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Freeze-cast composite scaffolds prepared from sol-gel derived 58S bioactive glass and polycaprolactone
Ceramics International 45 (2019) 9891–9900
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Freeze-cast composite scaffolds prepared from sol-gel derived 58S bioactive glass and polycaprolactone
T
Diogo M.M. dos Santosa, Sandhra M. de Carvalhoa, Marivalda M. Pereiraa, Manuel Houmardb, Eduardo H.M. Nunesa,∗ Universidade Federal de Minas Gerais, Departamento de Engenharia Metalúrgica e de Materiais, Avenida Presidente Antônio Carlos, 6627 – Pampulha. Escola de Engenharia, Bloco 2, Sala 2233. Minas Gerais, Belo Horizonte, CEP: 31270-901, Brazil b Universidade Federal de Minas Gerais, Departamento de Engenharia de Materiais e Construção, Avenida Presidente Antônio Carlos, 6627 – Pampulha. Escola de Engenharia. Minas Gerais, Belo Horizonte, CEP: 31270-901, Brazil a
A R T I C LE I N FO
A B S T R A C T
Keywords: Composites Porosity Mechanical properties Biomedical applications Scaffolds
This work deals with the preparation of freeze-cast scaffolds using sol-gel derived 58S bioactive glass and a hypoeutectic naphthalene-camphor mixture as the starting powder and freezing vehicle, respectively. After the freeze-casting step, samples were air sintered at 1250 °C for 2 h, which led to the crystallization of 58S. The obtained scaffolds were subsequently infiltrated with poly(ε-caprolactone) (PCL), a biodegradable polymer with potential application for bone tissue repair. The prepared materials were examined by helium pycnometry, laser granulometry, scanning electron microscopy (SEM), Archimedes tests, X-ray microtomography (micro-CT), Fourier transform infrared spectroscopy (FTIR), N2 adsorption, X-ray diffraction (XRD), and uniaxial compression tests. Samples cytotoxicity was evaluated by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) and LIVE/DEAD assays. Their biocompatibility was also examined after soaking in a simulated body fluid (SBF) solution at 37 °C for up to 14 days. It was observed that the infiltration of PCL into the 58S scaffolds greatly increased their mechanical stability. Moreover, it was shown that these composites displayed a high cell viability (above 70%), which reveals that they did not interfere in the production of osteoblast cells. A hydroxyapatite coating was observed on the samples surface upon soaking in SBF, reinforcing that they are biocompatible materials. As far as we know, this is the first time that freeze-cast scaffolds were obtained using sol-gel derived 58S particles and a naphthalene-camphor mixture. Besides, as the infiltration of PCL into freeze-cast bioactive glass scaffolds improved their mechanical stability without impairing their bioactivity, this is a promising approach to prepare samples for load-bearing applications in bone tissue engineering.
1. Introduction Bioactive glass (BG) was first prepared by Hench and co-workers in early 1970's [1]. At that time, the researchers observed that this material could allow the development of interfacial bonds between the implant and surrounding tissues in the human body. Since then, BG has been widely used in the repair and regeneration of damaged tissues and organs [2]. The glass sample originally obtained by Hench et al. [1] belongs to the SiO2eCaOeP2O5eNa2O system, showing 45 mol% SiO2, 24.5 mol% CaO, 6 mol% P2O5 and 24.5 mol% Na2O. This material was prepared by the traditional melt-quenching technique and it has been called 45S5 or Bioglass®. Na2O plays a key role in this approach because it acts as a network modifier, decreasing the melting point of the glass
∗
system and also improving its processability. Different compositional variations of 45S5 has been investigated, including 58S (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5), 70S30C (70 mol% SiO2, 30 mol% CaO), and 77S (80 mol% SiO2, 16 mol% CaO, 4 mol% P2O5) [3,4]. Among the techniques commonly used for obtaining BG, the sol-gel process stands out. This chemical route allows obtaining samples with tailored compositions and pore structures [5]. Moreover, it has also been reported that sol-gel derived glasses display larger porosities than melt-quenched materials [6]. The use of materials showing interconnected macroporous structures is imperative for regenerating damaged tissues. These macroporous solids, also called scaffolds, act as temporary templates assisting tissue repair. According to Jones [7], scaffolds should stimulate the
Corresponding author. E-mail addresses: [email protected] (M. Houmard), [email protected] (E.H.M. Nunes).
https://doi.org/10.1016/j.ceramint.2019.02.030 Received 6 January 2019; Received in revised form 4 February 2019; Accepted 4 February 2019 Available online 05 February 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
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2.2. Freeze-casting
natural regeneration of the human body, allowing the proliferation of cells and formation of blood vessels. Ideally, a scaffold should degrade over time, leaving tissues to remodel naturally. Several forming methods have been used in the fabrication of ceramic scaffolds, including stereolithography [8], gel casting [9], extrusion [10], sacrificial templating [11], robocasting [12,13], and freeze-casting [14]. Freezecasting is particularly attractive because it is an environmentally friendly and cost effective technique [15]. This method is based on the directional freezing of a slurry containing a finely dispersed ceramic powder. Upon sublimating the solvent, a highly ordered pore structure is obtained. The green body is subsequently sintered in order to obtain a mechanically stable scaffold [16]. It is well established that the morphology of the obtained pore structure is strongly associated with the solvent used in the freeze-casting process [15]. For instance, water gives rise to lamellar pores, whereas camphene and tert-butanol alcohol lead to dendritic and prismatic channels [17]. It was recently reported that naphthalene-camphor (Naph-Camp) mixtures can give rise to materials with tunable pore morphologies [18]. The high melting temperature and vapor pressure of Naph-Camp allow freezing and sublimating ceramic slurries under milder conditions when compared to water-based systems [19]. Furthermore, it was demonstrated in a previous study that freeze-cast alumina scaffolds obtained from a NaphCamp hypoeutectic mixture exhibited mean pore sizes above 80 μm, which could favor osteointegration [18]. In this work 58S particles were initially obtained by sol-gel process. These particles were examined by helium pycnometry, laser granulometry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), N2 adsorption, and X-ray diffraction (XRD). Macroporous scaffolds were subsequently prepared by the freezecasting approach using a hypoeutectic Naph-Camp mixture as the freezing vehicle. The obtained scaffolds were then infiltrated with poly (ε-caprolactone) (PCL), a biodegradable polymer with potential application for bone tissue repair [20–22]. The scaffolds were evaluated by Archimedes tests, X-ray microtomography (micro-CT), SEM, and uniaxial compression tests. Samples cytotoxicity was examined by MTT and LIVE/DEAD assays. Their biocompatibility was evaluated after soaking them in a simulated body fluid (SBF) solution at 37 °C for up to 14 days. As far as we know, this is the first time that freeze-cast scaffolds were obtained using sol-gel derived 58S particles and a NaphCamp mixture. Moreover, the infiltration of PCL into freeze-cast BG scaffolds is also a promising approach to prepare samples for loadbearing applications.
As discussed before, a Naph-Camp mixture was used as the solvent in this work. Both Naph and Camp were obtained from Aldrich and show purity above 96%. It was used a hypoeutectic mixture, exhibiting a 60 wt% Camp−40 wt% Naph composition. Naph-Camp was both frozen and sublimated at room temperature. A previous work of this research group [18] revealed that alumina-based samples prepared under these conditions displayed an expressive total porosity and mean pore size. The ceramic slurries were prepared as follows. Naph-Camp was initially mixed with Texaphor 963 (Cognis, Southampton Hampshire, UK) under stirring at 70 °C. Ball-milled 58S particles were then added and the as-prepared slurry was kept under stirring for additional 3 h. Next, the system was sonicated for 15 min for breaking particle agglomerates. The 58S loading in the obtained slurries ranged from 10 to 26 vol%, whereas the Texaphor concentration was fixed at 1 wt% of the 58S amount. The initial plan was to fabricate slurries with solid loadings in the range of 10–30 vol%. However, it was not possible to prepare samples with solid loadings above 26 vol% due to the high viscosity of the obtained slurry. The suspensions were then poured into cylindrical PTFE molds (10 mm in diameter × 30 mm in height) and cooled down to room temperature. After samples solidification, they were removed from the molds and kept under ambient conditions for up to 15 days in order to allow the sublimation of the Naph-Camp hypoeutectic mixture [18]. The green bodies were initially heat-treated in air at 400 °C for 1 h with a heating rate of 2 °C.min−1. The furnace temperature was then increased to 1250 °C for 2 h using the same heating rate. 2.3. Polymer infiltration PCL (Aldrich/97%/Mn = 80,000 g mol−1) was infiltrated into the freeze-cast samples as follows [24,25]. PCL pellets were initially diluted in acetone (Aldrich) under stirring at room temperature for up to 8 h. The concentration of PCL in the as-prepared solutions ranged from 5 to 20% (wt/vol), corresponding to 50–200 g L−1. Next, freeze-cast samples were immersed in the PCL solutions and vacuumed for 5 min in order to allow the infiltration of PCL into the scaffolds structure. The samples were then removed from the PCL-containing solutions and airdried at 60 °C overnight. The loading of PCL (vol%) incorporated into the scaffolds (VPCL) was assessed by Equation (1), where m0 represents the initial mass of the scaffold (g), m1 its mass after PCL impregnation and acetone evaporation (g), ρPCL the density of PCL (1.145 g cm−3), φ the total porosity assessed for the bare scaffold in Archimedes tests (%), and V the volume of the specimen evaluated by considering its dimensions (cm3) [25]:
2. Materials and methods 2.1. 58S sol-gel synthesis
m − m0 ⎞ VPCL = ⎜⎛ 1 ⎟ × 100%. ⎝ ρPCL.ϕ.V ⎠
58S was prepared using methodology similar to that one described by Pereira et al. [23]. Briefly, Milli-Q water and nitric acid (HNO3/ Aldrich/70%) were initially mixed at room temperature. The as-prepared solution exhibited a pH about 2. Tetraethyl orthosilicate (TEOS/ Aldrich/98%) was subsequently added and the solution was kept under stirring at room temperature for 60 min. The molar ratio of TEOS: H2O was adjusted to 12: 1. Triethyl phosphate (TEP/Aldrich/≥ 99.8%) and calcium nitrate tetrahydrate (Ca(NO3)2·4H2O/Vetec/≥ 98%) were also added. It is worth mentioning that Ca(NO3)2·4H2O was added 1 h after TEP. The as-prepared solution was then stirred at room temperature for 1 h. Next, it was poured into polytetrafluoroethylene (PTFE) molds and aged at 60 °C for 72 h. The obtained monoliths were powdered and airdried at a maximum temperature of 120 °C for up to 5 days. BG particles were subsequently heat-treated in air at 700 °C for 6 h with a heating rate of 1 °C.min−1. Afterwards, BG particles were ball-milled under dry conditions overnight.
