In vitro degradation behaviour of hybrid electrospun scaffolds of polycaprolactone and strontium-containing hydroxyapatite microparticles

Polymer Degradation and Stability 167 (2019) 21e32

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In vitro degradation behaviour of hybrid electrospun scaffolds of polycaprolactone and strontium-containing hydroxyapatite microparticles Elizaveta V. Melnik a, b, Svetlana N. Shkarina a, c, Sergei I. Ivlev d, Venera Weinhardt c, e, f, Tilo Baumbach c, e, Marina V. