In situ mineralization of nano-hydroxyapatite on bifunctional cellulose nanofiber/polyvinyl alcohol/sodium alginate hydrogel using 3D printing

https://doi.org/10.1016/j.ijbiomac.2020.05.181Get rights and content

Highlights

  • Bifunctional CNF with carboxyl and aldehyde moieties were prepared.

  • Bifunctional CNF were cross-linked with PVA.

  • 3D printed scaffolds were prepared using PVA-grafted CNF and sodium alginate.

  • In-situ mineralization of hydroxyapatite was performed by adding phosphate ions.

  • This system is promising for bone tissue engineering.

Abstract

This paper reports the manufacturing by 3D printing of scaffolds for in-situ mineralization of hydroxyapatite using aqueous suspensions of alginate and polyvinyl alcohol (PVA)-grafted cellulose nanofibers (CNF). Bifunctional CNF with carboxyl and aldehyde moieties were prepared from bleached bagasse pulp and crosslinked with PVA. Aqueous hydrogels for 3D printing were prepared by directly mixing PVA-grafted CNF with sodium alginate, with and without the addition of phosphate ions. A calcium chloride solution was sprayed during the printing process in order to partially crosslink alginate and to increase the dimensional stability of the printed gel. At the end of the printing process, the prepared scaffolds were dipped into a CaCl2 solution to: i) complete alginate crosslinking and ii) promote hydroxyapatite nucleation and growth by reaction with phosphate ions. In order to better understand the mechanisms governing manufacturing of scaffolds by 3D printing, the rheological behavior of alginate/PVA-grafted CNF and the mechanical properties of unit filaments obtained by direct hydrogel extrusion were investigated. The final scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). This study shows that 3D printed sodium alginate/PVA-grafted CNF hydrogels are promising scaffold materials for bone tissue engineering.

Introduction

In recent years, 3D printing became an encouraging technology for the production of nanocomposites for bone tissue engineering [1,2]. In-situ biomimetic mineralization of hydroxyapatite/3D scaffold is one of the most important applications of biomaterials to replace damaged hard tissues and repair bone defects [3]. Several biomaterials can be used for the preparation of bone tissue scaffolds. These biomaterials must meet certain requirements such as biocompatibility, biodegradability, appropriate mechanical strength and low cytotoxicity [4,5]. Synthetic polymers, such as aliphatic polyesters, have been extensively investigated and tested, like polylactic acid (PLA), polyglycolic acid (PGA), poly-ε-caprolactone (PCL), polydioxanone (PDO), and polytrimethylene carbonate (PTMC) [[6], [7], [8]]. Nevertheless, natural biopolymers are becoming a valid alternative for the substitution of synthetic polymers for manufacturing 3D scaffolds because of several advantages, such as biodegradability, availability, and low cost. Also, natural polymers have demonstrated the potential advantage of supporting cell function and adhesion. Some natural polymers are water soluble, which means that these polymers dissolve in cell-friendly medium, such as cell culture medium and phosphate-buffered saline, to form solutions/hydrogels [9]. Collagen, alginate, gelatin and chitosan which are interesting compounds for tissue engineering [5,[10], [11], [12]] are emerging as outstanding materials for the manufacturing of 3D scaffolds.

Alginate is a natural biopolymer, which consists of two monosaccharide units: glucuronic acid and mannuronic acid. It has been commonly used as scaffold in tissue engineering, drug delivery and cell encapsulation due its biocompatibility, biodegradability and easy gelation with divalent cations under normal physiological conditions [13]. Similarly, cellulose nanofibers (CNF) have attracted increased interest in scientific and industrial research because of their unique properties, including sustainability, biodegradability, biocompatibility, high specific surface area, high mechanical strength, and abundant availability [[14], [15], [16]]. In addition to their physical properties, CNFs can be easily derived from various cellulose sources (such as wood or plant fibers) by using different techniques, i.e. pure mechanical shearing of cellulose fibers or a combination of chemical, enzymatic pretreatments and mechanical disintegration [17]. Owing to their high mechanical properties and extremely low percolation threshold, CNFs have been effectively used as reinforcing agent for various synthetic and natural polymers [[18], [19], [20]]. Poly (vinyl alcohol) (PVA) is a biodegradable and environmentally friendly synthetic polymer, with good thermal stability, optical properties and high oxygen barrier properties. It is widely used in various applications such as water purification [21], packaging [22] and tissue engineering [23].