(1)
As it will be addressed further on, the impregnation step was only applied to scaffolds containing a 58S loading of 20 vol%. This decision was based on the fact that such samples displayed an interesting compromise between porosity and mechanical strength. Table 1 summarizes the samples prepared in this study. It is worth noting that they were labeled according to their preparation conditions. 2.4. Structural characterization Helium-pycnometry was conducted in a Quantachrome MVP-1 Multi Pycnometer with samples air-dried at 100 °C. Laser granulometry was performed in a Cilas 1064 granulometer using water as the dispersing medium. SEM was carried out in a Jeol JSM- 6360LV microscope at a 20 kV accelerating voltage using samples sputter-coated with a 10 nm-thick gold film. Energy dispersive spectroscopy (EDS) was performed using a Thermo Noran Quest system available in the Jeol 9892
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were incubated at 37 °C and 5% CO2 after adding 170 μL of MTT (5 mg mL−1) (Sigma-Aldrich) to them. After 4 h, 80 μL of an isopropyl alcohol−hydrochloric acid (4 vol%) solution was placed in each well. Next, 100 μL were removed from each well and transferred to a 96-well plate for measuring the absorbance (Abs) in a spectrophotometer (IMark, Bio-Rad) with a 595 nm filter. The values obtained were expressed as shown in Equation (3), where Abssamples is the absorbance measured for samples and cells, and Abscontrol the value observed for the control group:
Table 1 Samples obtained in this work. *Loading of 58S in the starting ceramic slurry. **Concentration of PCL in the acetone-based solution used in the infiltration step. Sample
Loading
Remarks
58S* (vol%)
PCL** (wt/vol)
BG10 BG20 BG26
10 20 26
0 0 0
PCL-free samples
BG20-PCL5 BG20-PCL12 BG20-PCL20
20 20 20
5 12.5 20
PCL-infiltrated scaffolds
Cell viability(%) =
4Vmes Sp
Abscontrol
× 100.
(3)
For LIVE/DEAD assay, SAOS cells were initially synchronized in a serum-free medium for 24 h. Next, 3 × 105 cells/well were seeded on the samples grown in 24-well plates. After 72 h the medium was aspirated, the cells were washed 3 times with PBS (Gibco BRL) and then treated for 30 min with a LIVE/DEAD viability/cytotoxicity kit (Life Technologies of Brazil, São Paulo) according to the supplier specifications. Images were obtained with an inverted optical microscope (Leica DMIL LED) and fluorescence was captured separately for calcein (530.0 ± 12.5 nm) and propidium iodide (645 ± 20 nm). Both MTT and LIVE/DEAD assays were conducted using BG20 and BG20-PCL12 scaffolds. Statistical tests were carried out in the analysis of the results obtained from the cytotoxicity assays (P < 0.05, N = 6, one way anova, Bonferroni test). SBF solution was prepared following the procedure suggested by Kokubo and Takadama [27]. It is well established that the SBF solution mimics the human blood plasma regarding the concentration of ions. The formation of a hydroxyapatite (HA) layer during the soaking of a material in SBF is an important reference to endorse its bioactivity because HA is responsible to both bind the implant to the bone and ensure biological functions during regeneration [2,10]. The tested materials were kept submerged in SBF at 37 °C for up to 14 days. Their concentration in the SBF solution was kept constant at 1.5 mg mL−1. The samples were examined by FTIR, SEM, EDS, and XRD.
JSM- 6360LV electron microscope. XRD was conducted in a PhilipsPanalytical PW 1710 diffractometer operating at 40 kV and 30 mA. XRD patterns were taken at a step size of 0.06° and using CuKα as the radiation source. The database of JCPDS (Joint Committee on Powder Diffraction Standards) was used as reference in these tests. N2 adsorption was carried out in a Quantachrome Nova 1200e apparatus, using samples degassed under vacuum at 150 °C for up to 12 h. The mean pore size (dp) was assessed by Equation (2), where Vmes and Sp represent, respectively, the specific mesoporous volume (cm3 g−1) and surface area (m2 g−1):
dp =
Abssamples
(2)
The specific surface area was evaluated by the multipoint BET (Brunauer-Emmett-Teller) method. FTIR was performed in a Bruker Alpha spectrometer at a resolution of 4 cm−1 and 128 scans. These tests were conducted with an attenuated total reflectance (ATR) accessory, using a diamond crystal as the reflection element. Micro-CT was performed in a Bruker SkyScan 1174 system at an X-ray voltage of 50 kV. The pixel size used in these experiments was about 10 μm. Archimedes tests were carried out in a Marte AD330 balance measuring the masses of samples in the dry, wet and submerged in water conditions. The incorporation of water into dry samples was performed by keeping them submerged in a water-containing beaker under vacuum for up to 10 min. Uniaxial compression tests were conducted at room temperature with a Shimadzu AGS-X universal testing machine at a cross-head displacement speed of 0.01 mm. seg−1. The load was applied perpendicularly to the central axis of the cylindrical scaffolds.
