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Enhanced water uptake of PHBV scaffolds with functionalized cellulose nanocrystals
Polymer Testing 79 (2019) 106079
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Enhanced water uptake of PHBV scaffolds with functionalized cellulose nanocrystals
T
Thaís Larissa do Amaral Montanheiroa,b,∗, Larissa Stieven Montagnab, Viorica Patruleac, Olivier Jordanc, Gerrit Borchardc, Renata Guimarães Ribasa, Tiago Moreira Bastos Camposa, Gilmar Patrocínio Thima, Ana Paula Lemesb a Laboratory of Plasmas and Process (LPP), Technological Institute of Aeronautics (ITA), Praça Marechal Eduardo Gomes, 50 - Vila das Acacias, São José dos Campos, SP, 12228-900, Brazil b Technology Laboratory of Polymers and Biopolymers (TecPBio), Federal University of São Paulo (Unifesp), Talim, 330 – Vila Nair, São José dos Campos, SP, 12231280, Brazil c School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1 Rue Michel Servet, 1205 Geneva, Switzerland
A R T I C LE I N FO
A B S T R A C T
Keywords: Scaffold Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Cellulose nanocrystal Water uptake Tissue engineering
Super hydrophilic scaffolds of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 3 wt % of acetylated (CNC-Ac) and PEGylated (CNC-PEG) cellulose nanocrystals (CNC) were prepared. PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG scaffolds were characterized with respect to their morphology by scanning electron microscopy (SEM) and X-ray microtomography. The crystallinity was evaluated by differential scanning calorimetry (DSC) and the mechanical properties by uniaxial compression tests. The presence of residual solvent was identified by gas chromatography (GC), wettability measured by static contact angle and aqueous adsorption by gravimetry. All the scaffolds showed porous morphology, being that, for neat PHBV the morphology was more regular with oriented pores. The porosity was reduced by 26% with the introduction of CNC-Ac and CNC-PEG, and the compression modulus increased by 25% and 72% for PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds, respectively, compared to neat PHBV. Even with lower porosities, PHBV/CNC-Ac and PHBV/CNC-PEG adsorbed 16% and 67% more water than PHBV scaffold, following the intraparticle diffusion model for all the samples. No residual solvents were found and the crystallinity was slightly increased upon addition of CNC-Ac and CNC-PEG. Therefore, the addition of CNC-Ac and CNC-PEG can improve both compressive modulus and water uptake, turning PHBV nanocomposite scaffolds suitable for tissue engineering applications.
1. Introduction Scaffolds for tissue engineering are fundamental for cell growth, tissue vascularization and the formation of new tissues [1]. A wide variety of materials are being studied in the field of tissue engineering, including ceramic and polymeric matrices. Multicomponent scaffolds with tunable properties have been recently reported [2–5]. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds have been shown to be good candidates for tissue engineering, drug delivery and implants [6] since they are biocompatible, non-toxic and their degradation by-products can be metabolized [7]. However, PHBV has some shortcomings, as its fragility and low mechanical properties
[8], which hinders its effective use. Different particles have been studied to modify the properties of PHBV matrix, such as carbon nanotubes [8–11], graphite nanosheets [12–14], clays [15,16], titanium dioxide [17] and cellulosic materials [18–22]. The properties of PHBV scaffolds reinforced with cellulose nanocrystals (CNC) are still few investigated though. In our previous study [22], we have shown that CNC addition to the PHBV matrix enhances the compression modulus and cellular proliferation. CNCs are hydrophilic needle-like crystals, with excellent mechanical properties, and their non-toxicity and biocompatibility make them interesting reinforcements for biomaterials [23–27]. CNC functionalization or modification of may improve compatibility with hydrophobic
∗ Corresponding author. Laboratory of Plasmas and Process (LPP), Technological Institute of Aeronautics (ITA), Praça Marechal Eduardo Gomes, 50 - Vila das Acacias, São José dos Campos, SP, 12228-900, Brazil. E-mail addresses: [email protected] (T.L.d.A. Montanheiro), [email protected] (L.S. Montagna), [email protected] (V. Patrulea), [email protected] (O. Jordan), [email protected] (G. Borchard), [email protected] (R.G. Ribas), [email protected] (T.M.B. Campos), [email protected] (G.P. Thim), [email protected] (A.P. Lemes).
https://doi.org/10.1016/j.polymertesting.2019.106079 Received 16 July 2019; Received in revised form 15 August 2019; Accepted 24 August 2019 Available online 26 August 2019 0142-9418/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Production of the scaffolds
matrices [26]. Acetylation and PEGylation of CNC have been demonstrated to reduce their hydrophilicity, without damaging their structure, and without affecting their cytocompatibility. Moreover, acetylation has demonstrated to induce cell proliferation over time and with increasing concentration [26]. PHBV is a partially hydrophobic matrix, and both acetylation and PEGylation decrease the number of hydrogen bond interactions between CNC. This aids in their deagglomeration into partially hydrophobic matrices such as PHBV, improving the dispersion. Therefore, both functionalizations were performed to improve CNC dispersion in the PHBV matrix, and PEG was chosen for being miscible with PHBV [18,28]. We assume that PEG embeds CNCs establishing hydrogen bond interactions along the PEG chains. Few studies have evaluated the water uptake of PHBV scaffolds, as well as the water uptake kinetics, has been slightly explored. Water uptake is a valuable property that will influence the hydrolytic degradation of the scaffolds [29,30], transport of nutrients [31], whereas cell attachment and proliferation will be favored for materials with improved hydrophilicity [32]. In this work, PHBV scaffolds were reinforced with 3 wt% of acetylated CNC (CNC-Ac) and PEGylated CNC (CNC-PEG), to evaluate their morphology, dispersion of CNCs, mechanical compression modulus and, mainly the water absorption kinetics. In our previous study [22], we have evaluated bare CNC addition into PHBV scaffolds (1, 2, and 3 wt%), and we observed better mechanical reinforcement and better cell proliferation on scaffolds with 3% of CNC. For this reason, we decided to evaluate the best concentration (3%) adding functionalized CNC to the PHBV scaffolds. To the best of our knowledge, this is the first time that a research work reports the production of PHBV scaffolds reinforced with functionalized CNC and that a water absorption kinetic study is performed.
