Biomimetic Mineralization of Three-Dimensional Printed Alginate

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Biomimetic Mineralization of 3D Printed Alginate/TEMPO-Oxidized Cellulose Nanofibril Scaffolds for Bone Tissue Engineering Ragab Esmail Abou-Zeid, Ramzi Khiari, Davide Beneventi, and Alain Dufresne Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01325 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 12, 2018

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Biomimetic Mineralization of 3D Printed Alginate/TEMPO-Oxidized Cellulose Nanofibril Scaffolds for Bone Tissue Engineering

Ragab E. Abouzeid, *†,‡ Ramzi Khiari, ‡,§,‖ Davide Beneventi, ‡ Alain Dufresne*‡

†

Cellulose and Paper Department, National Research Centre, Dokki, Giza, 12622, Egypt

‡

Univ. Grenoble Alpes, CNRS, Grenoble INP, LGP2, F-38000 Grenoble, France

§

University of Monastir, Faculty of Sciences, UR13 ES 63 - Research Unity of Applied

Chemistry & Environment, 5000 Monastir, Tunisia. ‖

Higher Institute of Technological Studies of Ksar Hellal, Department of Textile, Tunisia

KEYWORDS: Cellulose nanofibril; Alginate hydrogel; 3D printing; Biomimetic mineralization; Bone tissue engineering.

ABSTRACT 3D printed scaffolds were prepared by partial crosslinking of TEMPO-oxidized cellulose nanofibril/alginate hydrogel using calcium ions for printing the hydrogel while maintaining its shape, fidelity and preventing collapse of the filaments. The prepared scaffolds were fully crosslinked using calcium ions immediately after printing to provide the rigidity of the hydrogel and give it long-term stability. The composition of the prepared pastes was adjusted in view of

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the description of the hydrogel and 3D printing parameters. The rheological properties, in terms of thixotropic behavior and viscosity recovery of hydrogels, were investigated by performing steady shear rate experiments. The results show that the viscosity recovery for pure alginate hydrogel was only about 16 % of the initial value, while it was 66% when adding cellulose nanofibrils to alginate. Consequently, the shape of the pure alginate scaffold was soft and easy to collapse contrarily to the composite scaffold. The biomimetic mineralization process of printed scaffolds using simulated body fluid, mimicking the inorganic composition of human blood plasma, was performed and the hydroxyapatite nucleation on the hydrogel was confirmed. The strength properties of the fabricated scaffolds in terms of compressive strength analysis were also investigated and discussed. The results show that the alginate/TEMPO-oxidized cellulose nanofibril system may be a promising 3D printing scaffold for bone tissue engineering. INTRODUCTION The 3D printing technology or additive manufacturing is a process aimed to the rapid production of complex 3D structures that integrate seamlessly with computer-assisted design (CAD) software. It was initially designed by Charles Hall in 1986.1 Recently, this technology triggered an ever increasing interest for the production of customer and applications used in industry, including motor vehicle, aviation assembling, and therapeutic applications.2,3 The use of 3D printing in biomedical applications paves the way to the mass customization of surgical parts, drilling guides and the production on-demand of various types of implants.3-5 During the last decade, many combinations of natural biopolymers and inorganic materials have been examined to develop new biomaterials with interesting performances. For example, an expansive number of papers have been given to enhancing the shape fidelity, mechanical and biological properties of scaffolds prepared by 3D printing. In all cases, these biomaterials should be biocompatibile,


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biodegradable, and have appropriate mechanical properties and low cytotoxicity.6,7 Aliphatic polyesters (polylactic acid (PLA), polyglycolic acid (PGA), poly(ε-caprolactone) (PCL), polydioxanone (PDO) and poly(trimethylene carbonate) (PTMC)),8-10 and natural biopolymers like collagen, alginate, gelatin, and chitosan are promising substrates for tissue engineering application.7 Sodium Alginate (SA) is a natural biopolymer, which consists of two monosaccharide units, i.e. -D-mannuronate and -L-guluronate guluronic acids. It has been widely utilized as a scaffold in tissue engineering, drug delivery, and cell encapsulation. The ease of gelation of alginate with divalent cations under normal physiological conditions is one of its most important features besides biocompatibility and biodegradability properties.11 Alginate has been used extensively in the formulation of extrudable mixtures for 3D printing because of its ability to enhance the viscosity of polymer solutions. On the other hand, alginate has the ability to form a gel under mild conditions, and does not require heat to form, by addition of calcium salts to the alginate solution. Calcium ions replace sodium ions in alginate and binding long chain alginate molecules, resulting in the formation of a 3D gel assembly called "egg box", ionoreversible and non-thermoreversible.12,13 However, alginate hydrogel suffers from a lack of compressive strength and has a low modulus and low dry matter content to be used as a bio-ink for bone tissue engineering. The lower concentration of polymer with higher absorbed water of the hydrogel induces its collapse after 3D printing. The challenge is to improve its viscoelastic properties, which maintain the shape fidelity after printing. Therefore, the hydrogel must move inside the needle during the 3D printing and maintain its form after printing. Despite the fact that the viscosity of alginate can be tuned by changing its concentration and molecular weight, its rheological behavior is not adequate for achieving shape reliability while printing. In such cases,


