Avidin-Conjugated Nanofibrillar Cellulose Hydrogel Functionalized with Biotinylated Fibronectin and Vitronectin Promotes 3D Culture of Fibroblasts

Jenni Leppiniemi; Zeeshan Mutahir; Alexander Dulebo; Piia Mikkonen; Markus Nuopponen; Paula Turkki; Vesa P. Hytönen

doi:10.1021/acs.biomac.1c00579

Avidin-Conjugated Nanofibrillar Cellulose Hydrogel Functionalized with Biotinylated Fibronectin and Vitronectin Promotes 3D Culture of Fibroblasts

Jenni Leppiniemi^*
, Zeeshan Mutahir
, Alexander Dulebo
, Piia Mikkonen
, Markus Nuopponen
, Paula Turkki
, Vesa P. Hytönen

^*Corresponding author for this work

Department of Clinical Chemistry

Research output: Contribution to journal › Article › Scientific › peer-review

11 Citations (Scopus)

64 Downloads (Pure)

Abstract

The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a well-defined 3D environment instructing cellular behavior. To establish a platform to produce tailored hydrogels, we conjugated avidin (Avd) to anionic nanofibrillar cellulose (aNFC) and demonstrated the use of the resulting Avd-NFC hydrogel for 3D cell culture, where Avd-NFC allows easy functionalization via biotinylated molecules. Avidin was successfully conjugated to nanocellulose and remained functional, as demonstrated by electrophoresis and titration with fluorescent biotin. Rheological analysis indicated that Avd-NFC retained shear-thinning and gel-forming properties. Topological characterization using AFM revealed the preserved fiber structure and confirmed the binding of biotinylated vitronectin (B-VN) on the fiber surface. The 3D cell culture experiments with mouse embryonic fibroblasts demonstrated the performance of Avd-NFC hydrogels functionalized with biotinylated fibronectin (B-FN) and B-VN. Cells cultured in Avd-NFC hydrogels functionalized with B-FN or B-VN formed matured integrin-mediated adhesions, indicated by phosphorylated focal adhesion kinase. We observed significantly higher cell proliferation rates when biotinylated proteins were bound to the Avd-NFC hydrogel compared to cells cultured in Avd-NFC alone, indicating the importance of the presence of adhesive sites for fibroblasts. The versatile Avd-NFC allows the easy functionalization of hydrogels with virtually any biotinylated molecule and may become widely utilized in 3D cell/tissue culture applications.

Original language	English
Pages (from-to)	4122-4137
Journal	Biomacromolecules
DOIs	https://doi.org/10.1021/acs.biomac.1c00579
Publication status	Published - 11 Oct 2021
Publication type	A1 Journal article-refereed

Funding

This research was supported by the Academy of Finland (grants 290506 and 331946 to V.P.H.) and a grant of the Finnish Cultural Foundation to J.L. In addition, UPM Biomedicals funded part of the work of J.L. We acknowledge the Tampere Facility of Protein Services and Tampere Imaging Facility, both affiliated with the Biocenter Finland, for their services. We thank Ulla Kiiskinen, Niklas Kähkönen, and Merja Jokela (Tampere University) and the application laboratory personnel at the UPM North European Research Center for excellent technical support. We also thank MSc Latifeh Azizi for her advice on cell culture experiments and PhD Rolle Rahikainen for discussions and useful advice in setting up the 3D cell culture.

Publication forum classification

Publication forum level 2

ASJC Scopus subject areas

Bioengineering
Biomaterials
Polymers and Plastics
Materials Chemistry

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acs.biomac.1c00579-1Final published version, 5.93 MBLicence: CC BY

https://urn.fi/URN:NBN:fi:tuni-202110257805Licence: CC BY

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@article{4d4ec22c003f49bf887ffec10de42a3d,

title = "Avidin-Conjugated Nanofibrillar Cellulose Hydrogel Functionalized with Biotinylated Fibronectin and Vitronectin Promotes 3D Culture of Fibroblasts",

abstract = "The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a well-defined 3D environment instructing cellular behavior. To establish a platform to produce tailored hydrogels, we conjugated avidin (Avd) to anionic nanofibrillar cellulose (aNFC) and demonstrated the use of the resulting Avd-NFC hydrogel for 3D cell culture, where Avd-NFC allows easy functionalization via biotinylated molecules. Avidin was successfully conjugated to nanocellulose and remained functional, as demonstrated by electrophoresis and titration with fluorescent biotin. Rheological analysis indicated that Avd-NFC retained shear-thinning and gel-forming properties. Topological characterization using AFM revealed the preserved fiber structure and confirmed the binding of biotinylated vitronectin (B-VN) on the fiber surface. The 3D cell culture experiments with mouse embryonic fibroblasts demonstrated the performance of Avd-NFC hydrogels functionalized with biotinylated fibronectin (B-FN) and B-VN. Cells cultured in Avd-NFC hydrogels functionalized with B-FN or B-VN formed matured integrin-mediated adhesions, indicated by phosphorylated focal adhesion kinase. We observed significantly higher cell proliferation rates when biotinylated proteins were bound to the Avd-NFC hydrogel compared to cells cultured in Avd-NFC alone, indicating the importance of the presence of adhesive sites for fibroblasts. The versatile Avd-NFC allows the easy functionalization of hydrogels with virtually any biotinylated molecule and may become widely utilized in 3D cell/tissue culture applications. ",

author = "Jenni Leppiniemi and Zeeshan Mutahir and Alexander Dulebo and Piia Mikkonen and Markus Nuopponen and Paula Turkki and Hyt{\"o}nen, \{Vesa P.\}",