For neat PHBV scaffolds, PHBV was solubilized in dioxane at 70 °C under stirring for 2 h to give 6% w/v solution. The solution was sonicated in a Hielscher UP200S ultrasonic processor (200 W, 24 kHz; HielscherUltrasonics, Germany) with 40% of amplitude for 2 min and then 4 mL aliquots of the solutions were poured into flasks and cooled down −43 °C during 30 min. The samples were freeze-dried at −86 °C during 24 h in a Labconco Free Zone 2.5 Plus Lyophilizer (USA). For PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds, the desired amount of CNC-Ac and CNC-PEG to give 3 wt % (WCNC/WPHBV) concentration was added to PHBV and dioxane, and the same methodology used for neat PHBV scaffolds was followed. 2.4. Characterization 2.4.1. Morphology and porosity The fracture surface morphology of the scaffolds was observed by scanning electron microscopy (SEM) using an Inspect S50 – FEI Company® (USA) microscope, operating at 7,5 keV with detectors of secondary electrons. The samples were fractured at room temperature, fixed on aluminum stubs and covered with gold. The dispersion of CNC and the porosity were determined by X-Ray Microtomography (SkyScan 1272 Bruker microCT, Belgium) with a pixel resolution of 7,4 μm. The X-ray source was defined as 20 kV energy and 175 μA of current. Images of the cross sections of the scaffolds were obtained with a XIMEA xiRAY16 (2419 dpi) camera. Porosity values of the scaffolds were obtained using CTAn software (Bruker). Rectangular cuttings of 2000 × 2000 μm and with fixed height were made in different regions of the scaffolds. The position in the x, y and z-axes was varied for both the cutting and calculation of the porosity values. 2.4.2. Differential scanning calorimetry (DSC) The crystallinity degree of the scaffolds was obtained by DSC using a TA Instruments Q2000 apparatus (TA Instruments, USA). Samples were sealed in an aluminum DSC pan and heated from room temperature to 200 °C at 10 °C min−1 under nitrogen atmosphere with a flow rate of 20 mL min−1. The % degree of crystallinity (Xc) was calculated according to the relation: Xc(%)/100 = ΔHm1/WPHBV * ΔHm0, where ΔHm1 is the total melting enthalpy on heating, WPHBV is the weight fraction of PHBV in the nanocomposite (0.97), and ΔHm0 is the theoretical melting heat value of 100% crystalline PHB (146 J g−1). This value can be considered for PHBV because of the low HV content [33,34].
2. Experimental 2.1. Materials Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was provided by PHB Industrial, Brazil, with 3.76 mol% of hydroxyvalerate units and average molecular weight (Mw) of 187.000 g mol−1. Dioxane 1,4 from Synth (Brazil) was used as solvent. Cellulose nanocrystals (CNC) were produced by acid hydrolysis and supplied by CelluForce (Canada). These CNCs, as reported in our previous publications, had needle like shape with average length of 91 ± 26 nm and average diameter of 7 ± 1 nm [22]. Glacial acetic and hydrochloric acid (Merck, Germany) were used for the acetylation reaction. Polyethylene glycol (Synth PEG1500) was used for the adsorption. Dulbecco's modified Eagle medium (DMEM) and all other biological reagents were purchased from Life Technologies (Switzerland). Amicon Ultra-15 Centrifugal Filter Units with a cut-off of 10 kDa were obtained from Merck Millipore (Germany). For gas chromatography, N, N-dimethylformamide (DMF) 99.8% extra dry from Acros Organics (Belgium) was used as blank solvent.
2.4.3. Gas chromatography (GC) Evaluation of residual solvent was made by gas chromatography. A Gas chromatograph (Agilent technologies GC Agilent 6850, USA) equipped with a flame ionization detector, a Headspace sampler (Agilent 7694 headspace) was used to load the samples. Column: DB624, 30 m, 0.32 mm ID, 1.80 μm and Helium gas was used as a carrier. Samples were previously solubilized (0,01 g/mL) in DMF. The test was made according to the European Pharmacopoeia Methods of Analysis [35].
2.2. Cellulose nanocrystals functionalization 2.4.4. Uniaxial compression Compressive mechanical properties of the scaffolds were determined using a TA.XT Plus texture analyzer from Stable Micro System (United Kingdom) with a speed of 1.2 mm min−1 using an aluminum probe with 20 mm diameter and samples with 12 mm diameter and 24 mm high. Five replicates were conducted. The compressive modulus on the most linear part of the stress-strain curve was determined.
CNCs were acetylated and PEGylated according to the methodology previously reported by Montanheiro et al. [26], where acetylation was performed with acetic acid and hydrochloric acid, and adsorption of PEG was made using an aqueous solution containing 4% of PEG. Acetylated CNCs were labeled as CNC-Ac and PEGylated CNCs were labeled as CNC-PEG. CNC-Ac acetylation degree was 71%. Both functionalizations were characterized by transmission electron microscopy, infrared spectroscopy, X-ray diffraction, DLS, zeta potential, thermogravimetry, and cell viability. Fig. 1 shows a scheme of acetylation and PEGylation.
2.4.5. Contact angle Static contact-angle measurements of the scaffolds were examined in air at room temperature using a Ramé-Hart Model 500 (USA). 2
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Fig. 1. Scheme of acetylation (left) and PEGylation (right) of CNC.
determined by plotting t versus (t/qt): the slope is related to the value of qe, and the intercept is related to k2. The intraparticle adsorption-diffusion model can be expressed by Eq. (4) [39,40]:
Contact-angle values were automatically calculated using DSA software. Measurements were made by dropping 10 μL of water on a pressed disk made with the samples, which were previously frozen in liquid nitrogen and ground into powder. Contact-angle values were obtained using the average of seven measurements.
qt = kint0.5 + C
where qt is the mass of adsorbed water at time t, kin is a kinetic constant, which is directly related to the intraparticle diffusion parameter and C is the thickness of the boundary layer. The values of the parameters kin and C can be determined by a plot of qt versus t0.5, where kin is the slope and C is the intercept. This model describes the water adsorption kinetics in PHBV scaffolds.
2.4.6. Water uptake Dry scaffolds (Wd) were immersed in distilled water for 600 h at 37 °C. On selected intervals, they were removed from the water, blotted dry on filter paper to remove excess water, weighed and returned to the water, following the previous mentioned methodology [48]. The weighed scaffolds were denominated (Ww). The water uptake was obtained using Eq. (1):
WU (%) =
(Ww − Wd) x 100 Wd
2.4.7. Statistical analysis All data were expressed as means ± standard deviation (SD) and were analyzed using analysis of variance (ANOVA) and Tukey–Kramer test P < 0.05 was considered significant.