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the 3D printing of alginate hydrogel has been enhanced by mixing with other biopolymer such as gelatin,12,14 or printing with a conciliatory supporting polymer.15 Cellulose nanofibrils (CNF) have gained increased interest for biomedical applications because of their unique properties including sustainability, biodegradability, biocompatibility, high surface area, good strength properties and abundant availability.16,17 CNFs can be prepared from natural cellulose (such as wood or plant fibers) using high mechanical shearing,18 and also different pretreatment methods can be carried out to facilitate the defibrillation process such as oxidation, and acidic or enzymatic treatment.19,21 CNFs have been used in combination with alginate to prepare 3D macroporous scaffolds for adipose tissue22 or cell encapsulation.23 TEMPO-oxidized CNFs (T-CNFs) were used as support for crosslinked alginate sponge through the participation of the new TEMPO-induced carboxyl groups in the construction of the crosslinked network.24 CNF and alginate have been used as bio-ink for 3D bioprinting of human chondrocytes.25 The printability, shape fidelity and cell viability were developed by using alginate and high-molecular-weight water-soluble carboxymethyl cellulose (CMC) to produce a 3D functional living tissue scaffold in-vitro.26 Furthermore, alginate hydrogel dedicated to bone tissue engineering has been modified by combination with inorganic materials such as hydroxyapatite (HAp) to improve the mechanical properties and cell-attachment properties of the scaffolds by direct mixing of alginate with HAp nanoparticles.16,27 In this case, the direct mixing of HAp powder with alginate resulted in a lack of homogeneity and restricted bioactivity of the polymer network was achieved. In-situ mineralization is another effective method for the preparation of polymer/HAp composite scaffolds.27-29 In this method, alginate powder was dissolved in a solution containing phosphate ions and during the crosslinking with calcium ions to form the final form of the scaffold, calcium


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ions also reacting with phosphate ions to form HAp. However, this type of polymer/HAp scaffold lacks sufficient porosity for cell penetration and growth of new tissue. The third method for preparing polymer/HAp scaffold is the biomimetic mineralization of HAp by dipping the scaffold into simulated body fluid (SBF) which has been widely used to prepare biopolymer/HAp scaffold.30-36 SBF contains ions at concentrations almost like those found in plasma. This type of mineralization is performed under biological conditions (temperature, pressure, and pH) forming HAp on the surface of the scaffold that has similar chemical structure and material properties to those of bone mineral. The present work focuses on the use of 3D printing technique for preparing porous scaffolds to replace damaged hard tissue and repair bone defects. It introduces a green preparing strategy for the advancement of 3D printing scaffold from T-CNF/SA/HAp hybrid materials for bone tissue engineering applications. T-CNF/SA hydrogel has been never used for 3D extrusion printing. It was achieved in two steps. Firstly, partial crosslinking of the hydrogel was performed before printing using calcium chloride. This step is used to improve the printability of the gel during 3D printing, and to prevent the filament from collapsing and maintain the shape fidelity of the 3D scaffold. The second step was achieved after printing, and consisted in the complete crosslinking using calcium chloride solution to ensure the rigidity of the alginate hydrogel after printing. On the other hand, the biomimetic mineralization of hydroxyapatite in SBF solution was studied as the effect of the combination of T-CNF and alginate. T-CNF contains carboxylate groups which play an important role in the nucleation of HAp. The efficiency of carboxyl groups in inducing the nucleation of HAp on the surface of T-CNF in SBF has been reported.37 EXPERIMENTAL SECTION