In this study, we developed nanocellulose reinforced polymers with the objective of achieving a completely bio-based, new structural material for additive manufacturing, which is a new area of research. The development of viscoelastic inks that can be readily extruded, and yet form self-supporting features after exiting the nozzle, is challenging. PVA was crosslinked with bifunctional cellulose nanofibers (BF-CNF) bearing both carboxyl and aldehyde groups, in order to form stable cyclic acetal bonds with the hydroxyl groups of PVA. PVA-grafted CNFs were mixed with alginate to form more flexible and softer 3D printed scaffolds than those made with pure alginate. To the best our knowledge, such alginate/PVA-grafted BF-CNF/SA hydrogel has never been used for 3D extrusion printing. It was achieved in three steps. The first step consisted in the TEMPO oxidation of cellulose fibers and the generation of carboxyl groups at C6 position. The second step involved the mechanical defibrillation of oxidized fibers and the production of cellulose nanofibers (T-CNF). In the last step, periodate oxidation of T-CNF induced the formation of dialdehyde moieties in position C2 and C3 with the cleavage of the C2-C3 bond. The obtained bifunctional cellulose nanofibers (BF-CNF) were used as a potential cross-linker for PVA with the formation of stable acetal bonds between the hydroxyl groups of PVA and the aldehyde groups of BF-CNF. The addition of BF-CNF clearly improves the stiffness of the hydrogel, which was found to be homogenous.

In order to evaluate bone precursor nucleation and growth, calcium phosphate was in situ mineralized on 3D porous scaffolds prepared with alginate/PVA-grafted CNFs hydrogels. The effect of pH (4 or 8) on the formation of calcium phosphate was also investigated showing that, in addition to their excellent dimensional stability, the developed scaffolds are homogeneously coated with a mineral layer. The 3D printed sodium alginate/PVA grafted CNF hydrogels turn to be promising scaffold materials for bone tissue engineering.

Section snippets

Materials

Cellulose nanofibers were prepared from bleached bagasse pulp which was supplied from Quena Company of Paper Industry (Egypt). All the chemical reagents (hydrolyzed polyvinyl alcohol (PVA), sodium alginate (SA), sodium periodate (NaIO4), disodium hydrogen phosphate (Na2HPO4), sodium bromide (NaBr) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were purchased from Sigma Aldrich and used without further purification.

Preparation of bifunctional cellulose nanofibers (BF-CNF)

TEMPO-oxidized bleached bagasse pulp was prepared according to a previously

Results and discussion

Scheme 1 presents the different steps for the production of bifunctional cellulose nanofibers (BF-CNF) using chemical and mechanical treatments. The first step consisted in the TEMPO oxidation of cellulose fibers and the generation of carboxyl groups at C6 position. The second step corresponded to the mechanical defibrillation of oxidized fibers and the production of cellulose nanofibers (T-CNF). In the last step, periodate oxidation of T-CNF induced the formation of dialdehyde moieties in

Conclusions

In this work, bifunctional cellulose nanofibers (BF-CNF) were prepared with reactive carboxyl and aldehyde groups and used for the crosslinking of PVA. Crosslinked PVA/alginate scaffolds were fabricated in the presence of Na2HPO4 via 3D printing and a subsequent in situ mineralization of calcium phosphate during scaffold crosslinking by immersion in a CaCl2 solution. The Young's modulus of cross-linked PVA/alginate scaffolds was increased from 0.54 ± 0.01 GPa for 0% BF-CNF to 2.76 ± 0.26 GPa

Declaration of competing interests

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.

Acknowledgments

This work was financially supported by the Embassy of France in Egypt − Institut Français d'Egypte (IFE) and Science & Technology Development Fund (STDF) in Egypt (Project No. 30663) as well as to the “PHC-UTIQUE CMCU” (project number 18G1132) for the financial support. LGP2 is part of the LabEx Tec 21 (Investissements d'Avenir - grant agreement n°ANR-11-LABX-0030) and of the PolyNat Carnot Institut (Investissements d'Avenir - grant agreement n°ANR-11-CARN-030-01).

References (34)

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