3. Results and discussion 3.1. As-prepared 58S particles Fig. 1a shows the N2 adsorption isotherm assessed for as-prepared
2.5. Biological tests Samples cytotoxicity was evaluated using immortalized human osteosarcoma cells (SAOS, ATCC® HTB-85) by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LIVE/DEAD assays. SAOS cells were purchased from the Rio de Janeiro cell bank (BCRJ, Federal University of Rio de Janeiro, Brazil). Prior to culture initiation, test samples were UV irradiated for 60 min on each side. SAOS cells were cultured at 37 °C in a 5% CO2 environment for 72 h in Dulbecco's modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS) (10 vol%), streptomycin sulfate (10 mg mL−1), penicillin G sodium (10 units. mL−1), and amphotericin-b (0.025 mg mL−1) supplied by Gibco BRL (USA). For MTT assay, samples were prepared according to ISO 10993-5 specifications [26]. Cell populations were initially synchronized in a serum-free medium for 24 h. After this period, 3 × 105 cells/well were seeded onto samples grown in 24-well plates. The cells were used for experiments on passage 48. Controls were used with the cells and DMEM containing 10 vol% FBS. It was used as positive control 1 vol% Triton X-100 in phosphate buffered saline (PBS, Gibco BRL, USA), and as a negative control chips of sterile polypropylene Eppendorf tubes (1 mg mL−1). After 72 h, the medium was aspirated and replaced by 210 μL culture medium with serum. Cells
Fig. 1. (a) N2 adsorption isotherm of as-prepared 58S. (b) Particle size distribution of 58S before and after ball-milling. 9893
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observed in the FTIR spectrum of 58S after sintering. The bands at 560 and 600 cm−1 are ascribed to the bending mode of PeO bonds, which has been associated with the presence of TCP [33]. The absorption bands at 710, 915 and 980 cm−1 can be related to pseudowollastonite, whereas those ones at 790 and 1060 cm−1 can be due to wollastonite [33,36]. The sharp band at 420 cm−1 was attributed to the bending mode of PeOeP bonds [37]. As a result, the additional bands observed in the FTIR spectrum of 58S after sintering reveal the formation of wollastonite, pseudowollastonite and TCP, which is in agreement with XRD (Fig. 2a). Therefore, the sintering of 58S at 1250 °C for 2 h led to the formation of crystalline phases in its structure. It is well established that the crystallization of BG decreases its bioactivity [38,39]. Nonetheless, several works report the use of glass-ceramics for load-bearing applications in bone tissue engineering [33,40–43]. The sintering of green bodies is imperative for improving the mechanical strength of freeze-cast materials. Previous tests revealed that samples heat-treated at temperatures below 1250 °C showed a poor mechanical stability, crumbling upon handling. As it will be addressed further on, the scaffolds prepared in this work displayed an expressive mechanical strength, which may favor their application in bone tissue regeneration. Thus, in spite of the crystallization of 58S, a sintering step at 1250 °C for 2 h was employed in this study for increasing the mechanical strength of the prepared scaffolds. SEM micrographs of as-prepared and sintered 58S particles are depicted in Fig. 3a. Particles showing irregular shapes are observed for the as-prepared sample. The sintering of this material caused the coalescence of 58S particles, leading to the formation of necks among them. Moreover, the sintered particles exhibited rounded shapes, whereas faceted corners were observed for as-prepared 58S. Fig. 3b shows the N2 adsorption isotherm collected for 58S after sintering. This material exhibited a type-III isotherm, which is commonly associated with macroporous materials [28]. The specific surface area, pore volume and mean pore size exhibited by this sample were 0.6 m2 g−1, 8 × 10−4 cm3 g−1 and 4.8 nm, respectively. These values are significantly smaller than those evaluated for the as-prepared material (282 m2 g−1, 0.45 cm3 g−1 and 6.4 nm). It was also evaluated a linear shrinkage about 20% of the scaffolds when their dimension before and
58S particles. This material showed both a type-IV isotherm and a H2 hysteresis loop, revealing that it exhibits a mesoporous network [28]. 58S displayed specific surface area, pore volume and mean pore size around 282 m2 g−1, 0.45 cm3 g−1 and 6.4 nm, respectively. These values are compatible with those reported by Sepulveda et al. [29] for solgel derived 58S samples. Fig. 1b depicts the particle size distribution of 58S particles before and after ball-milling. It can be clearly observed that ball-milling greatly decreased the granulometry of 58S, shifting its particle size distribution towards smaller values. Indeed, the mean particle size of 58S in the as-prepared and ball-milled conditions was about 220 and 18 μm, respectively. It is well established that the size of the starting powder used in freeze-casting shows a great effect on the prepared scaffold. According to Deville [15], the presence of large particles or particle agglomerates is detrimental to the homogeneity and properties of the final structure. Therefore, ball-milling is imperative for preparing freeze-cast scaffolds from particles obtained by the sol-gel route used in this study. Lins et al. [30] modified the chemical route first reported by Pereira et al. [23] in order to obtain nanosized BG particles. Nonetheless, only small amounts of BG can be obtained by this approach. It would be impractical to follow this procedure due to the expressive amount of material used in this work. 3.2. Effect of sintering on 58S Fig. 2a displays XRD patterns of 58S before and after sintering. No diffraction peak was observed for as-prepared 58S, revealing that it shows an amorphous structure. On the other hand, sharp diffraction peaks are noted in the XRD pattern of 58S after sintering. α-cristobalite (SiO2) and calcium metasilicate (CaSiO3 – wollastonite and pseudowollastonite) were the major crystalline phases observed in this pattern. Alpha and beta tricalcium phosphate (α- and β-TCP) were also detected. Fig. 2b exhibits the FTIR spectra of 58S in the as-prepared and sintered conditions. As-prepared 58S showed strong absorption bands centered at about 460 and 1030 cm−1, which are ascribed to SieO bonds [31,32]. The shoulder around 936 cm−1 can be related to nonbridging oxygen of SieOeCa bonds [33], whereas that one at 1200 cm−1 is due to SieOeSi bonds [34,35]. Additional features are
Fig. 2. (a) XRD pattern and (b) FTIR spectrum of 58S before and after sintering. The JCPDS files used for identifying the crystalline phases observed in the XRD patterns are indicated in Fig. 2a. 9894
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concentration of open pores are essential in the regeneration of damaged tissues [7]. The compressive strengths of these scaffolds are shown in Fig. 4b. One notices that the larger the sample porosity, the smaller its mechanical strength is. For instance, BG10, BG20 and BG26 exhibited compressive strengths of 0.15 ± 0.04, 1.52 ± 0.30 and 4.10 ± 0.65 MPa, respectively. Besides increasing the mechanical strength of the prepared scaffolds, the increase of the solid loading also decreased the strength variation. The Weibull modulus [44] evaluated for BG10, BG20 and BG26 was 3.3, 4.9 and 5.9, respectively. It is well known that the larger the Weibull modulus, the smaller the variability of mechanical strength within a group of samples [45]. Fig. 5a exhibits the pore size distribution assessed by micro-CT of scaffolds obtained with different 58S loadings. It can be observed that the larger the 58S loading, the finer the pore structure is. BG26, BG20 and BG10 displayed mean pore sizes around 45, 72 and 120 μm, respectively. This behavior is also observed in Fig. 5b, which shows SEM micrographs of these samples. Large pores exhibiting sizes up to 250 and 150 μm were respectively observed for BG10 and BG20, whereas BG26 displayed pores smaller than 80 μm. Fig. 6 shows micro-CT images obtained for BG10, BG20 and BG26. Again, it can be observed that BG26 showed a fine pore structure, whereas BG10 exhibited large pores in its structure. BG20 displayed an intermediate behavior in terms of porosity, where lamellar pores are observed. Based on the results described so far, it was decided to use BG20 as support for infiltrating PCL. This sample showed total porosity and mean pore size around 64% and 72 μm, respectively. Moreover, it displayed a significant mechanical strength (about 1.5 MPa), which should allow its application in bone tissue engineering [46]. On one hand, BG26 exhibited expressive mechanical strength (around 4 MPa). On the other hand, it displayed a fine pore structure as shown in Figs. 5 and 6. This fine porosity could decrease the scaffold bioactivity because it is likely to hinder the tissue ingrowth, nutrient transport, and angiogenesis [46]. BG10 exhibited a large porosity (≈79%) but a low compressive strength (≈0.15 MPa). This poor mechanical stability inhibits the use of BG10 for bone tissue applications.
Fig. 3. SEM micrographs of 58S in the as-prepared and sintered conditions. The scale bars shown in these images correspond to 10 μm.
after sintering was measured. These results point out that the sintering program used in this work effectively caused the densification of the 58S scaffolds. 3.3. Freeze-casting Fig. 4a exhibits the total, open, and closed porosities of scaffolds prepared using different 58S loadings. It can be clearly observed that the larger the 58S loading, the smaller the porosity of the prepared scaffold. BG10, BG20 and BG26 displayed, respectively, total porosities of 79.0 ± 3.5, 64.4 ± 1.5 and 59.2 ± 1.0%. It is well established that the pore network of freeze-cast specimens is a direct replica of the solvent structure [15]. Thus, it is expected that the lower the concentration of solids in a slurry, the larger should be the porosity of the material obtained after freeze-drying [16]. This behavior is in agreement with the trend observed in Fig. 4a. It is also worth highlighting the expressive concentration of open pores in the scaffolds prepared herein. This is an important finding because scaffolds displaying a significant
3.4. PCL-infiltrated samples As aforementioned, the volume of PCL infiltrated into the BG scaffolds was assessed by Equation (1). It was observed that acetone solutions showing PCL concentrations of 5, 12.5 and 20% (wt/vol) gave rise to samples with polymer loadings of 3.4 ± 0.4, 14.1 ± 1.0 and 29.0 ± 2.3 vol%, respectively. Fig. 7a depicts the scaffolds porosity as a function of the PCL loading. One observes that the larger the PCL concentration, the smaller the scaffold porosity. Fig. 7b displays SEM micrographs of the fracture surface for BG20 and PCL-containing scaffolds. PCL fibers bridging BG particles are noticed in these images, revealing that the polymer successfully penetrated into the scaffolds structure. Again, it can be observed a round shape of the BG particles, revealing that the sintering step was successfully conducted. Fig. 8a exhibits the stress-strain curves obtained for BG20 and PCLinfiltrated scaffolds, whereas Fig. 8b depicts the assessed compressive strength. The incorporation of PCL increased the mechanical stability of BG20; this behavior was more pronounced for BG20-PCL12 and BG20PCL20. No apparent strengthening effect was observed for BG20-PCL5, possibly due to the small concentration of PCL in this material (3.4 ± 0.4 vol%). However, although BG20 and BG20-PCL5 displayed similar compressive strengths, the latter exhibited a higher toughness in terms of the energy absorbed upon fracture. The so-called fracture toughness is associated with the area under the stress-strain curve. For comparison purposes, it was evaluated the area under the stress-strain curves shown in Fig. 8a by considering a strain up to 0.15. BG20, BG20PCL5, BG20-PCL12 and BG20-PCL20 displayed values around 0.8 × 105, 1 × 105, 2.7 × 105 and 3.8 × 105 J m−3, respectively. These results quantitatively reinforce the increase of the fracture toughness of
Fig. 4. (a) Total, open and closed porosities of scaffolds obtained from slurries containing different 58S loadings. (b) Compressive strength of the 58S scaffolds prepared herein as a function of the total porosity. The dashed straight lines connecting the data points are used as a guide to the eyes only. 9895
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Fig. 5. (a) Pore size distribution evaluated by micro-CT and (b) SEM micrographs of 58S scaffolds prepared from slurries with distinct solid loadings. The scale bars observed in SEM micrographs represent 1 mm.
Fig. 6. Micro-CT images of BG10, BG20 and BG26. The dark regions are pores whereas the white ones represent the solid phase. The scale bars shown in these images correspond to 1 mm.