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Materials. Bleached fibers extracted from bagasse were supplied by Qena Pulp and Paper Industry, Qena, Egypt. Several chemical products were purchased from Sigma-Aldrich, namely: EMPO ((CH2)3(CMe2)2NO), sodium hypochlorite (NaOCl), sodium hydroxide (NaOH), sodium chlorite (NaClO2), acetic acid (CH3COOH) and sodium bromide (NaBr). Sodium alginate and calcium chloride (CaCl2) of laboratory grade were also purchased from Sigma-Aldrich and used without further treatment. For the preparation of SBF, reagent grade chemicals were purchased from Sigma-Aldrich and used as received (NaCl (16.07 g), NaHCO3 (0.71 g), KCl (0.45 g), K2HPO4⋅3H2O (0.462 g), MgCl2⋅6H2O (0.622 g), Na2SO4 (0.144 g), CaCl2⋅2H2O (0.76 g), and Tris(hydroxymethyl) aminomethane (12.236 g) in 1 L of deionized water were used). The pH value was adjusted to 7.4 at 37°C. Preparation of TEMPO-oxidized cellulose nanofibrils. TEMPO-oxidized cellulose nanofibrils (T-CNF) were prepared from the bleached bagasse pulp according to the method reported by Saito et al.38 Briefly, the fibers obtained from bagasse pulp (5 g) were suspended in water (500 mL) containing TEMPO (0.08 g, 0.5 mmol) and NaBr (0.8 g, 8 mmol). Then 50 mL of NaOCl was put to the suspension under stirring and the pH was adjusted to ten. Finally, the pH was kept to 7 and the modified fibers were centrifuged at 7000 rpm. The obtained fibers were additionally purified by repeated water addition, dispersion, and centrifugation. Finally, the modified fibers were purified by dialysis for 1 week against deionized water. T-CNF was prepared using Masuko grinder as mechanical defibrillation treatment and their carboxylate content was found to be 1.2±0.4 mmol.g-1 using conduction volumetric analysis. Preparation of T-CNF/alginate hydrogels. Five printing paste formulations were prepared from T-CNF, SA and a mixture thereof (pure T-CNF, Pure SA, 70% T-CNF/30% SA, 50% TCNF/50% SA and 30% T-CNF/70% SA). T-CNF was utilized as a source of perspective gel in


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which the solid content was 2%. The codification and composition of the different printing paste formulations are reported in Table 1. The alginate powder was mixed directly with T-CNF under vigorous stirring for 2 h. After that, the formulations were partially crosslinked with an aqueous calcium chloride solution (3% based on dry alginate content). It aimed in forming paste hydrogels with appropriate elastic properties that can course through the needle and hold their structure after printing. Before printing, all the hydrogels were kept in a refrigerator at 6°C. Table 1. Codification and composition of the printing paste formulations. Sample

T-CNF/alginate T-CNF Alginate Calcium chloride Water content (wt%) (wt%) (wt%) (wt% of dry alginate) (wt%) CNF100 100/0 2.0 0.0 0.00 98.00 CNF70 70/30 2.0 1.0 0.03 96.97 CNF50 50/50 2.0 2.0 0.06 95.94 CNF30 30/70 2.0 4.7 0.14 93.19 CNF0 0/100 0.0 2.0 0.06 97.94 Preparation of 3D printed T-CNF/SA scaffolds. T-CNF/SA scaffolds with dimensions of 30×30×20 mm were printed using Leapfrog 3D device connected with a screw-pump paste extruder (Wasp, claystruder). The printing process factors for extruding the hydrogels are collected in Table 2. Table 2. Printing process factors. Factor Nozzle diameters Pressure Printing speed Primarily Layer height Extrusion width Interior fill percentage

Value 0.5 mm 0.5 bar 1000 mm.min-1 0.35 mm 0.6 mm 50 %

The prepared hydrogels were extruded at room temperature using a needle 0.5 mm in diameter, a pressure of 0.5 bar at a steady dispensing head speed of 1000 mm.min-1. The width of the


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filament and the layer height were set at 0.6 mm and 0.35 mm, respectively. The preformed scaffolds (Figure 1A) were soaked in 0.5 mol.L-1 CaCl2 aqueous solution for 20 min to achieve the full crosslinking, and then washed with deionized water three times. Finally, the scaffolds were freeze-dried for use in further experiments. The paste composition was optimized based on the properties of the hydrogel in terms of mechanical and rheological properties, as well as by 3D printing experiments. The best formulation was 50% T-CNF and 50% alginate partially crosslinked with 3% calcium chloride based on the alginate content. After that, the scaffold was printed in different shapes and designs from the optimum hydrogel formulation such as a cylinder, half-bone, boat, and human ear (Figure 1B).