(1)
To evaluate the mechanism of water uptake, different adsorption kinetic models were investigated, by fitting the experimental data to the pseudo-first-order, pseudo-second-order, and intraparticle diffusion models. The first two equations represent a chemical mechanism of water adsorption, whereas the last one is related to physical adsorption [36]. The pseudo-first-order model describes the initial phase of adsorption given by Eq. (2) [37,38]:
kt log(q e − qt) = logq e − 1 2.303
3. Results and discussion 3.1. Morphology and porosity Fig. 2 shows the fracture surface micrographs for neat PHBV, PHBV/ CNC-Ac, and PHBV/CNC-PEG scaffolds obtained by SEM. We observe that neat PHBV scaffolds have more regular sized and oriented pores. In contrast, scaffolds with CNC-Ac and CNC-PEG could keep porosity, but with more irregular pores shape, less oriented and less homogeneous in size and distribution. Since the regular pores are attributed to freezing direction and solvent elimination path [41], and the pore distribution is due to the behavior during phase separation [42,43], we may assume that adding CNC-Ac and CNC-PEG influenced the solvent elimination path and the behavior during phase separation. SEM analysis was performed especially to evaluate the overall morphology of the scaffolds and pores distribution, once even higher magnifications are not able to show the dispersion of CNC into the scaffolds. The presence of CNC agglomerates and the scaffolds' porosity were evaluated using X-ray microtomography. Fig. 3 shows the average porosity value and a representative slice cut from the scaffolds. The color scale reflects the differences in scaffold density. It may be
(2)
Where, qt and qe are the mass of adsorbed water at time t and at equilibrium, respectively, and k1 is the rate constant of pseudo-firstorder adsorption process. The constants qe and k1 can be graphically determined by the plot log(qe-qt) versus t, where the slope is related to k1 and the intercept is log(qe). This is the most appropriate model as it describes the whole process of adsorption. The pseudo-second-order model can mathematically be described by Eq. (3) [37–39]:
t 1 t = + qt qe k2q 2e
(4)
(3)
Where k2 is the pseudo-second-order rate constant and qt and qe were previously described. The constants of Eq. (3) can be graphically
Fig. 2. –Fracture surface morphology of (A) neat PHBV, (B) PHBV/CNC-Ac, and (C) PHBV/CNC-PEG scaffolds with 600x of magnification. 3
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Fig. 3. Porosity values and microtomography rectangular slice from PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds.
observed that neat PHBV has a homogeneous structure, with subtle density differences, attributed to the porosity of the material. PHBV/ CNC-Ac and PHBV/CNC-PEG scaffolds show disordered morphology, which is in accordance with SEM images from Fig. 2, and besides that, shows areas of higher density materials. This density difference is attributed to CNC-Ac and CNC-PEG agglomerates, and relates to the disordered porous morphology. The porosity was reduced by about 26% with the introduction of CNC-Ac and CNC-PEG. As reported in our previous work, the addition of 3 wt% of bare CNC also reduced the porosity of PHBV scaffolds by about 28%, due to changes in the solution viscosity in the moment of phase separation [22], as also observed here after the introduction of CNC-Ac and CNC-PEG. 3.2. Differential scanning calorimetry DSC analyses were performed to determine the thermal behavior and crystallinity degree of PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG scaffolds. Fig. 4 presents the first heating curves for the three samples. Table 1 shows the values of crystalline melting temperature (Tm), crystalline melting enthalpy (ΔHm) and crystallinity degree (Xc). Only the first heating was evaluated to show the thermal properties related to the components and production process of the scaffolds, once in the second heating scan the thermal history is eliminated and the porous structure is destroyed. According to the data in Table 1 and Fig. 4, it can be seen that the addition of CNC-Ac and CNC-PEG did not cause significant changes in the melting and crystallization behavior of the scaffolds. Both scaffolds CNC-Ac and CNC-PEG showed a slight increase in the crystallinity degree compared to neat PHBV. The slight increase in the crystallinity degree observed for the scaffolds with CNC-Ac and CNC-PEG is lower than expected after the introduction of nanoparticles into a polymer matrix. This behavior may be attributed to the fast freezing rate or to a non-efficient dispersion of CNC-Ac and CNC-PEG into PHBV, as observed in Fig. 3. This result concerns about the components and the production process of the scaffolds, which are evaluated together on the first heating scan. The first heating reflects the crystallinity of the material as it would be used in the body, considering the components and the production process. Some works have reported the production and characterization by casting or electrospinning of PHBV and CNC nanocomposites [18,21,28,44]. All these studies report, in general, the nucleating effect of CNC in the PHBV matrix. However, the production of PHBV scaffolds with functionalized CNC was not reported yet.
Fig. 4. DSC curves from first heating for neat PHBV, PHBV/CNC-Ac, and PHBV/ CNC-PEG scaffolds. Table 1 Values of melting temperature (Tm), melting enthalpy (ΔHm) and crystallinity degree (Xc) obtained in the first heating scan for the scaffolds. Sample
Tm1 (°C)
Tm2 (°C)
ΔHm (J)
Xc (%)
PHBV PHBV/CNC-Ac PHBV/CNC-PEG
131 131 131
170 171 170
80.5 81.9 82.8
55 58 58
3.3. Gas chromatography Organic solvents are routinely used during the synthesis of drugs, excipients, or during the formulation of products. These solvents are not 4
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Fig. 5. Chromatograms showing retention time for solvents (DMF (blank) and Dioxane) investigated on neat PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds.
desired in the final product, mainly because of their toxicity, odor, and taste [45,46]. Evaluating the presence of residual solvents in scaffolds was performed using gas chromatography. The presence of dioxane in neat PHBV, PHBV/NCC-Ac, and PHBV/NCC-PEG scaffolds was investigated. Dioxane was chosen to be analyzed because it was the solvent used for scaffolds’ production. DMF was selected as the standard and as the diluent of the samples (blank) because of its ability to efficiently solubilize a wide variety of substances, and because of its boiling point (153 °C) not to be near the boiling point of dioxane (101 °C). Fig. 5 shows the chromatograms of DMF, DMF and dioxane, neat PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG, respectively. All samples show a peak at retention time of approximately 27 min. This retention time refers to DMF, the solvent used to solubilize the samples for testing. Chromatogram B, referring to dioxane + DMF, shows a peak at 18.302 min, besides the peak attributed to DMF, which is assigned to dioxane. It can be stated, from the chromatograms of Fig. 5, that no scaffold sample showed residual dioxane, once the peak at about 18 min of retention time was not detectable. Therefore, it can be concluded that the methodology used for the scaffolds production is efficient to remove all residual solvent from the sample, making them suitable for use, according to the standards of the European Pharmacopoeia [35].
3.4. Uniaxial compression Fig. 6. Compression modulus obtained for the scaffolds. Results are given as mean ± SD (n = 5). One-way ANOVA, significance levels: *p ≤ 0.05, and ***p < 0.001.