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Fig. 7. (a) Porosity as a function of the PCL loading in BG scaffolds. (b) SEM micrographs of the fracture surface for PCL-containing samples. The scaffolds were fractured under liquid nitrogen for promoting the brittle failure of PCL. The porosities given in Fig. 7a were assessed from Archimedes and gravimetric tests. The scale bars displayed in Figure 7b correspond to 30 μm.
3.5. Biocompatibility tests SAOS cells have been widely used as an experimental model for osteoblasts because their cultures are easy to handle and proliferate. In addition, the ability of SAOS cells to generate a mineralized matrix when stimulated, makes them a suitable experimental model to study mineralized tissues [47]. Fig. 9a show images obtained from the LIVE/ DEAD assay. The LIVE/DEAD assay is a two-color fluorescence cell viability test based on the simultaneous determination of live (green luminescence) and dead (red luminescence) cells. It can be observed that BG20 and BG20-PCL12 displayed behavior similar to the control group in terms of the green fluorescence (live cells). However, more red dots (dead cells) were noted for BG20 and BG20-PCL12. Fig. 9b displays the results obtained from the MTT assay. BG20 scaffolds exhibited greater cell viability than the control group, whereas BG20-PCL12 showed a similar behavior to the latter one. Both BG20 and BG20PCL12 displayed cell viability above 70%, which reveals that they did not interfere in the production of SAOS cells. The smaller cell viability displayed by BG20-PCL12 when compared to BG20 can be related to the presence of PCL in the former. As depicted in Fig. 7a, the infiltration of PCL into the BG scaffolds decreased their porosity. It is well established that the biological response of BG is strongly related to its porosity [48]. Thus, the decrease in porosity observed when PCL was infiltrated into the 58S scaffolds can explain the smaller cell viability exhibited by BG20-PCL12 when compared to BG20. Moreover, one must take into account the significant decrease in the specific surface area (280 against 0.6 m2 g−1), pore volume (0.45 against 8 × 10−4 cm3 g−1) and mean pore size (6.4 against 4.8 nm) of 58S when it was sintered at 1250 °C. The small porosity shown by 58S after sintering may account to the biological response exhibited by BG20 and BG20-PCL12. It has been reported that the larger the porosity of BG, the higher its bioactivity is [49]. Fig. 10 depicts SEM micrographs and EDS spectra of BG20 and BG20-PCL12 after soaking in SBF for 14 days. For reference purposes, data ascribed to as-prepared BG20 are also shown. Cauliflower-like nodules are noticed in the micrographs of BG20 and BG20-PCL12 after the soaking in SBF, which are commonly observed for HA [50]. One observes that such nodules are not present in the structure of as-prepared BG20. The EDS spectra displayed in Fig. 10 reveals that the Ca/P ratio is different for as-prepared or SBF-soaked materials. Indeed, this ratio was about 7 for as-prepared BG, and a value around 2–3 was measured for specimens after soaking in SBF. This change in the Ca/P ratio indicates the formation of HA in the structure of the SBF-soaked
Fig. 8. (a) Typical stress-strain curves obtained for BG20, BG20-PCL5, BG20PCL12 and BG20-PCL20. (b) Compressive strength as a function of the PCL loading in BG scaffolds.
the 58S scaffolds when PCL is infiltrated into them. BG20-PCL12 and BG20-PCL20 displayed a remarkable strengthening effect in terms of both compressive strength and fracture toughness. It can be noted that this behavior is more evident the higher the PCL loading in the scaffold. The smoother shape of the stress-strain curves obtained for PCL-infiltrated samples also indicates that the composite retained some significant mechanical resistance even after multiple cracking events. According to Martínez-Vázquez et al. [24], the incorporation of polymers into ceramic structures leads to the transference of loading from the ceramic to polymer phase, which improves the mechanical strength of the scaffold. Moreover, the polymer can fill pre-existing defects in the scaffold structure, bridging ceramic particles and increasing the stress needed to propagate cracks. The bridging of BG particles by PCL is clearly observed in Fig. 7b. Moreover, besides improving the compressive strength and fracture toughness of the bare scaffold (BG20), the incorporation of PCL also increased its Weibull modulus from 4.9 to about 9.7.
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Fig. 9. Results obtained from (a) LIVE/DEAD and (b) MTT assays. The green and red luminescences correspond to live and dead cells, respectively. The scale bars shown in Fig. 9a are equivalent to 100 μm. The dashed line exhibited in Fig. 9b is associated with a cell viability of 70%. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
sintered 58S. The freeze-cast scaffolds displayed a highly interconnected and open macroporous structure. The increase of the 58S loading in the starting slurry resulted in samples with larger compressive strengths. The infiltration of PCL into the 58S scaffolds increased their mechanical stability in terms of both compressive strength and fracture toughness. This behavior is more evident the higher the PCL loading is. PCL fibers bridging BG particles were noticed in the SEM micrographs of PCL-infiltrated scaffolds. It appears that PCL can fill preexisting defects in the scaffold structure, bridging ceramic particles and increasing the stress needed to propagate cracks. Cytotoxicity tests revealed that the PCL-infiltrated scaffolds did not interfere in the production of osteoblast cells. Moreover, it was observed the growth of HA
samples. The formation of HA on the tested samples was also confirmed by FTIR and XRD (not shown in this work). These results attest the biocompatible behavior of BG20 and BG20-PCL12, which is in line with the MTT and LIVE/DEAD assays (Fig. 9). 4. Conclusions It was observed that the sintering of 58S at 1250 °C for 2 h greatly decreased its specific surface area, pore volume and mean pore size. Moreover, this heat treatment also led to the crystallization of this material. α-cristobalite, calcium metasilicate (wollastonite and pseudowollastonite), alpha and beta tricalcium phosphate were observed in
Fig. 10. SEM micrographs and EDS spectra obtained for BG20 and BG20-PCL12 after soaking in SBF for 14 days. The data collected for as-prepared BG20 are also exhibited for comparison purposes. The scale bars exhibited in the SEM micrographs correspond to either 10 or 30 μm. 9898
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on the surface of a PCL-infiltrated scaffold upon soaking in SBF for 14 days. The findings described in this work suggest that the composite scaffolds prepared herein are promising candidates for application in bone tissue repair.
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Acknowledgements The authors thank the financial support from JHS Biomaterials, CAPES (PROEX), CNPq (301423/2018), FAPEMIG (APQ-00852-17) and PRPq-UFMG (05/2016). We also thank LAMPAC (Prof. Tulio Matencio, Welerson Lima and Renata Martins), INCT-Acqua (Ilda Batista) for the great support to this study. Patrícia Azevedo, Samuel Lima, Leonardo Mayer and Prof. Dagoberto Brandão are greatly acknowledged for performing SEM, XRD and compression tests. Prof. Paulo Brandão, Eric Mazzer, Isabel Batista and Fábio Almeida are also appreciated. References [1] L.L. Hench, R.J. Splinter, W.C. Allen, T.K. Greenlee, Bonding mechanisms at the interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 5 (1971) 117–141, https://doi.org/10.1002/jbm.820050611. [2] M.N. Rahaman, D.E. Day, B. Sonny Bal, Q. Fu, S.B. Jung, L.F. Bonewald, A.P. Tomsia, Bioactive glass in tissue engineering, Acta Biomater. 7 (2011) 2355–2373, https://doi.org/10.1016/j.actbio.2011.03.016. [3] L. Ji, W. Qiao, K. Huang, Y. Zhang, H. Wu, S. Miao, H. Liu, Y. Dong, A. Zhu, D. Qiu, Synthesis of nanosized 58S bioactive glass particles by a three-dimensional ordered macroporous carbon template, Mater. Sci. Eng. C 75 (2017) 590–595, https://doi. org/10.1016/j.msec.2017.02.107. [4] C.-L. Huang, W. Fang, I.-H. Chen, T.-Y. Hung, Manufacture and biomimetic mineral deposition of nanoscale bioactive glasses with mesoporous structures using sol-gel methods, Ceram. Int. 44 (2018) 17224–17229, https://doi.org/10.1016/j.ceramint. 2018.06.180. [5] M.M. Pereira, J.R. Jones, L.L. Hench, Bioactive glass and hybrid scaffolds prepared by sol–gel method for bone tissue engineering, Adv. Appl. Ceram. 104 (2005) 35–42, https://doi.org/10.1179/174367605225011034. [6] G. Kaur, G. Pickrell, N. Sriranganathan, V. Kumar, D. Homa, Review and the state of the art: sol–gel and melt quenched bioactive glasses for tissue engineering, J. Biomed. Mater. Res. B Appl. Biomater. 104 (2016) 1248–1275, https://doi.org/10. 1002/jbm.b.33443. [7] J.R. Jones, Reprint of: review of bioactive glass: from Hench to hybrids, Acta Biomater. 