Figure 1. (A) Fabrication process for 3D printing scaffolds from T-CNF/SA hydrogels, (B) scaffold printed in different forms and designs from the optimum hydrogel formulation. Rheological properties. The rheological properties of all the pastes were determined using MCR 301 rheometer (Anton Paar). A parallel plate 25 mm in diameter was used and the gap


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between both plates was set to 1 mm. A cover was used to prevent water evaporation during measurements, and the temperature was maintained at 23°C. The samples were equilibrated 1 min to remove any previous shear history and shear rates ranging from 10-3 to 103 s-1 were applied. Thixotropic tests were investigated by fixing the shear rate at 1000 s-1 for 100 s before a sudden drop to 0.1 s-1. The rheological behavior in terms of viscosity trends as well as stress response was evaluated as a function of time. Oscillatory tests were used to determine the viscoelastic characteristics of all mixtures. The linear viscoelastic region (LVR) was determined by amplitude sweep tests conducted at the frequency of 1 Hz and strain ranging from 0.001 to 100%. The frequency sweep was performed at 0.1% strain from 0.1 to 10 Hz. The evolution of the storage modulus (G’), loss modulus (G”), complex modulus (G*), and loss angle tangent (tan δ = G”/G’) as a function of strain, frequency, or stress was analyzed. The analyses were conducted in triplicate at 25°C. Mechanical testing. The mechanical properties of dry scaffolds were evaluated in terms of compressive using an Instron model 5569 with a load cell of 5 kN and a crosshead speed of 1.0 mm.min-1. Scaffolds with a size of 15×15×15 mm were printed with 50 % filing for the compressive tests. All tests were established of 50% strain in z-direction. From the obtained stress-strain curve, the storage modulus was determinate. At least, 10 samples were measured on average and standard deviations were then calculated. Mineralization. As reported previously36, the biomimetic mineralization of calcium phosphate was established. In fact, double concentrated simulated body fluid (2xSBF) was prepared and used to enhance the precipitation of calcium phosphate. CNF50 scaffold was used to study the biomimetic mineralization and the mineralization process was conveyed for 14 days under stirring at room temperature. SBF was changed every 48h with adjusting pH to 7.4 after


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centrifugation and check regularly to decrease the problems of SBF stabilization. The sample was purified by centrifugation and washing five times with deionized water and ethanol. Finally, the product was dried at room temperature for further characterization. Fourier transform infrared spectroscopy. Fourier transform infrared spectra (FT-IR) were obtained using Perkin Elmer FT-IR spectrometer (Perkin Elmer, USA) and KBr discs in the range 4000-500 cm−1 with a resolution of 4 cm-1 and an accumulation of 16 scans per analysis. Morphological investigation. Atomic force microscopy (AFM Multimode - DI, Veeco, Instrumentation Group) in tapping mode with Multi 130 tips was used to characterize T-CNF and study the morphology of the prepared T-CNF/SA hydrogels. Moreover, the morphological features of the scaffolds before and after mineralization were determined using scanning electron microscopy (FEI-Quanta 2000, ESEMTM) equipped with EDX Unit device namely: Energy Dispersive-ray Analyses. Thermogravimetric analysis. The thermal behavior was established by PerkinElmer TGA thermogravimetric analyzer. The TGA analysis was done under O2 condition from 25 to 800°C with a heating rate of 10oC.min-1. Synchrotron X-ray powder diffraction. X-ray powder diffraction data of a fine powder from prepared scaffold before and after mineralization were collected at room temperature at the MCX beamline at Elettra-Sincrotrone Trieste, Trieste, Italy.39,40 The sample was contained in a thinwalled borosilicate glass capillary with a diameter of 1.0 mm. The wavelength used for data collection was 0.827 Å and the data were collected in the 2θ range 3-50°. In order to improve the particle distribution statistics, the capillary was rotated during data collection. Using Eq. 1 described by Scherrer,41 the crystallite size was evaluted from the XRD curves. 0.9 λ

Crystallite size D(hkl) = 2Bcos θ(hkl)

(1)


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where λ is the X-ray wavelength of the monochromatic X-ray beam (0.827 Å), B is FWHM (Full Width at Half Maximum) of XRD peak, θ (hkl) is the XRD peak position, one half of 2θ. In this work, the (002) reflection peak from the XRD pattern was used to calculate the crystallite size of hydroxyapatite. The crystallinity index of the prepared T-CNF was calculated using the Segal’s Equation (Eq. 2).42 𝐼 𝑎𝑚

Crystalinity index = (1 ― 𝐼 200)

(2)

where I200 represents the maximum intensity of the (002) lattice diffraction peak and Iam is the intensity scattered by the amorphous material. Even if this method of determination of the crystallinity is not the most accurate compared to deconvolution approach or Rietveld analysis, it is widely used for cellulosic materials. RESULTS AND DISCUSSION Characterization of T-CNF. Bagasse residue is a good source of cellulosic fibers compared to other annual biomasses.43 This widely available residue can be seriously considered as an important cellulosic source for nanocellulose production due to its high cellulose content (around 70%). TEMPO-CNF (T-CNF) was produced as mentioned in the Experimental Section. The morphology of T-CNF was investigated using AFM in tapping mode. Figure 2A shows that the diameter of T-CNF varies from 3 to 10 nm and the length is in the micrometer range and this confirmed with the diameter measurement distribution (Figure 2A*). These observations are in agreement with our previous work44 and confirm again that T-CNF has a uniform structure due to the formation of carboxylate groups on its surface. The FT-IR spectrum for T-CNF is shown in Figure 2B. From this spectrum different signals can be observed: (i)

A broad peaks at 3350 cm−1 corresponding to OH stretching vibrations.