Compressive modulus of PHBV, PHBVCNC-Ac and PHBV/CNC-PEG scaffolds was determined in the linear region of the stress-strain curve; values are shown in Fig. 6. Compressive modulus of PHBV scaffold was increased of about 25% with the addition of 3 wt % of CNC-Ac and about 72% with the addition of 3 wt% of CNC-PEG. CNC and its derivatives are well known to increase tensile modulus in PHBV nanocomposites; however, the effect of CNC in the compressive modulus of PHBV scaffold is poorly reported. In our previous work [22], we reported an increase of 63% in the compressive modulus of
PHBV scaffold reinforced with 3 wt% of bare CNC. The effect of modified or functionalized CNC into PHBV scaffold has not yet been investigated. Nevertheless, PHBV/CNC-PEG scaffolds exhibited a trend of obtaining an additional increase of about 5% in the compressive modulus of PHBV/CNC-PEG scaffold, compared to bare CNC 5
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reach CNC-Ac and CNC-PEG. For this reason, the difference in water absorption is initially observed only after the first 10 h. The effect of CNC-Ac and CNC-PEG in the hydrophilicity of the scaffolds is not significant until water molecules permeate the polymer chains. All the samples were tested against the pseudo-first-order, pseudosecond-order, and intraparticle diffusion models to evaluate which one better fits. The graphical results are presented in Fig. 8-B, 8-C, and 8-D for PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG, respectively, and the obtained constants are shown in Table 2. The model that better fits for the three samples is the intraparticle diffusion model, especially over the first 240 h, suggesting similar physical mechanism of water absorption, in which water molecules permeate the scaffolds through the pores. Intraparticle diffusion rate constant (kin) increases from 24.3 mg/(g h½) in PHBV to 31.3 and 47.0 mg/(g h½) with the addition of CNC-Ac and CNC-PEG, respectively. This supports an enhanced rate of adsorption, which is linked to the addition of hydrophilic molecules into the scaffold. Application of this model to our experimental data gave a good fit plot with a correlation coefficient R2 > 0.98008 as shown in Table 2. Both samples containing CNC-Ac and CNC-PEG had lower values of porosity, but this fact was not enough to restrain the water adsorption in the scaffolds, which was much higher for samples PHBV/CNC-Ac and PHBV/CNC-PEG. Acetylation of CNC partially substitutes the –OH groups from cellulose by COCH3 from acetyl, but even though CNC-Ac remains hydrophilic, as already shown by our previous study [26], and for this reason, PHBV/CNC-Ac sample showed improved water absorption compared to neat PHBV. PEG is only adsorbed to CNC surface, then, besides all OH surface groups of CNC remain free, PEG is highly hydrophilic, which helps to increase the water absorption into the PHBV/CNC-PEG scaffold. For this reason, PHBV/CNC-PEG scaffold showed a higher percentage of water absorption. However, the kinetics of water absorption was not changed for any sample, meaning that CNC-Ac and CNC-PEG favor the permeation of water molecules into the scaffold. Herein, samples PHBV/CNC-Ac and PHBV/CNC-PEG showed less oriented structures and higher water uptake. Therefore, scaffolds with higher water uptake would have enhanced ability to transport waste and nutrients, and to store growth factors [31], being potential candidates for tissue engineering materials.
(13.46 MPa). It is expected that, up to the percolation threshold, CNC has homogeneous dispersion into the polymer matrix, and for higher concentrations, CNC agglomeration occurs, inducing stress concentration [25]. In this work, we may assume that both CNC-Ac and CNC-PEG were not homogeneously dispersed, as observed in Fig. 3. CNC-Ac, even still increasing the compressive modulus compared to neat PHBV, showed reduced modulus compared to the same scaffold produced with bare CNC. On the other hand, CNC-PEG could, even if slightly, increase the compressive modulus. This behavior may be justified by a plasticizer effect of PEG, which reduced the fragility of PHBV, delaying the collapse of the pores. However, the increase in compression modulus may not be solely attributed to the reinforcing effect of CNC-Ac and CNC-PEG, once these scaffolds had their porosity reduced. The reduction in the porosity may be the main affecting factor for the increase in the compression modulus [22,31,47]. 3.5. Contact angle The contact angle of pressed disks made from the scaffolds was obtained to evaluate the influence of CNC-Ac and CNC-PEG in the hydrophilicity of PHBV. Fig. 7 shows the average result and the standard deviation. The average value of the contact angle was not affected by the addition of 3 wt% CNC-Ac and CNC-PEG. CNC-Ac and CNC-PEG are hydrophilic materials, which may contribute to slightly increasing the hydrophilicity of the PHBV matrix. In addition, CNCs may not be exposed on the surface of the scaffolds, but embedded by the PHBV matrix, which would leave unchanged the contact angle. 3.6. Water uptake kinetics To evaluate water uptake kinetics, the scaffolds were immersed in water for 600 h, and their mass was measured in predefined times. Fig. 8-A shows the percentage of water absorbed for samples PHBV (496%), PHBV/CNC-Ac (578%), and PHBV/CNC-PEG (829%). Even with lower porosity, PHBV/CNC-Ac and PHBV-CNC-PEG scaffolds adsorbed about 16% and 67% more water than neat PHBV, respectively. All the samples achieved adsorption equilibrium around 360 h. At the initial stage of water adsorption, all samples showed similar values, which is in accordance with the values of the contact angle. It is necessary that water molecules permeate into the PHBV chains, and then
4. Conclusion PHBV scaffolds were produced by thermally induced phase separation and reinforced with acetylated and PEGylated CNC. The scaffolds were residual solvent-free from the producing step. The addition of CNC-Ac and CNC-PEG reduced the porosity and changed the overall morphology of the scaffolds. Despite low porosity, samples produced with CNC-Ac and CNC-PEG showed improved water adsorption compared to neat PHBV scaffold. Mechanical compression modulus was improved by 25% for PHBV/CNC-Ac scaffold, and by 72% for PHBV/CNC-PEG. Water absorption was 16% higher for PHBV/CNC-Ac and 67% for PHBV/CNC-PEG scaffolds, compared to neat PHBV. The kinetics mechanism was not changed after introducing CNC-Ac and CNC-PEG and the intraparticle diffusion model prevailed. The reduction in porosity, which could be seen as a disadvantage for a scaffold, caused a positive impact on the compression modulus and was overcome by the introduction of CNC-Ac and CNC-PEG, which facilitate the diffusion of water inside the scaffolds. The results of contact angle combined with water uptake showed that the hydrophilic behavior of CNC is only noticed in the scaffolds after a certain time, since PHBV embeds CNCs, and the water molecules need to diffuse between the PHBV chains until they reach the CNCs. Overall, hydrophilic nanocomposites with reinforced mechanical and improved water adsorption properties were described, potentially useful for biomedical applications.
Fig. 7. Contact angle values of neat PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds. 6
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Fig. 8. Results of water uptake (A) and kinetics fittings of PHBV (B), PHBV/CNC-Ac (C) and PHBV/CNC-PEG (D). Table 2 Kinetics studies of water uptake to PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG. SAMPLES
PHBV PHBV/CNC-Ac PHBV/CNC-PEG
Pseudo First-Order Model
Pseudo Second-Order Model
Intraparticle Diffusion Model
k1 (h−1)
qe (mg g−1)
R2
k2(h−1)
qe (mg g−1)
R2
kin (mg/(g h1/2))
R2
8.71E-03 1.41E-02 6.70E-03
469.53 477.33 861.12
0.94117 0.95577 0.98526
2.01612 2.28E-05 5.92E-06
547.06 600.21 1095.61
0.95830 0.97935 0.98916
24.31310 31.28397 46.96668
0.98008 0.98169 0.99008
Compliance with ethical standards
Acknowledgments
Funding
We would like to thank LNNano for the use of X-Ray Microtomography facility. Tayeb Jbilou (University of Geneva, Switzerland) is acknowledged for performing GC analysis.
This study was funded by the Brazilian Funding institutions CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (476131/2013-8 and 153640/2016-2) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (2013/27064-9 and 2017/ 24873-4).
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Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.