23 (2015) S53–S82, https://doi.org/10.1016/j.actbio.2015.07.019. [8] B. Thavornyutikarn, P. Tesavibul, K. Sitthiseripratip, N. Chatarapanich, B. Feltis, P.F.A. Wright, T.W. Turney, Porous 45S5 Bioglass®-based scaffolds using stereolithography: effect of partial pre-sintering on structural and mechanical properties of scaffolds, Mater. Sci. Eng. C 75 (2017) 1281–1288, https://doi.org/10.1016/j. msec.2017.03.001. [9] H. Elsayed, A.R. Romero, G. Molino, C.V. Brovarone, E. Bernardo, Bioactive glassceramic foam scaffolds from “inorganic gel casting” and sinter-crystallization, Materials (Basel) 10 (2018) 171–186, https://doi.org/10.3390/ma11030349. [10] M. Elbadawi, Z.J. Wally, I.M. Reaney, Porous hydroxyapatite-bioactive glass hybrid scaffolds fabricated via ceramic honeycomb extrusion, J. Am. Ceram. Soc. 101 (2018) 3541–3556, https://doi.org/10.1111/jace.15514. [11] S.L. McNamara, J. Rnjak-Kovacina, D.F. Schmidt, T.J. Lo, D.L. Kaplan, Silk as a biocohesive sacrificial binder in the fabrication of hydroxyapatite load bearing scaffolds, Biomaterials 35 (2014) 6941–6953, https://doi.org/10.1016/j. biomaterials.2014.05.013. [12] M. Houmard, Q. Fu, E. Saiz, A.P. Tomsia, Sol-gel method to fabricate CaP scaffolds by robocasting for tissue engineering, J. Mater. Sci. Mater. Med. 23 (2012) 921–930, https://doi.org/10.1007/s10856-012-4561-2. [13] M. Houmard, Q. Fu, M. Genet, E. Saiz, A.P. Tomsia, On the structural, mechanical, and biodegradation properties of HA/β-TCP robocast scaffolds, J. Biomed. Mater. Res. B Appl. Biomater. 101 (2013) 1233–1242, https://doi.org/10.1002/jbm.b. 32935. [14] R. Liu, T. Xu, C. an Wang, A review of fabrication strategies and applications of porous ceramics prepared by freeze-casting method, Ceram. Int. 42 (2016) 2907–2925, https://doi.org/10.1016/j.ceramint.2015.10.148. [15] S. Deville, Freeze-casting of porous ceramics: a review of current achievements and issues, Adv. Eng. Mater. 10 (2008) 155–169, https://doi.org/10.1002/adem. 200700270. [16] D.F. Souza, E.H.M. Nunes, D.S. Pimenta, D.C.L. Vasconcelos, J.F. Nascimento, W. Grava, M. Houmard, W.L. Vasconcelos, Synthesis and structural evaluation of freeze-cast porous alumina, Mater. Char. 96 (2014) 183–195, https://doi.org/10. 1016/j.matchar.2014.08.009. [17] K.L. Scotti, D.C. Dunand, Freeze casting – a review of processing, microstructure and properties via the open data repository, FreezeCasting.net, Prog. Mater. Sci. 94 (2018) 243–305, https://doi.org/10.1016/j.pmatsci.2018.01.001. [18] L.D. Lacerda, D.F. Souza, E.H.M. Nunes, M. Houmard, Macroporous alumina structures tailored by freeze-casting using naphthalene–camphor as freezing vehicle, Ceram. Int. 44 (2018) 16010–16016, https://doi.org/10.1016/j.ceramint. 2018.06.036.
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Freeze-cast composite scaffolds prepared from sol-gel derived 58S bioactive glass and polycaprolactone
T
Diogo M.M. dos Santosa, Sandhra M. de Carvalhoa, Marivalda M. Pereiraa, Manuel Houmardb, Eduardo H.M. Nunesa,∗ Universidade Federal de Minas Gerais, Departamento de Engenharia Metalúrgica e de Materiais, Avenida Presidente Antônio Carlos, 6627 – Pampulha. Escola de Engenharia, Bloco 2, Sala 2233. Minas Gerais, Belo Horizonte, CEP: 31270-901, Brazil b Universidade Federal de Minas Gerais, Departamento de Engenharia de Materiais e Construção, Avenida Presidente Antônio Carlos, 6627 – Pampulha. Escola de Engenharia. Minas Gerais, Belo Horizonte, CEP: 31270-901, Brazil a
A R T I C LE I N FO
A B S T R A C T
Keywords: Composites Porosity Mechanical properties Biomedical applications Scaffolds
This work deals with the preparation of freeze-cast scaffolds using sol-gel derived 58S bioactive glass and a hypoeutectic naphthalene-camphor mixture as the starting powder and freezing vehicle, respectively. After the freeze-casting step, samples were air sintered at 1250 °C for 2 h, which led to the crystallization of 58S. The obtained scaffolds were subsequently infiltrated with poly(ε-caprolactone) (PCL), a biodegradable polymer with potential application for bone tissue repair. The prepared materials were examined by helium pycnometry, laser granulometry, scanning electron microscopy (SEM), Archimedes tests, X-ray microtomography (micro-CT), Fourier transform infrared spectroscopy (FTIR), N2 adsorption, X-ray diffraction (XRD), and uniaxial compression tests. Samples cytotoxicity was evaluated by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) and LIVE/DEAD assays. Their biocompatibility was also examined after soaking in a simulated body fluid (SBF) solution at 37 °C for up to 14 days. It was observed that the infiltration of PCL into the 58S scaffolds greatly increased their mechanical stability. Moreover, it was shown that these composites displayed a high cell viability (above 70%), which reveals that they did not interfere in the production of osteoblast cells. A hydroxyapatite coating was observed on the samples surface upon soaking in SBF, reinforcing that they are biocompatible materials. As far as we know, this is the first time that freeze-cast scaffolds were obtained using sol-gel derived 58S particles and a naphthalene-camphor mixture. Besides, as the infiltration of PCL into freeze-cast bioactive glass scaffolds improved their mechanical stability without impairing their bioactivity, this is a promising approach to prepare samples for load-bearing applications in bone tissue engineering.
1. Introduction Bioactive glass (BG) was first prepared by Hench and co-workers in early 1970's [1]. At that time, the researchers observed that this material could allow the development of interfacial bonds between the implant and surrounding tissues in the human body. Since then, BG has been widely used in the repair and regeneration of damaged tissues and organs [2]. The glass sample originally obtained by Hench et al. [1] belongs to the SiO2eCaOeP2O5eNa2O system, showing 45 mol% SiO2, 24.5 mol% CaO, 6 mol% P2O5 and 24.5 mol% Na2O. This material was prepared by the traditional melt-quenching technique and it has been called 45S5 or Bioglass®. Na2O plays a key role in this approach because it acts as a network modifier, decreasing the melting point of the glass
∗
system and also improving its processability. Different compositional variations of 45S5 has been investigated, including 58S (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5), 70S30C (70 mol% SiO2, 30 mol% CaO), and 77S (80 mol% SiO2, 16 mol% CaO, 4 mol% P2O5) [3,4]. Among the techniques commonly used for obtaining BG, the sol-gel process stands out. This chemical route allows obtaining samples with tailored compositions and pore structures [5]. Moreover, it has also been reported that sol-gel derived glasses display larger porosities than melt-quenched materials [6]. The use of materials showing interconnected macroporous structures is imperative for regenerating damaged tissues. These macroporous solids, also called scaffolds, act as temporary templates assisting tissue repair. According to Jones [7], scaffolds should stimulate the
Corresponding author. E-mail addresses: [email protected] (M. Houmard), [email protected] (E.H.M. Nunes).
https://doi.org/10.1016/j.ceramint.2019.02.030 Received 6 January 2019; Received in revised form 4 February 2019; Accepted 4 February 2019 Available online 05 February 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
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2.2. Freeze-casting
natural regeneration of the human body, allowing the proliferation of cells and formation of blood vessels. Ideally, a scaffold should degrade over time, leaving tissues to remodel naturally. Several forming methods have been used in the fabrication of ceramic scaffolds, including stereolithography [8], gel casting [9], extrusion [10], sacrificial templating [11], robocasting [12,13], and freeze-casting [14]. Freezecasting is particularly attractive because it is an environmentally friendly and cost effective technique [15]. This method is based on the directional freezing of a slurry containing a finely dispersed ceramic powder. Upon sublimating the solvent, a highly ordered pore structure is obtained. The green body is subsequently sintered in order to obtain a mechanically stable scaffold [16]. It is well established that the morphology of the obtained pore structure is strongly associated with the solvent used in the freeze-casting process [15]. For instance, water gives rise to lamellar pores, whereas camphene and tert-butanol alcohol lead to dendritic and prismatic channels [17]. It was recently reported that naphthalene-camphor (Naph-Camp) mixtures can give rise to materials with tunable pore morphologies [18]. The high melting temperature and vapor pressure of Naph-Camp allow freezing and sublimating ceramic slurries under milder conditions when compared to water-based systems [19]. Furthermore, it was demonstrated in a previous study that freeze-cast alumina scaffolds obtained from a NaphCamp hypoeutectic mixture exhibited mean pore sizes above 80 μm, which could favor osteointegration [18]. In this work 58S particles were initially obtained by sol-gel process. These particles were examined by helium pycnometry, laser granulometry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), N2 adsorption, and X-ray diffraction (XRD). Macroporous scaffolds were subsequently prepared by the freezecasting approach using a hypoeutectic Naph-Camp mixture as the freezing vehicle. The obtained scaffolds were then infiltrated with poly (ε-caprolactone) (PCL), a biodegradable polymer with potential application for bone tissue repair [20–22]. The scaffolds were evaluated by Archimedes tests, X-ray microtomography (micro-CT), SEM, and uniaxial compression tests. Samples cytotoxicity was examined by MTT and LIVE/DEAD assays. Their biocompatibility was evaluated after soaking them in a simulated body fluid (SBF) solution at 37 °C for up to 14 days. As far as we know, this is the first time that freeze-cast scaffolds were obtained using sol-gel derived 58S particles and a NaphCamp mixture. Moreover, the infiltration of PCL into freeze-cast BG scaffolds is also a promising approach to prepare samples for loadbearing applications.