(ii)

A signal assigned at 1247 cm-1 which confirms to C=O stretching vibration.


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A peak at 1640 cm−1 associated with the O–H bending vibration of absorbed water with some contributions from carboxylate groups.

(iv)

A signal at 1058 cm-1 corresponding to C–O stretching vibrations.

(v)

A new band appears at 1750 cm-1 corresponding to stretching of carbonyl groups (C=O) resulting from TEMPO reaction.

C

(200) (110) (004) 1750 (C=O) 2900 (C–H) 3350 (O–H)

1058 (C-O-C)

1640 (O-H) adsorbed water

B


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Figure 2. (A) AFM image and (A*) diameter distribution obtained from Nasoscope analysis software, (B) FT-IR spectrum, and (C) XRD pattern for T-CNF. The XRD analysis for T-CNF is presented in Figure 2C. From this figure, two major diffraction peaks (110) and (200) are observed for T-CNF, which are characteristic of native cellulose. The peak observed around 18° is indexed to (004) plane. These diffraction peaks are observed at lower diffraction angles than usually reported. However, it is worth noting that the wavelength used for XRD experiments (0.827 Å) is lower than for Cu Kα radiation (1.5418 Å) usually used. By applying a correction factor of 1.5418/0.827, the typical positions of the diffraction peaks for cellulose I are observed. The crystallinity index for T-CNF was calculated using the Segal method42 and a value of 0.75 was obtained. Characterization of printing pastes. The morphology of the prepared T-CNF/SA printing hydrogels was investigated by AFM. Figure 3 shows the images obtained for pure SA (panel A) and CNF50 (panel B). From this micrograph, it can be noted that alginate and T-CNF exhibit a good compatibility and no phase separation can be observed which indicates the good interaction between alginate and T-CNF as expected. A

B


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Figure 3. AFM images for (A) CNF0 and (B) CNF50 hydrogel. The printability and fidelity of T-CNF/SA hydrogels was characterized by determining their thixotropic properties and recovery times that were investigated by applying a steady shear rate, the complete test consisting of 3 steps. During the first step (1), the shear rate was fixed to 0.1 s-1 and applied for 100 s, which simulated the initial state before printing. In the second step (2), the shear rate was suddenly increased to 1000 s-1 for 100 s, simulating the printing conditions. Finally, the shear rate was suddenly reduced to 0.1 s-1 for 100 s (which corresponds to the third step (3)) to simulate a condition similar to the final state of the hydrogel after printing. Figure 4 shows the thixotropic behavior for the prepared hydrogels. The hydrogels that give high fidelity shape and printability should be highly thixotropic, which means that the viscosity of the hydrogel should rapidly drop when applying a shear force and quickly recover after the shear force is removed. It is also important to know how crosslinking of the hydrogel can recover before the next layer starts to be printed.

Figure 4. Thixotropic behavior for the different T-CNF/SA hydrogels: CNF0 (), CNF30 (), CNF50 (×), CNF70 (), and CNF100 (). For pure alginate (CNF0), the initial viscosity was 5.89×105 mPa·s, and it decreased to 75.9 mPa·s upon increasing the shear rate to 1000 s-1 (step 2). After that, when the shear rate suddenly


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decreased to 0.1 s-1 the viscosity increased to 0.95×105 mPa·s, and therefore the recovery of the viscosity was only about 16% of the initial value. For CNF50 the initial viscosity value was 1.49×105 mPa·s at step 1, then the viscosity was sharply decreased to 536 mPa·s when increasing the shear rate to 1000 s-1 and suddenly recovered a viscosity of 0.99 E×105 mPa·s at step 3. The recovery was therefore 66% of the initial value. The initial/final viscosity, recovery and corresponding photographs for all printing pastes are summarized in Table 3. It can be seen that the recovery of the past progressively increased by adding T-CNF to the alginate hydrogel up to 50% T-CNF and then decreased for higher T-CNF contents. This is confirmed from the photographs in Table 3 that show that the shape of the pure alginate (CNF0) scaffold was soft and easy to collapse, whereas the printed CNF50 filaments were uniform in width and the shape was more stable than for the other images. Table 3. Initial (step 1, ηintial) and final (step 3, ηfinal) viscosity, recovery and corresponding photographs during thixotropic tests.