Conflict of interest The authors declare that they have no conflict of interest. 7
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Enhanced water uptake of PHBV scaffolds with functionalized cellulose nanocrystals
T
Thaís Larissa do Amaral Montanheiroa,b,∗, Larissa Stieven Montagnab, Viorica Patruleac, Olivier Jordanc, Gerrit Borchardc, Renata Guimarães Ribasa, Tiago Moreira Bastos Camposa, Gilmar Patrocínio Thima, Ana Paula Lemesb a Laboratory of Plasmas and Process (LPP), Technological Institute of Aeronautics (ITA), Praça Marechal Eduardo Gomes, 50 - Vila das Acacias, São José dos Campos, SP, 12228-900, Brazil b Technology Laboratory of Polymers and Biopolymers (TecPBio), Federal University of São Paulo (Unifesp), Talim, 330 – Vila Nair, São José dos Campos, SP, 12231280, Brazil c School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1 Rue Michel Servet, 1205 Geneva, Switzerland
A R T I C LE I N FO
A B S T R A C T
Keywords: Scaffold Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Cellulose nanocrystal Water uptake Tissue engineering
Super hydrophilic scaffolds of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 3 wt % of acetylated (CNC-Ac) and PEGylated (CNC-PEG) cellulose nanocrystals (CNC) were prepared. PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG scaffolds were characterized with respect to their morphology by scanning electron microscopy (SEM) and X-ray microtomography. The crystallinity was evaluated by differential scanning calorimetry (DSC) and the mechanical properties by uniaxial compression tests. The presence of residual solvent was identified by gas chromatography (GC), wettability measured by static contact angle and aqueous adsorption by gravimetry. All the scaffolds showed porous morphology, being that, for neat PHBV the morphology was more regular with oriented pores. The porosity was reduced by 26% with the introduction of CNC-Ac and CNC-PEG, and the compression modulus increased by 25% and 72% for PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds, respectively, compared to neat PHBV. Even with lower porosities, PHBV/CNC-Ac and PHBV/CNC-PEG adsorbed 16% and 67% more water than PHBV scaffold, following the intraparticle diffusion model for all the samples. No residual solvents were found and the crystallinity was slightly increased upon addition of CNC-Ac and CNC-PEG. Therefore, the addition of CNC-Ac and CNC-PEG can improve both compressive modulus and water uptake, turning PHBV nanocomposite scaffolds suitable for tissue engineering applications.
1. Introduction Scaffolds for tissue engineering are fundamental for cell growth, tissue vascularization and the formation of new tissues [1]. A wide variety of materials are being studied in the field of tissue engineering, including ceramic and polymeric matrices. Multicomponent scaffolds with tunable properties have been recently reported [2–5]. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds have been shown to be good candidates for tissue engineering, drug delivery and implants [6] since they are biocompatible, non-toxic and their degradation by-products can be metabolized [7]. However, PHBV has some shortcomings, as its fragility and low mechanical properties
[8], which hinders its effective use. Different particles have been studied to modify the properties of PHBV matrix, such as carbon nanotubes [8–11], graphite nanosheets [12–14], clays [15,16], titanium dioxide [17] and cellulosic materials [18–22]. The properties of PHBV scaffolds reinforced with cellulose nanocrystals (CNC) are still few investigated though. In our previous study [22], we have shown that CNC addition to the PHBV matrix enhances the compression modulus and cellular proliferation. CNCs are hydrophilic needle-like crystals, with excellent mechanical properties, and their non-toxicity and biocompatibility make them interesting reinforcements for biomaterials [23–27]. CNC functionalization or modification of may improve compatibility with hydrophobic
∗ Corresponding author. Laboratory of Plasmas and Process (LPP), Technological Institute of Aeronautics (ITA), Praça Marechal Eduardo Gomes, 50 - Vila das Acacias, São José dos Campos, SP, 12228-900, Brazil. E-mail addresses: [email protected] (T.L.d.A. Montanheiro), [email protected] (L.S. Montagna), [email protected] (V. Patrulea), [email protected] (O. Jordan), [email protected] (G. Borchard), [email protected] (R.G. Ribas), [email protected] (T.M.B. Campos), [email protected] (G.P. Thim), [email protected] (A.P. Lemes).
https://doi.org/10.1016/j.polymertesting.2019.106079 Received 16 July 2019; Received in revised form 15 August 2019; Accepted 24 August 2019 Available online 26 August 2019 0142-9418/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Production of the scaffolds
matrices [26]. Acetylation and PEGylation of CNC have been demonstrated to reduce their hydrophilicity, without damaging their structure, and without affecting their cytocompatibility. Moreover, acetylation has demonstrated to induce cell proliferation over time and with increasing concentration [26]. PHBV is a partially hydrophobic matrix, and both acetylation and PEGylation decrease the number of hydrogen bond interactions between CNC. This aids in their deagglomeration into partially hydrophobic matrices such as PHBV, improving the dispersion. Therefore, both functionalizations were performed to improve CNC dispersion in the PHBV matrix, and PEG was chosen for being miscible with PHBV [18,28]. We assume that PEG embeds CNCs establishing hydrogen bond interactions along the PEG chains. Few studies have evaluated the water uptake of PHBV scaffolds, as well as the water uptake kinetics, has been slightly explored. Water uptake is a valuable property that will influence the hydrolytic degradation of the scaffolds [29,30], transport of nutrients [31], whereas cell attachment and proliferation will be favored for materials with improved hydrophilicity [32]. In this work, PHBV scaffolds were reinforced with 3 wt% of acetylated CNC (CNC-Ac) and PEGylated CNC (CNC-PEG), to evaluate their morphology, dispersion of CNCs, mechanical compression modulus and, mainly the water absorption kinetics. In our previous study [22], we have evaluated bare CNC addition into PHBV scaffolds (1, 2, and 3 wt%), and we observed better mechanical reinforcement and better cell proliferation on scaffolds with 3% of CNC. For this reason, we decided to evaluate the best concentration (3%) adding functionalized CNC to the PHBV scaffolds. To the best of our knowledge, this is the first time that a research work reports the production of PHBV scaffolds reinforced with functionalized CNC and that a water absorption kinetic study is performed.
