Elsevier

Materials Letters

Volume 264, 1 April 2020, 127326
Materials Letters

Cross-linked gelatin-nanocellulose scaffolds for bone tissue engineering

https://doi.org/10.1016/j.matlet.2020.127326Get rights and content

Highlights

  • Porous scaffolds were produced from wood-based nanocellulose and gelatin (Gel-CNF).

  • Gel-CNF was cross-linked by either DHT, or a combination of HMDA, genipin, and DHT.

  • Gel-CNF hydrogels are cytocompatible with no adverse biological effects.

  • The porous hydrogel scaffolds promote osteogenic differentiation of MSCs.

  • The novel scaffolds hold potential for bone tissue engineering applications.

Abstract

Wood-based cellulose nanofibrils (CNFs) have, in addition to high specific surface area and high surface reactivity, ability to mimic nanostructured collagen in bone extracellular matrix. These properties make CNFs promising materials for bone tissue engineering (BTE). The CNFs degrade slowly in vivo. By blending and cross-linking gelatin (Gel) with CNFs, scaffolds were produced with tuned degradation rate and enhanced mechanical properties, more suitable for BTE applications. This in vitro study aimed to examine initial biological responses of human bone marrow mesenchymal stem cells to cross-linked Gel-CNF scaffolds. The scaffolds were fabricated from 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNF blended with Gel and cross-linked either by dehydrothermal treatment (DHT) or by a combination of hexamethylenediamine, genipin, and DHT. CNF scaffolds without cross-linking served as control. The produced scaffolds supported cell attachment, spreading, and osteogenic differentiation. However, the early cell attachment after 1 day and the expression of RUNX2 and SPP1 genes after 7 days were highest in the CNF scaffolds. The results suggest that cross-linked Gel-CNF are cytocompatible and holds potential for BTE applications.

Introduction

Recent advances in the field of bone tissue engineering (BTE) present encouraging approaches to solve challenges associated with current bone augmentation techniques [1]. Hydrogels are a group of polymers that can absorb water up to thousand folds their dry weight, creating a gel-like environment mimicking stem cell niche [1]. Recently, a promising class of hydrogels have been produced from wood-based cellulose nanofibrils (CNFs). The CNFs have, due to their nanostructure, both high specific surface area and high surface reactivity, but are slowly degradable in vivo [2]. Insufficient degradation rate will cause the host to respond with a foreign body reaction [16]. A new hydrogel has been developed by blending gelatin (Gel) with CNF, to better meet requirements for BTE [6]. Mechanical properties and the degradation rate of the Gel-CNF hydrogel were further adjusted with different cross-linking approaches [6].

In this study, Gel-CNF hydrogels were cross-linked by either dehydrothermal treatment (DHT), or by a combination of hexamethylenediamine (HMDA), genipin, and DHT. The DHT is a physical treatment that includes high temperature and vacuum to remove the water from the matrix, resulting in the formation of intermolecular cross-links [4]. The HMDA is a diamine which is commonly used with biomaterials containing carboxyl groups, creating covalent bonds between oxidized CNFs [5]. The biodegradable molecule genipin reacts spontaneously with amino acids, and can cross-link the gelatin chains [6]. However, there is a risk of introducing cytotoxicity to the material when using chemical reagents as cross-linkers.

The present study was undertaken to investigate the osteogenic potential of these cross-linked scaffolds in vitro. Human bone marrow mesenchymal stem cells (hBMSCs) were cultured on scaffolds and their initial biological responses studied in terms of cell attachment, viability, and osteogenic differentiation.

Section snippets

Preparation of scaffolds

Oxidized CNFs were prepared by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) according to Saito et al. [7]. To create interconnected porosity by ice crystal formation, the CNF suspension was frozen at −20 °C for 24 h before freeze-drying for another 24 h. Porous Gel-CNF scaffolds cross-linked by DHT (Gel-CNF1) and porous Gel-CNF scaffolds cross-linked by HMDA, genipin, and DHT (Gel-CNF2) were prepared according to Campodini et al. [6].

Scaffolds structure and porosity

Assessment of the 3D architecture was performed by

Results and discussion

Architecture of the scaffolds strongly influences their mechanical properties. The freeze-drying method used, produced hydrogel scaffolds with variable porosity and interconnected pores as shown by µ-CT (Fig. 1A-C) and SEM (Fig. 1D-F). Both total porosity and pore sizes of CNF scaffolds were higher than that of the Gel-CNF samples, regardless the method of cross-linking (Fig. 1, Table 1). Previously, Campodoni et al. reported that porous scaffolds from Gel-CNF hydrogels, cross-linked by either

Conclusion

The cross-linking methods used to modify mechanical properties and degradation rates of the Gel-CNF scaffolds did not generate any adverse biological responses to hBMSCs and supported their osteogenic differentiation. The results of this study suggest that the cross-linked Gel-CNF hydrogels are promising materials for BTE.

CRediT authorship contribution statement

Ingeborg Elisabeth Carlström: Writing - original draft, Writing - review & editing. Ahmad Rashad: Conceptualization, Methodology, Writing - review & editing, Supervision. Elisabetta Campodoni: Conceptualization, Methodology, Writing - review & editing. Monica Sandri: Writing - review & editing. Kristin Syverud: Conceptualization, Writing - review & editing. Anne Isine Bolstad: Methodology, Writing - review & editing, Supervision. Kamal Mustafa: Conceptualization, Methodology, Writing - review &

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work has been funded by the Research Council of Norway through the NORCEL project, (Grant no. 228147) and Trond Mohn Foundation (TMS, project no. BFS2018TMT10).

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These authors contributed equally to this study.

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