As discussed before, a Naph-Camp mixture was used as the solvent in this work. Both Naph and Camp were obtained from Aldrich and show purity above 96%. It was used a hypoeutectic mixture, exhibiting a 60 wt% Camp−40 wt% Naph composition. Naph-Camp was both frozen and sublimated at room temperature. A previous work of this research group [18] revealed that alumina-based samples prepared under these conditions displayed an expressive total porosity and mean pore size. The ceramic slurries were prepared as follows. Naph-Camp was initially mixed with Texaphor 963 (Cognis, Southampton Hampshire, UK) under stirring at 70 °C. Ball-milled 58S particles were then added and the as-prepared slurry was kept under stirring for additional 3 h. Next, the system was sonicated for 15 min for breaking particle agglomerates. The 58S loading in the obtained slurries ranged from 10 to 26 vol%, whereas the Texaphor concentration was fixed at 1 wt% of the 58S amount. The initial plan was to fabricate slurries with solid loadings in the range of 10–30 vol%. However, it was not possible to prepare samples with solid loadings above 26 vol% due to the high viscosity of the obtained slurry. The suspensions were then poured into cylindrical PTFE molds (10 mm in diameter × 30 mm in height) and cooled down to room temperature. After samples solidification, they were removed from the molds and kept under ambient conditions for up to 15 days in order to allow the sublimation of the Naph-Camp hypoeutectic mixture [18]. The green bodies were initially heat-treated in air at 400 °C for 1 h with a heating rate of 2 °C.min−1. The furnace temperature was then increased to 1250 °C for 2 h using the same heating rate. 2.3. Polymer infiltration PCL (Aldrich/97%/Mn = 80,000 g mol−1) was infiltrated into the freeze-cast samples as follows [24,25]. PCL pellets were initially diluted in acetone (Aldrich) under stirring at room temperature for up to 8 h. The concentration of PCL in the as-prepared solutions ranged from 5 to 20% (wt/vol), corresponding to 50–200 g L−1. Next, freeze-cast samples were immersed in the PCL solutions and vacuumed for 5 min in order to allow the infiltration of PCL into the scaffolds structure. The samples were then removed from the PCL-containing solutions and airdried at 60 °C overnight. The loading of PCL (vol%) incorporated into the scaffolds (VPCL) was assessed by Equation (1), where m0 represents the initial mass of the scaffold (g), m1 its mass after PCL impregnation and acetone evaporation (g), ρPCL the density of PCL (1.145 g cm−3), φ the total porosity assessed for the bare scaffold in Archimedes tests (%), and V the volume of the specimen evaluated by considering its dimensions (cm3) [25]:
2. Materials and methods 2.1. 58S sol-gel synthesis
m − m0 ⎞ VPCL = ⎜⎛ 1 ⎟ × 100%. ⎝ ρPCL.ϕ.V ⎠
58S was prepared using methodology similar to that one described by Pereira et al. [23]. Briefly, Milli-Q water and nitric acid (HNO3/ Aldrich/70%) were initially mixed at room temperature. The as-prepared solution exhibited a pH about 2. Tetraethyl orthosilicate (TEOS/ Aldrich/98%) was subsequently added and the solution was kept under stirring at room temperature for 60 min. The molar ratio of TEOS: H2O was adjusted to 12: 1. Triethyl phosphate (TEP/Aldrich/≥ 99.8%) and calcium nitrate tetrahydrate (Ca(NO3)2·4H2O/Vetec/≥ 98%) were also added. It is worth mentioning that Ca(NO3)2·4H2O was added 1 h after TEP. The as-prepared solution was then stirred at room temperature for 1 h. Next, it was poured into polytetrafluoroethylene (PTFE) molds and aged at 60 °C for 72 h. The obtained monoliths were powdered and airdried at a maximum temperature of 120 °C for up to 5 days. BG particles were subsequently heat-treated in air at 700 °C for 6 h with a heating rate of 1 °C.min−1. Afterwards, BG particles were ball-milled under dry conditions overnight.
(1)
As it will be addressed further on, the impregnation step was only applied to scaffolds containing a 58S loading of 20 vol%. This decision was based on the fact that such samples displayed an interesting compromise between porosity and mechanical strength. Table 1 summarizes the samples prepared in this study. It is worth noting that they were labeled according to their preparation conditions. 2.4. Structural characterization Helium-pycnometry was conducted in a Quantachrome MVP-1 Multi Pycnometer with samples air-dried at 100 °C. Laser granulometry was performed in a Cilas 1064 granulometer using water as the dispersing medium. SEM was carried out in a Jeol JSM- 6360LV microscope at a 20 kV accelerating voltage using samples sputter-coated with a 10 nm-thick gold film. Energy dispersive spectroscopy (EDS) was performed using a Thermo Noran Quest system available in the Jeol 9892
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were incubated at 37 °C and 5% CO2 after adding 170 μL of MTT (5 mg mL−1) (Sigma-Aldrich) to them. After 4 h, 80 μL of an isopropyl alcohol−hydrochloric acid (4 vol%) solution was placed in each well. Next, 100 μL were removed from each well and transferred to a 96-well plate for measuring the absorbance (Abs) in a spectrophotometer (IMark, Bio-Rad) with a 595 nm filter. The values obtained were expressed as shown in Equation (3), where Abssamples is the absorbance measured for samples and cells, and Abscontrol the value observed for the control group:
Table 1 Samples obtained in this work. *Loading of 58S in the starting ceramic slurry. **Concentration of PCL in the acetone-based solution used in the infiltration step. Sample
Loading
Remarks
58S* (vol%)
PCL** (wt/vol)
BG10 BG20 BG26
10 20 26
0 0 0
PCL-free samples
BG20-PCL5 BG20-PCL12 BG20-PCL20
20 20 20
5 12.5 20
PCL-infiltrated scaffolds
Cell viability(%) =
4Vmes Sp
Abscontrol
× 100.
(3)
For LIVE/DEAD assay, SAOS cells were initially synchronized in a serum-free medium for 24 h. Next, 3 × 105 cells/well were seeded on the samples grown in 24-well plates. After 72 h the medium was aspirated, the cells were washed 3 times with PBS (Gibco BRL) and then treated for 30 min with a LIVE/DEAD viability/cytotoxicity kit (Life Technologies of Brazil, São Paulo) according to the supplier specifications. Images were obtained with an inverted optical microscope (Leica DMIL LED) and fluorescence was captured separately for calcein (530.0 ± 12.5 nm) and propidium iodide (645 ± 20 nm). Both MTT and LIVE/DEAD assays were conducted using BG20 and BG20-PCL12 scaffolds. Statistical tests were carried out in the analysis of the results obtained from the cytotoxicity assays (P < 0.05, N = 6, one way anova, Bonferroni test). SBF solution was prepared following the procedure suggested by Kokubo and Takadama [27]. It is well established that the SBF solution mimics the human blood plasma regarding the concentration of ions. The formation of a hydroxyapatite (HA) layer during the soaking of a material in SBF is an important reference to endorse its bioactivity because HA is responsible to both bind the implant to the bone and ensure biological functions during regeneration [2,10]. The tested materials were kept submerged in SBF at 37 °C for up to 14 days. Their concentration in the SBF solution was kept constant at 1.5 mg mL−1. The samples were examined by FTIR, SEM, EDS, and XRD.
JSM- 6360LV electron microscope. XRD was conducted in a PhilipsPanalytical PW 1710 diffractometer operating at 40 kV and 30 mA. XRD patterns were taken at a step size of 0.06° and using CuKα as the radiation source. The database of JCPDS (Joint Committee on Powder Diffraction Standards) was used as reference in these tests. N2 adsorption was carried out in a Quantachrome Nova 1200e apparatus, using samples degassed under vacuum at 150 °C for up to 12 h. The mean pore size (dp) was assessed by Equation (2), where Vmes and Sp represent, respectively, the specific mesoporous volume (cm3 g−1) and surface area (m2 g−1):
dp =
Abssamples
(2)
The specific surface area was evaluated by the multipoint BET (Brunauer-Emmett-Teller) method. FTIR was performed in a Bruker Alpha spectrometer at a resolution of 4 cm−1 and 128 scans. These tests were conducted with an attenuated total reflectance (ATR) accessory, using a diamond crystal as the reflection element. Micro-CT was performed in a Bruker SkyScan 1174 system at an X-ray voltage of 50 kV. The pixel size used in these experiments was about 10 μm. Archimedes tests were carried out in a Marte AD330 balance measuring the masses of samples in the dry, wet and submerged in water conditions. The incorporation of water into dry samples was performed by keeping them submerged in a water-containing beaker under vacuum for up to 10 min. Uniaxial compression tests were conducted at room temperature with a Shimadzu AGS-X universal testing machine at a cross-head displacement speed of 0.01 mm. seg−1. The load was applied perpendicularly to the central axis of the cylindrical scaffolds.