For neat PHBV scaffolds, PHBV was solubilized in dioxane at 70 °C under stirring for 2 h to give 6% w/v solution. The solution was sonicated in a Hielscher UP200S ultrasonic processor (200 W, 24 kHz; HielscherUltrasonics, Germany) with 40% of amplitude for 2 min and then 4 mL aliquots of the solutions were poured into flasks and cooled down −43 °C during 30 min. The samples were freeze-dried at −86 °C during 24 h in a Labconco Free Zone 2.5 Plus Lyophilizer (USA). For PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds, the desired amount of CNC-Ac and CNC-PEG to give 3 wt % (WCNC/WPHBV) concentration was added to PHBV and dioxane, and the same methodology used for neat PHBV scaffolds was followed. 2.4. Characterization 2.4.1. Morphology and porosity The fracture surface morphology of the scaffolds was observed by scanning electron microscopy (SEM) using an Inspect S50 – FEI Company® (USA) microscope, operating at 7,5 keV with detectors of secondary electrons. The samples were fractured at room temperature, fixed on aluminum stubs and covered with gold. The dispersion of CNC and the porosity were determined by X-Ray Microtomography (SkyScan 1272 Bruker microCT, Belgium) with a pixel resolution of 7,4 μm. The X-ray source was defined as 20 kV energy and 175 μA of current. Images of the cross sections of the scaffolds were obtained with a XIMEA xiRAY16 (2419 dpi) camera. Porosity values of the scaffolds were obtained using CTAn software (Bruker). Rectangular cuttings of 2000 × 2000 μm and with fixed height were made in different regions of the scaffolds. The position in the x, y and z-axes was varied for both the cutting and calculation of the porosity values. 2.4.2. Differential scanning calorimetry (DSC) The crystallinity degree of the scaffolds was obtained by DSC using a TA Instruments Q2000 apparatus (TA Instruments, USA). Samples were sealed in an aluminum DSC pan and heated from room temperature to 200 °C at 10 °C min−1 under nitrogen atmosphere with a flow rate of 20 mL min−1. The % degree of crystallinity (Xc) was calculated according to the relation: Xc(%)/100 = ΔHm1/WPHBV * ΔHm0, where ΔHm1 is the total melting enthalpy on heating, WPHBV is the weight fraction of PHBV in the nanocomposite (0.97), and ΔHm0 is the theoretical melting heat value of 100% crystalline PHB (146 J g−1). This value can be considered for PHBV because of the low HV content [33,34].
2. Experimental 2.1. Materials Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was provided by PHB Industrial, Brazil, with 3.76 mol% of hydroxyvalerate units and average molecular weight (Mw) of 187.000 g mol−1. Dioxane 1,4 from Synth (Brazil) was used as solvent. Cellulose nanocrystals (CNC) were produced by acid hydrolysis and supplied by CelluForce (Canada). These CNCs, as reported in our previous publications, had needle like shape with average length of 91 ± 26 nm and average diameter of 7 ± 1 nm [22]. Glacial acetic and hydrochloric acid (Merck, Germany) were used for the acetylation reaction. Polyethylene glycol (Synth PEG1500) was used for the adsorption. Dulbecco's modified Eagle medium (DMEM) and all other biological reagents were purchased from Life Technologies (Switzerland). Amicon Ultra-15 Centrifugal Filter Units with a cut-off of 10 kDa were obtained from Merck Millipore (Germany). For gas chromatography, N, N-dimethylformamide (DMF) 99.8% extra dry from Acros Organics (Belgium) was used as blank solvent.
2.4.3. Gas chromatography (GC) Evaluation of residual solvent was made by gas chromatography. A Gas chromatograph (Agilent technologies GC Agilent 6850, USA) equipped with a flame ionization detector, a Headspace sampler (Agilent 7694 headspace) was used to load the samples. Column: DB624, 30 m, 0.32 mm ID, 1.80 μm and Helium gas was used as a carrier. Samples were previously solubilized (0,01 g/mL) in DMF. The test was made according to the European Pharmacopoeia Methods of Analysis [35].
2.2. Cellulose nanocrystals functionalization 2.4.4. Uniaxial compression Compressive mechanical properties of the scaffolds were determined using a TA.XT Plus texture analyzer from Stable Micro System (United Kingdom) with a speed of 1.2 mm min−1 using an aluminum probe with 20 mm diameter and samples with 12 mm diameter and 24 mm high. Five replicates were conducted. The compressive modulus on the most linear part of the stress-strain curve was determined.
CNCs were acetylated and PEGylated according to the methodology previously reported by Montanheiro et al. [26], where acetylation was performed with acetic acid and hydrochloric acid, and adsorption of PEG was made using an aqueous solution containing 4% of PEG. Acetylated CNCs were labeled as CNC-Ac and PEGylated CNCs were labeled as CNC-PEG. CNC-Ac acetylation degree was 71%. Both functionalizations were characterized by transmission electron microscopy, infrared spectroscopy, X-ray diffraction, DLS, zeta potential, thermogravimetry, and cell viability. Fig. 1 shows a scheme of acetylation and PEGylation.
2.4.5. Contact angle Static contact-angle measurements of the scaffolds were examined in air at room temperature using a Ramé-Hart Model 500 (USA). 2
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Fig. 1. Scheme of acetylation (left) and PEGylation (right) of CNC.
determined by plotting t versus (t/qt): the slope is related to the value of qe, and the intercept is related to k2. The intraparticle adsorption-diffusion model can be expressed by Eq. (4) [39,40]:
Contact-angle values were automatically calculated using DSA software. Measurements were made by dropping 10 μL of water on a pressed disk made with the samples, which were previously frozen in liquid nitrogen and ground into powder. Contact-angle values were obtained using the average of seven measurements.
qt = kint0.5 + C
where qt is the mass of adsorbed water at time t, kin is a kinetic constant, which is directly related to the intraparticle diffusion parameter and C is the thickness of the boundary layer. The values of the parameters kin and C can be determined by a plot of qt versus t0.5, where kin is the slope and C is the intercept. This model describes the water adsorption kinetics in PHBV scaffolds.
2.4.6. Water uptake Dry scaffolds (Wd) were immersed in distilled water for 600 h at 37 °C. On selected intervals, they were removed from the water, blotted dry on filter paper to remove excess water, weighed and returned to the water, following the previous mentioned methodology [48]. The weighed scaffolds were denominated (Ww). The water uptake was obtained using Eq. (1):
WU (%) =
(Ww − Wd) x 100 Wd
2.4.7. Statistical analysis All data were expressed as means ± standard deviation (SD) and were analyzed using analysis of variance (ANOVA) and Tukey–Kramer test P < 0.05 was considered significant.
(1)
To evaluate the mechanism of water uptake, different adsorption kinetic models were investigated, by fitting the experimental data to the pseudo-first-order, pseudo-second-order, and intraparticle diffusion models. The first two equations represent a chemical mechanism of water adsorption, whereas the last one is related to physical adsorption [36]. The pseudo-first-order model describes the initial phase of adsorption given by Eq. (2) [37,38]:
kt log(q e − qt) = logq e − 1 2.303
3. Results and discussion 3.1. Morphology and porosity Fig. 2 shows the fracture surface micrographs for neat PHBV, PHBV/ CNC-Ac, and PHBV/CNC-PEG scaffolds obtained by SEM. We observe that neat PHBV scaffolds have more regular sized and oriented pores. In contrast, scaffolds with CNC-Ac and CNC-PEG could keep porosity, but with more irregular pores shape, less oriented and less homogeneous in size and distribution. Since the regular pores are attributed to freezing direction and solvent elimination path [41], and the pore distribution is due to the behavior during phase separation [42,43], we may assume that adding CNC-Ac and CNC-PEG influenced the solvent elimination path and the behavior during phase separation. SEM analysis was performed especially to evaluate the overall morphology of the scaffolds and pores distribution, once even higher magnifications are not able to show the dispersion of CNC into the scaffolds. The presence of CNC agglomerates and the scaffolds' porosity were evaluated using X-ray microtomography. Fig. 3 shows the average porosity value and a representative slice cut from the scaffolds. The color scale reflects the differences in scaffold density. It may be
(2)
Where, qt and qe are the mass of adsorbed water at time t and at equilibrium, respectively, and k1 is the rate constant of pseudo-firstorder adsorption process. The constants qe and k1 can be graphically determined by the plot log(qe-qt) versus t, where the slope is related to k1 and the intercept is log(qe). This is the most appropriate model as it describes the whole process of adsorption. The pseudo-second-order model can mathematically be described by Eq. (3) [37–39]:
t 1 t = + qt qe k2q 2e
(4)
(3)
Where k2 is the pseudo-second-order rate constant and qt and qe were previously described. The constants of Eq. (3) can be graphically
Fig. 2. –Fracture surface morphology of (A) neat PHBV, (B) PHBV/CNC-Ac, and (C) PHBV/CNC-PEG scaffolds with 600x of magnification. 3
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Fig. 3. Porosity values and microtomography rectangular slice from PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds.
