7314 (2018). https://doi.org/10.1007/s10570-018-2058-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10570-018-2058-y