3. Results and discussion 3.1. As-prepared 58S particles Fig. 1a shows the N2 adsorption isotherm assessed for as-prepared
2.5. Biological tests Samples cytotoxicity was evaluated using immortalized human osteosarcoma cells (SAOS, ATCC® HTB-85) by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LIVE/DEAD assays. SAOS cells were purchased from the Rio de Janeiro cell bank (BCRJ, Federal University of Rio de Janeiro, Brazil). Prior to culture initiation, test samples were UV irradiated for 60 min on each side. SAOS cells were cultured at 37 °C in a 5% CO2 environment for 72 h in Dulbecco's modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS) (10 vol%), streptomycin sulfate (10 mg mL−1), penicillin G sodium (10 units. mL−1), and amphotericin-b (0.025 mg mL−1) supplied by Gibco BRL (USA). For MTT assay, samples were prepared according to ISO 10993-5 specifications [26]. Cell populations were initially synchronized in a serum-free medium for 24 h. After this period, 3 × 105 cells/well were seeded onto samples grown in 24-well plates. The cells were used for experiments on passage 48. Controls were used with the cells and DMEM containing 10 vol% FBS. It was used as positive control 1 vol% Triton X-100 in phosphate buffered saline (PBS, Gibco BRL, USA), and as a negative control chips of sterile polypropylene Eppendorf tubes (1 mg mL−1). After 72 h, the medium was aspirated and replaced by 210 μL culture medium with serum. Cells
Fig. 1. (a) N2 adsorption isotherm of as-prepared 58S. (b) Particle size distribution of 58S before and after ball-milling. 9893
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observed in the FTIR spectrum of 58S after sintering. The bands at 560 and 600 cm−1 are ascribed to the bending mode of PeO bonds, which has been associated with the presence of TCP [33]. The absorption bands at 710, 915 and 980 cm−1 can be related to pseudowollastonite, whereas those ones at 790 and 1060 cm−1 can be due to wollastonite [33,36]. The sharp band at 420 cm−1 was attributed to the bending mode of PeOeP bonds [37]. As a result, the additional bands observed in the FTIR spectrum of 58S after sintering reveal the formation of wollastonite, pseudowollastonite and TCP, which is in agreement with XRD (Fig. 2a). Therefore, the sintering of 58S at 1250 °C for 2 h led to the formation of crystalline phases in its structure. It is well established that the crystallization of BG decreases its bioactivity [38,39]. Nonetheless, several works report the use of glass-ceramics for load-bearing applications in bone tissue engineering [33,40–43]. The sintering of green bodies is imperative for improving the mechanical strength of freeze-cast materials. Previous tests revealed that samples heat-treated at temperatures below 1250 °C showed a poor mechanical stability, crumbling upon handling. As it will be addressed further on, the scaffolds prepared in this work displayed an expressive mechanical strength, which may favor their application in bone tissue regeneration. Thus, in spite of the crystallization of 58S, a sintering step at 1250 °C for 2 h was employed in this study for increasing the mechanical strength of the prepared scaffolds. SEM micrographs of as-prepared and sintered 58S particles are depicted in Fig. 3a. Particles showing irregular shapes are observed for the as-prepared sample. The sintering of this material caused the coalescence of 58S particles, leading to the formation of necks among them. Moreover, the sintered particles exhibited rounded shapes, whereas faceted corners were observed for as-prepared 58S. Fig. 3b shows the N2 adsorption isotherm collected for 58S after sintering. This material exhibited a type-III isotherm, which is commonly associated with macroporous materials [28]. The specific surface area, pore volume and mean pore size exhibited by this sample were 0.6 m2 g−1, 8 × 10−4 cm3 g−1 and 4.8 nm, respectively. These values are significantly smaller than those evaluated for the as-prepared material (282 m2 g−1, 0.45 cm3 g−1 and 6.4 nm). It was also evaluated a linear shrinkage about 20% of the scaffolds when their dimension before and
58S particles. This material showed both a type-IV isotherm and a H2 hysteresis loop, revealing that it exhibits a mesoporous network [28]. 58S displayed specific surface area, pore volume and mean pore size around 282 m2 g−1, 0.45 cm3 g−1 and 6.4 nm, respectively. These values are compatible with those reported by Sepulveda et al. [29] for solgel derived 58S samples. Fig. 1b depicts the particle size distribution of 58S particles before and after ball-milling. It can be clearly observed that ball-milling greatly decreased the granulometry of 58S, shifting its particle size distribution towards smaller values. Indeed, the mean particle size of 58S in the as-prepared and ball-milled conditions was about 220 and 18 μm, respectively. It is well established that the size of the starting powder used in freeze-casting shows a great effect on the prepared scaffold. According to Deville [15], the presence of large particles or particle agglomerates is detrimental to the homogeneity and properties of the final structure. Therefore, ball-milling is imperative for preparing freeze-cast scaffolds from particles obtained by the sol-gel route used in this study. Lins et al. [30] modified the chemical route first reported by Pereira et al. [23] in order to obtain nanosized BG particles. Nonetheless, only small amounts of BG can be obtained by this approach. It would be impractical to follow this procedure due to the expressive amount of material used in this work. 3.2. Effect of sintering on 58S Fig. 2a displays XRD patterns of 58S before and after sintering. No diffraction peak was observed for as-prepared 58S, revealing that it shows an amorphous structure. On the other hand, sharp diffraction peaks are noted in the XRD pattern of 58S after sintering. α-cristobalite (SiO2) and calcium metasilicate (CaSiO3 – wollastonite and pseudowollastonite) were the major crystalline phases observed in this pattern. Alpha and beta tricalcium phosphate (α- and β-TCP) were also detected. Fig. 2b exhibits the FTIR spectra of 58S in the as-prepared and sintered conditions. As-prepared 58S showed strong absorption bands centered at about 460 and 1030 cm−1, which are ascribed to SieO bonds [31,32]. The shoulder around 936 cm−1 can be related to nonbridging oxygen of SieOeCa bonds [33], whereas that one at 1200 cm−1 is due to SieOeSi bonds [34,35]. Additional features are
Fig. 2. (a) XRD pattern and (b) FTIR spectrum of 58S before and after sintering. The JCPDS files used for identifying the crystalline phases observed in the XRD patterns are indicated in Fig. 2a. 9894
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concentration of open pores are essential in the regeneration of damaged tissues [7]. The compressive strengths of these scaffolds are shown in Fig. 4b. One notices that the larger the sample porosity, the smaller its mechanical strength is. For instance, BG10, BG20 and BG26 exhibited compressive strengths of 0.15 ± 0.04, 1.52 ± 0.30 and 4.10 ± 0.65 MPa, respectively. Besides increasing the mechanical strength of the prepared scaffolds, the increase of the solid loading also decreased the strength variation. The Weibull modulus [44] evaluated for BG10, BG20 and BG26 was 3.3, 4.9 and 5.9, respectively. It is well known that the larger the Weibull modulus, the smaller the variability of mechanical strength within a group of samples [45]. Fig. 5a exhibits the pore size distribution assessed by micro-CT of scaffolds obtained with different 58S loadings. It can be observed that the larger the 58S loading, the finer the pore structure is. BG26, BG20 and BG10 displayed mean pore sizes around 45, 72 and 120 μm, respectively. This behavior is also observed in Fig. 5b, which shows SEM micrographs of these samples. Large pores exhibiting sizes up to 250 and 150 μm were respectively observed for BG10 and BG20, whereas BG26 displayed pores smaller than 80 μm. Fig. 6 shows micro-CT images obtained for BG10, BG20 and BG26. Again, it can be observed that BG26 showed a fine pore structure, whereas BG10 exhibited large pores in its structure. BG20 displayed an intermediate behavior in terms of porosity, where lamellar pores are observed. Based on the results described so far, it was decided to use BG20 as support for infiltrating PCL. This sample showed total porosity and mean pore size around 64% and 72 μm, respectively. Moreover, it displayed a significant mechanical strength (about 1.5 MPa), which should allow its application in bone tissue engineering [46]. On one hand, BG26 exhibited expressive mechanical strength (around 4 MPa). On the other hand, it displayed a fine pore structure as shown in Figs. 5 and 6. This fine porosity could decrease the scaffold bioactivity because it is likely to hinder the tissue ingrowth, nutrient transport, and angiogenesis [46]. BG10 exhibited a large porosity (≈79%) but a low compressive strength (≈0.15 MPa). This poor mechanical stability inhibits the use of BG10 for bone tissue applications.