observed that neat PHBV has a homogeneous structure, with subtle density differences, attributed to the porosity of the material. PHBV/ CNC-Ac and PHBV/CNC-PEG scaffolds show disordered morphology, which is in accordance with SEM images from Fig. 2, and besides that, shows areas of higher density materials. This density difference is attributed to CNC-Ac and CNC-PEG agglomerates, and relates to the disordered porous morphology. The porosity was reduced by about 26% with the introduction of CNC-Ac and CNC-PEG. As reported in our previous work, the addition of 3 wt% of bare CNC also reduced the porosity of PHBV scaffolds by about 28%, due to changes in the solution viscosity in the moment of phase separation [22], as also observed here after the introduction of CNC-Ac and CNC-PEG. 3.2. Differential scanning calorimetry DSC analyses were performed to determine the thermal behavior and crystallinity degree of PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG scaffolds. Fig. 4 presents the first heating curves for the three samples. Table 1 shows the values of crystalline melting temperature (Tm), crystalline melting enthalpy (ΔHm) and crystallinity degree (Xc). Only the first heating was evaluated to show the thermal properties related to the components and production process of the scaffolds, once in the second heating scan the thermal history is eliminated and the porous structure is destroyed. According to the data in Table 1 and Fig. 4, it can be seen that the addition of CNC-Ac and CNC-PEG did not cause significant changes in the melting and crystallization behavior of the scaffolds. Both scaffolds CNC-Ac and CNC-PEG showed a slight increase in the crystallinity degree compared to neat PHBV. The slight increase in the crystallinity degree observed for the scaffolds with CNC-Ac and CNC-PEG is lower than expected after the introduction of nanoparticles into a polymer matrix. This behavior may be attributed to the fast freezing rate or to a non-efficient dispersion of CNC-Ac and CNC-PEG into PHBV, as observed in Fig. 3. This result concerns about the components and the production process of the scaffolds, which are evaluated together on the first heating scan. The first heating reflects the crystallinity of the material as it would be used in the body, considering the components and the production process. Some works have reported the production and characterization by casting or electrospinning of PHBV and CNC nanocomposites [18,21,28,44]. All these studies report, in general, the nucleating effect of CNC in the PHBV matrix. However, the production of PHBV scaffolds with functionalized CNC was not reported yet.
Fig. 4. DSC curves from first heating for neat PHBV, PHBV/CNC-Ac, and PHBV/ CNC-PEG scaffolds. Table 1 Values of melting temperature (Tm), melting enthalpy (ΔHm) and crystallinity degree (Xc) obtained in the first heating scan for the scaffolds. Sample
Tm1 (°C)
Tm2 (°C)
ΔHm (J)
Xc (%)
PHBV PHBV/CNC-Ac PHBV/CNC-PEG
131 131 131
170 171 170
80.5 81.9 82.8
55 58 58
3.3. Gas chromatography Organic solvents are routinely used during the synthesis of drugs, excipients, or during the formulation of products. These solvents are not 4
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Fig. 5. Chromatograms showing retention time for solvents (DMF (blank) and Dioxane) investigated on neat PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds.
desired in the final product, mainly because of their toxicity, odor, and taste [45,46]. Evaluating the presence of residual solvents in scaffolds was performed using gas chromatography. The presence of dioxane in neat PHBV, PHBV/NCC-Ac, and PHBV/NCC-PEG scaffolds was investigated. Dioxane was chosen to be analyzed because it was the solvent used for scaffolds’ production. DMF was selected as the standard and as the diluent of the samples (blank) because of its ability to efficiently solubilize a wide variety of substances, and because of its boiling point (153 °C) not to be near the boiling point of dioxane (101 °C). Fig. 5 shows the chromatograms of DMF, DMF and dioxane, neat PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG, respectively. All samples show a peak at retention time of approximately 27 min. This retention time refers to DMF, the solvent used to solubilize the samples for testing. Chromatogram B, referring to dioxane + DMF, shows a peak at 18.302 min, besides the peak attributed to DMF, which is assigned to dioxane. It can be stated, from the chromatograms of Fig. 5, that no scaffold sample showed residual dioxane, once the peak at about 18 min of retention time was not detectable. Therefore, it can be concluded that the methodology used for the scaffolds production is efficient to remove all residual solvent from the sample, making them suitable for use, according to the standards of the European Pharmacopoeia [35].
3.4. Uniaxial compression Fig. 6. Compression modulus obtained for the scaffolds. Results are given as mean ± SD (n = 5). One-way ANOVA, significance levels: *p ≤ 0.05, and ***p < 0.001.