ηintial (mPa·s) ηfinal (mPa·s) Recovery %

CNF100

CNF70

CNF50

CNF30

CNF0

0.39×105 0.15×105 38

0.36×105 0.15×105 42

1.49×105 0.99×105 66

3.49×105 1.73×105 50

5.89×105 0.95×105 16

Photograph

The rheological properties of the hydrogels were studied in order to further investigate the printability of T-CNF/SA pastes. Firstly, the viscosity was measured as a function of the shear rate as described in the Experimental Section. As shown in Figure 5a, all prepared hydrogels exhibit a shear-thinning behavior in the shear rate range 0.01-1000 s-1. This viscosity decrease significantly by increasing the shear rate is the most common behavior of non-Newtonian


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fluids.46,47. This behavior was ascribed to the assumption that as the shear rate increases, the hydrogel network becomes distributed, weakens and releases the entrapped liquid which resists flow and, thus, induces a decrease in the apparent viscosity. The highest viscosity value was observed for the pure alginate paste (CNF0) showing the strong impact of crosslinking. The lowest viscosity value was reported for the pure T-CNF hydrogel (CNF100) with value similar to others reported in the literature.48 Intermediate values are observed for other printing pastes. The viscosity of the CNF50 hydrogel was estimated as a function of time at various steady shear rates (0.01, 0.1, 1, 10, 100 and 1000 s-1) as shown in Figure 5B. The viscosity was constant (equilibrium) over long times during these constant shear rate experiments. A

B

C

D

Figure 5: (A) Viscosity vs. shear rate for the various hydrogel formulations at 23°C: CNF0 (), CNF30 (), CNF50 (×), CNF70 (), and CNF100 (), (B) viscosity vs. time for the CNF50


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hydrogel at different constant shear rates, (C) strain sweep study for the various hydrogels at 1 Hz: evolution of the storage modulus G' (filled symbols) and loss modulus G" (open symbols): CNF0 (,), CNF30 (,), CNF50 (,), CNF70 (,), and CNF100 (,), and (D) strain sweep study for CNF50 at 1 Hz: evolution of the storage modulus G' () and loss modulus G" (). Amplitude sweep experiments were carried out to determine the linear viscoelastic region (LVR) of the prepared hydrogels. These tests highlight the evolution of the elastic behavior and yield stress at constant frequency. The strain amplitude was determined for all hydrogels at a frequency of 1 Hz (Figure 5C). It can be noticed that for low strain values, the storage modulus (G') and the loss modulus (G") were independent of the strain amplitude and G' > G" for all samples, indicating that the material is highly structured. For higher strain values, a drop in G' is observed revealing the structural deformation and the transition of the hydrogel from elastic to viscous behavior (G" increases). The value of the strain at the transition from the LVR to the viscous region represents the critical strain (γc) which is defined at the point where G' shows 5% reduction from its LVR value (the end of the LVR).49 Increasing the strain value above the critical strain disrupts the network structure of the hydrogel which progressively shifts from solid- to liquid-like. For all hydrogels, as γc was less than 1.0 %, the strain value of 0.5% was selected to study the linear viscoelastic behavior of the prepared hydrogels to avoid structural breakdown. CNF50 appears to have a higher storage modulus (G') than other formulations, even CNF0 (pure SA). This is directly related to the extent of crosslinking and higher strength or mechanical rigidity of CNF50 compared to other formulations. In Figure 5D, it can be observed that the LVE for CNF50 extends to almost 1% at a frequency of 1 Hz as G' is independent of the strain from 0.01 to about 1% and G′ is higher than G″. Increasing the strain above the critical


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strain disrupts the network structure and G″ becomes higher than G′. In these conditions the gel starts to be destroyed and the material becomes gradually more fluid-like. The frequency sweep tests were carried out in the limit of LVR, i.e. below the critical strain at 0.5%. The frequency sweep test was used to determine the dependence of the elastic and viscous moduli on frequency. The resulting storage modulus (G') and loss modulus (G") for T-CNF/SA pastes as a function of frequency are shown in Figure 6A and 6B, respectively. All the paste formulations showed a clear gel-like behavior (G' > G"). The gel strength increased when increasing the T-CNF content, but started to decrease above 50% T-CNF, after which the gel strength stops increasing and start to decrease instead. The determination of the loss tangent or damping factor, tan δ, where δ is the phase angle (i.e. the ratio of G' to G", in term of frequency is reported in Figure 6C. The loss tangent values can be correlated to the internal network structure and the strength of the hydrogel. When tan δ is close to zero, the hydrogel is elastic and the CNF-SA network has a strongly cohesive-solid like structure. All the hydrogels containing CNF had a more solid-like structure than liquid-like as shown by through tan δ values below 0.25.