Fig. 3. SEM micrographs of 58S in the as-prepared and sintered conditions. The scale bars shown in these images correspond to 10 μm.
after sintering was measured. These results point out that the sintering program used in this work effectively caused the densification of the 58S scaffolds. 3.3. Freeze-casting Fig. 4a exhibits the total, open, and closed porosities of scaffolds prepared using different 58S loadings. It can be clearly observed that the larger the 58S loading, the smaller the porosity of the prepared scaffold. BG10, BG20 and BG26 displayed, respectively, total porosities of 79.0 ± 3.5, 64.4 ± 1.5 and 59.2 ± 1.0%. It is well established that the pore network of freeze-cast specimens is a direct replica of the solvent structure [15]. Thus, it is expected that the lower the concentration of solids in a slurry, the larger should be the porosity of the material obtained after freeze-drying [16]. This behavior is in agreement with the trend observed in Fig. 4a. It is also worth highlighting the expressive concentration of open pores in the scaffolds prepared herein. This is an important finding because scaffolds displaying a significant
3.4. PCL-infiltrated samples As aforementioned, the volume of PCL infiltrated into the BG scaffolds was assessed by Equation (1). It was observed that acetone solutions showing PCL concentrations of 5, 12.5 and 20% (wt/vol) gave rise to samples with polymer loadings of 3.4 ± 0.4, 14.1 ± 1.0 and 29.0 ± 2.3 vol%, respectively. Fig. 7a depicts the scaffolds porosity as a function of the PCL loading. One observes that the larger the PCL concentration, the smaller the scaffold porosity. Fig. 7b displays SEM micrographs of the fracture surface for BG20 and PCL-containing scaffolds. PCL fibers bridging BG particles are noticed in these images, revealing that the polymer successfully penetrated into the scaffolds structure. Again, it can be observed a round shape of the BG particles, revealing that the sintering step was successfully conducted. Fig. 8a exhibits the stress-strain curves obtained for BG20 and PCLinfiltrated scaffolds, whereas Fig. 8b depicts the assessed compressive strength. The incorporation of PCL increased the mechanical stability of BG20; this behavior was more pronounced for BG20-PCL12 and BG20PCL20. No apparent strengthening effect was observed for BG20-PCL5, possibly due to the small concentration of PCL in this material (3.4 ± 0.4 vol%). However, although BG20 and BG20-PCL5 displayed similar compressive strengths, the latter exhibited a higher toughness in terms of the energy absorbed upon fracture. The so-called fracture toughness is associated with the area under the stress-strain curve. For comparison purposes, it was evaluated the area under the stress-strain curves shown in Fig. 8a by considering a strain up to 0.15. BG20, BG20PCL5, BG20-PCL12 and BG20-PCL20 displayed values around 0.8 × 105, 1 × 105, 2.7 × 105 and 3.8 × 105 J m−3, respectively. These results quantitatively reinforce the increase of the fracture toughness of
Fig. 4. (a) Total, open and closed porosities of scaffolds obtained from slurries containing different 58S loadings. (b) Compressive strength of the 58S scaffolds prepared herein as a function of the total porosity. The dashed straight lines connecting the data points are used as a guide to the eyes only. 9895
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Fig. 5. (a) Pore size distribution evaluated by micro-CT and (b) SEM micrographs of 58S scaffolds prepared from slurries with distinct solid loadings. The scale bars observed in SEM micrographs represent 1 mm.
Fig. 6. Micro-CT images of BG10, BG20 and BG26. The dark regions are pores whereas the white ones represent the solid phase. The scale bars shown in these images correspond to 1 mm.
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Fig. 7. (a) Porosity as a function of the PCL loading in BG scaffolds. (b) SEM micrographs of the fracture surface for PCL-containing samples. The scaffolds were fractured under liquid nitrogen for promoting the brittle failure of PCL. The porosities given in Fig. 7a were assessed from Archimedes and gravimetric tests. The scale bars displayed in Figure 7b correspond to 30 μm.
3.5. Biocompatibility tests SAOS cells have been widely used as an experimental model for osteoblasts because their cultures are easy to handle and proliferate. In addition, the ability of SAOS cells to generate a mineralized matrix when stimulated, makes them a suitable experimental model to study mineralized tissues [47]. Fig. 9a show images obtained from the LIVE/ DEAD assay. The LIVE/DEAD assay is a two-color fluorescence cell viability test based on the simultaneous determination of live (green luminescence) and dead (red luminescence) cells. It can be observed that BG20 and BG20-PCL12 displayed behavior similar to the control group in terms of the green fluorescence (live cells). However, more red dots (dead cells) were noted for BG20 and BG20-PCL12. Fig. 9b displays the results obtained from the MTT assay. BG20 scaffolds exhibited greater cell viability than the control group, whereas BG20-PCL12 showed a similar behavior to the latter one. Both BG20 and BG20PCL12 displayed cell viability above 70%, which reveals that they did not interfere in the production of SAOS cells. The smaller cell viability displayed by BG20-PCL12 when compared to BG20 can be related to the presence of PCL in the former. As depicted in Fig. 7a, the infiltration of PCL into the BG scaffolds decreased their porosity. It is well established that the biological response of BG is strongly related to its porosity [48]. Thus, the decrease in porosity observed when PCL was infiltrated into the 58S scaffolds can explain the smaller cell viability exhibited by BG20-PCL12 when compared to BG20. Moreover, one must take into account the significant decrease in the specific surface area (280 against 0.6 m2 g−1), pore volume (0.45 against 8 × 10−4 cm3 g−1) and mean pore size (6.4 against 4.8 nm) of 58S when it was sintered at 1250 °C. The small porosity shown by 58S after sintering may account to the biological response exhibited by BG20 and BG20-PCL12. It has been reported that the larger the porosity of BG, the higher its bioactivity is [49]. Fig. 10 depicts SEM micrographs and EDS spectra of BG20 and BG20-PCL12 after soaking in SBF for 14 days. For reference purposes, data ascribed to as-prepared BG20 are also shown. Cauliflower-like nodules are noticed in the micrographs of BG20 and BG20-PCL12 after the soaking in SBF, which are commonly observed for HA [50]. One observes that such nodules are not present in the structure of as-prepared BG20. The EDS spectra displayed in Fig. 10 reveals that the Ca/P ratio is different for as-prepared or SBF-soaked materials. Indeed, this ratio was about 7 for as-prepared BG, and a value around 2–3 was measured for specimens after soaking in SBF. This change in the Ca/P ratio indicates the formation of HA in the structure of the SBF-soaked
Fig. 8. (a) Typical stress-strain curves obtained for BG20, BG20-PCL5, BG20PCL12 and BG20-PCL20. (b) Compressive strength as a function of the PCL loading in BG scaffolds.
the 58S scaffolds when PCL is infiltrated into them. BG20-PCL12 and BG20-PCL20 displayed a remarkable strengthening effect in terms of both compressive strength and fracture toughness. It can be noted that this behavior is more evident the higher the PCL loading in the scaffold. The smoother shape of the stress-strain curves obtained for PCL-infiltrated samples also indicates that the composite retained some significant mechanical resistance even after multiple cracking events. According to Martínez-Vázquez et al. [24], the incorporation of polymers into ceramic structures leads to the transference of loading from the ceramic to polymer phase, which improves the mechanical strength of the scaffold. Moreover, the polymer can fill pre-existing defects in the scaffold structure, bridging ceramic particles and increasing the stress needed to propagate cracks. The bridging of BG particles by PCL is clearly observed in Fig. 7b. Moreover, besides improving the compressive strength and fracture toughness of the bare scaffold (BG20), the incorporation of PCL also increased its Weibull modulus from 4.9 to about 9.7.
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Fig. 9. Results obtained from (a) LIVE/DEAD and (b) MTT assays. The green and red luminescences correspond to live and dead cells, respectively. The scale bars shown in Fig. 9a are equivalent to 100 μm. The dashed line exhibited in Fig. 9b is associated with a cell viability of 70%. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
sintered 58S. The freeze-cast scaffolds displayed a highly interconnected and open macroporous structure. The increase of the 58S loading in the starting slurry resulted in samples with larger compressive strengths. The infiltration of PCL into the 58S scaffolds increased their mechanical stability in terms of both compressive strength and fracture toughness. This behavior is more evident the higher the PCL loading is. PCL fibers bridging BG particles were noticed in the SEM micrographs of PCL-infiltrated scaffolds. It appears that PCL can fill preexisting defects in the scaffold structure, bridging ceramic particles and increasing the stress needed to propagate cracks. Cytotoxicity tests revealed that the PCL-infiltrated scaffolds did not interfere in the production of osteoblast cells. Moreover, it was observed the growth of HA
samples. The formation of HA on the tested samples was also confirmed by FTIR and XRD (not shown in this work). These results attest the biocompatible behavior of BG20 and BG20-PCL12, which is in line with the MTT and LIVE/DEAD assays (Fig. 9). 4. Conclusions It was observed that the sintering of 58S at 1250 °C for 2 h greatly decreased its specific surface area, pore volume and mean pore size. Moreover, this heat treatment also led to the crystallization of this material. α-cristobalite, calcium metasilicate (wollastonite and pseudowollastonite), alpha and beta tricalcium phosphate were observed in
Fig. 10. SEM micrographs and EDS spectra obtained for BG20 and BG20-PCL12 after soaking in SBF for 14 days. The data collected for as-prepared BG20 are also exhibited for comparison purposes. The scale bars exhibited in the SEM micrographs correspond to either 10 or 30 μm. 9898
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on the surface of a PCL-infiltrated scaffold upon soaking in SBF for 14 days. The findings described in this work suggest that the composite scaffolds prepared herein are promising candidates for application in bone tissue repair.
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