note = "Funding Information: This research was supported by the Academy of Finland (grants 290506 and 331946 to V.P.H.) and a grant of the Finnish Cultural Foundation to J.L. In addition, UPM Biomedicals funded part of the work of J.L. We acknowledge the Tampere Facility of Protein Services and Tampere Imaging Facility, both affiliated with the Biocenter Finland, for their services. We thank Ulla Kiiskinen, Niklas K{\"a}hk{\"o}nen, and Merja Jokela (Tampere University) and the application laboratory personnel at the UPM North European Research Center for excellent technical support. We also thank MSc Latifeh Azizi for her advice on cell culture experiments and PhD Rolle Rahikainen for discussions and useful advice in setting up the 3D cell culture. ",

year = "2021",

month = oct,

day = "11",

doi = "10.1021/acs.biomac.1c00579",

language = "English",

pages = "4122--4137",

journal = "Biomacromolecules",

issn = "1525-7797",

publisher = "American Chemical Society",

}

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T1 - Avidin-Conjugated Nanofibrillar Cellulose Hydrogel Functionalized with Biotinylated Fibronectin and Vitronectin Promotes 3D Culture of Fibroblasts

AU - Leppiniemi, Jenni

AU - Mutahir, Zeeshan

AU - Dulebo, Alexander

AU - Mikkonen, Piia

AU - Nuopponen, Markus

AU - Turkki, Paula

AU - Hytönen, Vesa P.

N1 - Funding Information: This research was supported by the Academy of Finland (grants 290506 and 331946 to V.P.H.) and a grant of the Finnish Cultural Foundation to J.L. In addition, UPM Biomedicals funded part of the work of J.L. We acknowledge the Tampere Facility of Protein Services and Tampere Imaging Facility, both affiliated with the Biocenter Finland, for their services. We thank Ulla Kiiskinen, Niklas Kähkönen, and Merja Jokela (Tampere University) and the application laboratory personnel at the UPM North European Research Center for excellent technical support. We also thank MSc Latifeh Azizi for her advice on cell culture experiments and PhD Rolle Rahikainen for discussions and useful advice in setting up the 3D cell culture.

PY - 2021/10/11

Y1 - 2021/10/11

N2 - The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a well-defined 3D environment instructing cellular behavior. To establish a platform to produce tailored hydrogels, we conjugated avidin (Avd) to anionic nanofibrillar cellulose (aNFC) and demonstrated the use of the resulting Avd-NFC hydrogel for 3D cell culture, where Avd-NFC allows easy functionalization via biotinylated molecules. Avidin was successfully conjugated to nanocellulose and remained functional, as demonstrated by electrophoresis and titration with fluorescent biotin. Rheological analysis indicated that Avd-NFC retained shear-thinning and gel-forming properties. Topological characterization using AFM revealed the preserved fiber structure and confirmed the binding of biotinylated vitronectin (B-VN) on the fiber surface. The 3D cell culture experiments with mouse embryonic fibroblasts demonstrated the performance of Avd-NFC hydrogels functionalized with biotinylated fibronectin (B-FN) and B-VN. Cells cultured in Avd-NFC hydrogels functionalized with B-FN or B-VN formed matured integrin-mediated adhesions, indicated by phosphorylated focal adhesion kinase. We observed significantly higher cell proliferation rates when biotinylated proteins were bound to the Avd-NFC hydrogel compared to cells cultured in Avd-NFC alone, indicating the importance of the presence of adhesive sites for fibroblasts. The versatile Avd-NFC allows the easy functionalization of hydrogels with virtually any biotinylated molecule and may become widely utilized in 3D cell/tissue culture applications.

AB - The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a well-defined 3D environment instructing cellular behavior. To establish a platform to produce tailored hydrogels, we conjugated avidin (Avd) to anionic nanofibrillar cellulose (aNFC) and demonstrated the use of the resulting Avd-NFC hydrogel for 3D cell culture, where Avd-NFC allows easy functionalization via biotinylated molecules. Avidin was successfully conjugated to nanocellulose and remained functional, as demonstrated by electrophoresis and titration with fluorescent biotin. Rheological analysis indicated that Avd-NFC retained shear-thinning and gel-forming properties. Topological characterization using AFM revealed the preserved fiber structure and confirmed the binding of biotinylated vitronectin (B-VN) on the fiber surface. The 3D cell culture experiments with mouse embryonic fibroblasts demonstrated the performance of Avd-NFC hydrogels functionalized with biotinylated fibronectin (B-FN) and B-VN. Cells cultured in Avd-NFC hydrogels functionalized with B-FN or B-VN formed matured integrin-mediated adhesions, indicated by phosphorylated focal adhesion kinase. We observed significantly higher cell proliferation rates when biotinylated proteins were bound to the Avd-NFC hydrogel compared to cells cultured in Avd-NFC alone, indicating the importance of the presence of adhesive sites for fibroblasts. The versatile Avd-NFC allows the easy functionalization of hydrogels with virtually any biotinylated molecule and may become widely utilized in 3D cell/tissue culture applications.

U2 - 10.1021/acs.biomac.1c00579

DO - 10.1021/acs.biomac.1c00579

M3 - Article

AN - SCOPUS:85116563489

SN - 1525-7797

SP - 4122

EP - 4137

JO - Biomacromolecules

JF - Biomacromolecules

ER -

Avidin-Conjugated Nanofibrillar Cellulose Hydrogel Functionalized with Biotinylated Fibronectin and Vitronectin Promotes 3D Culture of Fibroblasts

Abstract

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