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A

B

C

Figure 6. Frequency sweep tests for T-CNF/SA hydrogels: (A) storage modulus (G'), (B) loss modulus (G"), and (C) Tan δ vs. frequency for CNF0 (), CNF30 (), CNF50 (×), CNF70 (), and CNF100 (). Mechanical properties of printed scaffolds. The compressive strength properties were determined for all printed scaffolds and measured up to 50% compressive strain without breakage except for pure alginate that breaks for a compressive strain of 30%. The compressive stress and modulus for all prepared scaffolds are collected in Table 4. Table 4. Compressive strength properties for prepared scaffolds: compressive stress at 50% compressive strain and compressive modulu. Sample Compressive stress (MPa) Compressive modulus (MPa) CNF100 87 ±15 135±60 CNF30 422 ±35 1078±98


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CNF50 CNF70 CNF0

455 ±42 419 ± 65 392± 24*

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1104±75 1233±90 1511±120*

* determined at 30% compressive strain.

The compressive strength for pure CNF scaffold (CNF100) was much lower than that for pure alginate scaffold (CNF0) and also lower than that for all other formulations. The results show that CNF30, CNF50 and CNF70 display good mechanical strength compared to pure CNF and pure alginate as expected and are close to each other. The values for the compressive modulus are also reported in Table 4 which reflects the stiffness of the scaffold. It increased from 135 MPa for pure T-CNF to 1511 MPa for pure alginate. For other formulations, a progressive increase in the modulus value is observed when increasing the alginate content up to 50 wt%. This indicates that increasing the alginate concentration improves the stiffness of the scaffold. The photographs reported in Figure 7 show the aspect of the CNF50 scaffold before (panel A), during (panel B) and after (panel C) the compressive test. It can be seen that the scaffold has a good stiffness and it still maintains its bulk morphology without collapsing after undergoing 50% compressive strain.


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Figure 7. Aspect of the CNF50 scaffold (A) before, (B) during and (C) after the compressive test (50% compressive strain). Characterization of the mineralized scaffolds. The biomimetic mineralization of hydroxyapatite was characterized using FTIR analysis. The FTIR spectra for CNF0, CNF100 and CNF50 scaffolds before mineralization and for the mineralized CNF50 scaffold after mineralization are shown in Figure 8. After mineralization, characteristic absorption bands of P– O stretching vibration mode ν3 at wavenumbers 1160 and 1049 cm-1, O–P–O bending vibration mode ν4 at 550 and 600 cm-1, and P–O mode ν1 at 894 cm-1 are observed. These peaks are dedicated to the various vibration modes of PO4−3 groups which confirm the formation of hydroxyapatite.50 All other peaks corresponding to cellulose and alginate remained the same, even after mineralization.

Figure 8. FTIR spectra for (A) pure alginate (CNF0), (B) CNF100, (C) CNF50, and (D) mineralized CNF50. Figure 9A shows the XRD patterns obtained for CNF50 before and after mineralization in 2xSBF for 14 days. By comparing with the database cart of hydroxyaptite (Reference code: 01-


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073-1731), the XRD analysis for mineralized CNF50 clearly revealed the presence of the hydroxyapatite phase with a hexagonal crystal structure and main (100), (002), (210), (112), (202), (130), (222), (213), (004) and (304) planes of hydroxyapatite indexed in Figure 9A. The crystallite size, D(hkl), of hydroxyapatite can be detected from the XRD pattern using the Scherrer equation (Eq. 1). The average size of hydroxyapatite crystallites was estimated to be 25.4 nm. This crystallite size is similar to that reported elsewhere for HAp crystals using biomimetic mineralization in SBF and is silmilar to the size of natural apatite in bone tissue.41,51,52

Figure 9. (A) XRD patterns and (B) TGA curves for CNF50 before and after mineralization. The TGA thermograms for the CNF50 scaffold before and after mineralization in SBF are shown in Figure 9B. The TGA values indicated that the final char content for CNF50 before and after mineralization was 12.4 and 32.5%, respectively that represents the whole decomposition of the organic materials at 800oC. The approximate hydroxyapatite content in the mineralized scaffold can be therefore estimated around 20.1%. The morphology of CNF50 mineralization was examined utilizing FEG-SEM (Fig. 10). Before mineralization a smooth cell structure is observed (Fig. 10A and 10C) and a homogeneous