Compressive modulus of PHBV, PHBVCNC-Ac and PHBV/CNC-PEG scaffolds was determined in the linear region of the stress-strain curve; values are shown in Fig. 6. Compressive modulus of PHBV scaffold was increased of about 25% with the addition of 3 wt % of CNC-Ac and about 72% with the addition of 3 wt% of CNC-PEG. CNC and its derivatives are well known to increase tensile modulus in PHBV nanocomposites; however, the effect of CNC in the compressive modulus of PHBV scaffold is poorly reported. In our previous work [22], we reported an increase of 63% in the compressive modulus of
PHBV scaffold reinforced with 3 wt% of bare CNC. The effect of modified or functionalized CNC into PHBV scaffold has not yet been investigated. Nevertheless, PHBV/CNC-PEG scaffolds exhibited a trend of obtaining an additional increase of about 5% in the compressive modulus of PHBV/CNC-PEG scaffold, compared to bare CNC 5
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reach CNC-Ac and CNC-PEG. For this reason, the difference in water absorption is initially observed only after the first 10 h. The effect of CNC-Ac and CNC-PEG in the hydrophilicity of the scaffolds is not significant until water molecules permeate the polymer chains. All the samples were tested against the pseudo-first-order, pseudosecond-order, and intraparticle diffusion models to evaluate which one better fits. The graphical results are presented in Fig. 8-B, 8-C, and 8-D for PHBV, PHBV/CNC-Ac, and PHBV/CNC-PEG, respectively, and the obtained constants are shown in Table 2. The model that better fits for the three samples is the intraparticle diffusion model, especially over the first 240 h, suggesting similar physical mechanism of water absorption, in which water molecules permeate the scaffolds through the pores. Intraparticle diffusion rate constant (kin) increases from 24.3 mg/(g h½) in PHBV to 31.3 and 47.0 mg/(g h½) with the addition of CNC-Ac and CNC-PEG, respectively. This supports an enhanced rate of adsorption, which is linked to the addition of hydrophilic molecules into the scaffold. Application of this model to our experimental data gave a good fit plot with a correlation coefficient R2 > 0.98008 as shown in Table 2. Both samples containing CNC-Ac and CNC-PEG had lower values of porosity, but this fact was not enough to restrain the water adsorption in the scaffolds, which was much higher for samples PHBV/CNC-Ac and PHBV/CNC-PEG. Acetylation of CNC partially substitutes the –OH groups from cellulose by COCH3 from acetyl, but even though CNC-Ac remains hydrophilic, as already shown by our previous study [26], and for this reason, PHBV/CNC-Ac sample showed improved water absorption compared to neat PHBV. PEG is only adsorbed to CNC surface, then, besides all OH surface groups of CNC remain free, PEG is highly hydrophilic, which helps to increase the water absorption into the PHBV/CNC-PEG scaffold. For this reason, PHBV/CNC-PEG scaffold showed a higher percentage of water absorption. However, the kinetics of water absorption was not changed for any sample, meaning that CNC-Ac and CNC-PEG favor the permeation of water molecules into the scaffold. Herein, samples PHBV/CNC-Ac and PHBV/CNC-PEG showed less oriented structures and higher water uptake. Therefore, scaffolds with higher water uptake would have enhanced ability to transport waste and nutrients, and to store growth factors [31], being potential candidates for tissue engineering materials.
(13.46 MPa). It is expected that, up to the percolation threshold, CNC has homogeneous dispersion into the polymer matrix, and for higher concentrations, CNC agglomeration occurs, inducing stress concentration [25]. In this work, we may assume that both CNC-Ac and CNC-PEG were not homogeneously dispersed, as observed in Fig. 3. CNC-Ac, even still increasing the compressive modulus compared to neat PHBV, showed reduced modulus compared to the same scaffold produced with bare CNC. On the other hand, CNC-PEG could, even if slightly, increase the compressive modulus. This behavior may be justified by a plasticizer effect of PEG, which reduced the fragility of PHBV, delaying the collapse of the pores. However, the increase in compression modulus may not be solely attributed to the reinforcing effect of CNC-Ac and CNC-PEG, once these scaffolds had their porosity reduced. The reduction in the porosity may be the main affecting factor for the increase in the compression modulus [22,31,47]. 3.5. Contact angle The contact angle of pressed disks made from the scaffolds was obtained to evaluate the influence of CNC-Ac and CNC-PEG in the hydrophilicity of PHBV. Fig. 7 shows the average result and the standard deviation. The average value of the contact angle was not affected by the addition of 3 wt% CNC-Ac and CNC-PEG. CNC-Ac and CNC-PEG are hydrophilic materials, which may contribute to slightly increasing the hydrophilicity of the PHBV matrix. In addition, CNCs may not be exposed on the surface of the scaffolds, but embedded by the PHBV matrix, which would leave unchanged the contact angle. 3.6. Water uptake kinetics To evaluate water uptake kinetics, the scaffolds were immersed in water for 600 h, and their mass was measured in predefined times. Fig. 8-A shows the percentage of water absorbed for samples PHBV (496%), PHBV/CNC-Ac (578%), and PHBV/CNC-PEG (829%). Even with lower porosity, PHBV/CNC-Ac and PHBV-CNC-PEG scaffolds adsorbed about 16% and 67% more water than neat PHBV, respectively. All the samples achieved adsorption equilibrium around 360 h. At the initial stage of water adsorption, all samples showed similar values, which is in accordance with the values of the contact angle. It is necessary that water molecules permeate into the PHBV chains, and then
4. Conclusion PHBV scaffolds were produced by thermally induced phase separation and reinforced with acetylated and PEGylated CNC. The scaffolds were residual solvent-free from the producing step. The addition of CNC-Ac and CNC-PEG reduced the porosity and changed the overall morphology of the scaffolds. Despite low porosity, samples produced with CNC-Ac and CNC-PEG showed improved water adsorption compared to neat PHBV scaffold. Mechanical compression modulus was improved by 25% for PHBV/CNC-Ac scaffold, and by 72% for PHBV/CNC-PEG. Water absorption was 16% higher for PHBV/CNC-Ac and 67% for PHBV/CNC-PEG scaffolds, compared to neat PHBV. The kinetics mechanism was not changed after introducing CNC-Ac and CNC-PEG and the intraparticle diffusion model prevailed. The reduction in porosity, which could be seen as a disadvantage for a scaffold, caused a positive impact on the compression modulus and was overcome by the introduction of CNC-Ac and CNC-PEG, which facilitate the diffusion of water inside the scaffolds. The results of contact angle combined with water uptake showed that the hydrophilic behavior of CNC is only noticed in the scaffolds after a certain time, since PHBV embeds CNCs, and the water molecules need to diffuse between the PHBV chains until they reach the CNCs. Overall, hydrophilic nanocomposites with reinforced mechanical and improved water adsorption properties were described, potentially useful for biomedical applications.
Fig. 7. Contact angle values of neat PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG scaffolds. 6
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Fig. 8. Results of water uptake (A) and kinetics fittings of PHBV (B), PHBV/CNC-Ac (C) and PHBV/CNC-PEG (D). Table 2 Kinetics studies of water uptake to PHBV, PHBV/CNC-Ac and PHBV/CNC-PEG. SAMPLES
PHBV PHBV/CNC-Ac PHBV/CNC-PEG
Pseudo First-Order Model
Pseudo Second-Order Model
Intraparticle Diffusion Model
k1 (h−1)
qe (mg g−1)
R2
k2(h−1)
qe (mg g−1)
R2
kin (mg/(g h1/2))
R2
8.71E-03 1.41E-02 6.70E-03
469.53 477.33 861.12
0.94117 0.95577 0.98526
2.01612 2.28E-05 5.92E-06
547.06 600.21 1095.61
0.95830 0.97935 0.98916
24.31310 31.28397 46.96668
0.98008 0.98169 0.99008
Compliance with ethical standards
Acknowledgments
Funding
We would like to thank LNNano for the use of X-Ray Microtomography facility. Tayeb Jbilou (University of Geneva, Switzerland) is acknowledged for performing GC analysis.
This study was funded by the Brazilian Funding institutions CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (476131/2013-8 and 153640/2016-2) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (2013/27064-9 and 2017/ 24873-4).
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Conflict of interest The authors declare that they have no conflict of interest. 7
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