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distribution between T-CNF and SA is reported as no T-CNF aggregates are observed. This homogeneity results from the strong interaction between T-CNF and SA and also from the crosslinking with Ca2+. After 14 days of immersion in SBF, calcium phosphate deposition was clearly observed on the surface of the nanofibers as shown in Figures 10B and 10D. Furthermore, from EDX analysis, Ca and P peaks were detected in mineralized scaffolds as well as O and C signals (Fig. 10F). Ca and P result from the formation of calcium phosphate on the scaffold. For the scaffold before mineralization, stronger carbon and oxygen peaks were observed, but a peak associated to the presence of Ca was also detected (Fig. 10E). It results from the addition of CaCl2 used for crosslinking. For the mineralized scaffold, the Ca/P ratio was 1.78 ± 0.2 which is slightly higher than the stoichiometric ratio of hydroxyapatite (1.67). This is due to the crosslinking of the scaffold with calcium ions, suggesting the formation of hydroxyapatite which was confirmed as well as by XRD experiments.


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Figure 10. FEG-SEM for CNF50 before mineralization (A and C) and after mineralization (B and D), and corresponding EDX analyses (E and F). CONCLUSIONS In this study, new T-CNF/SA hydrogel structures were developed for extrusion-based 3D printing technique. They were prepared through partial crosslinking of the hydrogel before printing using calcium chloride with concentration of 3% based on alginate dry weight. All prepared pastes show highly thixotropic behavior and the hydrogel with 50% T-CNF and 50 %


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alginate hydrogel (CNF50) displayed the highest fidelity in the reproduction of the digital object and the best printability. T-CNF/SA scaffolds exhibited excellent mechanical properties compared to pure SA and pure T-CNF under 50% compressive strain without breakage. The compressive strength was increased from 87 to 455 MPa for CNF100 and CNF50, respectively, whereas the elastic modulus increased from 135 to 1511 for CNF100 and CNF0, respectively. The biomimetic mineralization of hydroxyapatite through dipping in SBF was studied and the materialization of hydroxyapatite was confirmed using several characterization techniques such as FTIR, XRD, TGA and FEG-SEM. The approximate amount of hydroxyapatite in the mineralized scaffolds was estimated to be 20.1%. Overall, it can be concluded that mineralized T-CNF/SA 3D printed scaffolds could be a promising material for bone tissue engineering application. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest ACKNOWLEDGMENT This work was financially supported by : (i) the Embassy of France in Egypt – Institut Français d’Egypte (IFE) and Science & Technology Development Fund (STDF) in Egypt for the financial


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support (project No. 30663). (ii) 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). (iii)The “PHC Utique” program of the French Ministry of Foreign Affairs and Ministry of higher education and research and the Tunisian Ministry of higher education and scientific research in the CMCU project number 18G1132. ABBREVIATIONS T-CNF, TEMPO-oxidized cellulose nanofibril; SA, sodium alginate ; LVR, linear viscoelastic region; G', storage Modulus; G", loss Modulus; HAp, hydroxyapatite; SBF, simulated body fluid; PLA, polylactic acid; PGA, poly (glycolic acid); PCL, poly(ε-caprolactone); PDO, poly(dioxanone); PTMC, poly(trimethylene carbonate). REFERENCES (1) Hull, C. W. Apparatus for Production of Three-Dimensional Objects by Stereolithography. US Pat. 4,575,330 1986, 1-16. (2) Irvine, S. A.; Venkatraman, S. S. Bioprinting and Differentiation of Stem Cells. Molecules 2016, 21, 1188. (3) Murphy, S. V.; Atala, A. 3D Bioprinting of Tissues and Organs. Nat. Biotechnol. 2014, 32, 773-785. (4) Rengier, F.; Mehndiratta, A.; Von Tengg-Kobligk, H.; Zechmann, C.M.; Unterhinninghofen, R.; Kauczor, H.U.; Giesel, F.L. 3D Printing Based on Imaging Data: Review of Medical Applications. Int. J. Comput. Ass. Rad. 2010, 5, 335-341. (5) Mironov, V.; Kasyanov, V.; Markwald, R.R. Organ Printing: From Bioprinter to Organ Biofabrication Line. Curr. Opin. Biotechnol. 2011, 22, 667-673.


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Biomimetic Mineralization of 3D Printed Alginate/TEMPO-Oxidized Cellulose Nanofibril Scaffolds for Bone Tissue Engineering

Ragab E. Abouzeid, *†,‡ Ramzi Khiari, ‡,§,‖ Davide Beneventi, ‡ Alain Dufresne*‡

Table of Contents Graphic


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Biomimetic Mineralization of Three-Dimensional Printed Alginate

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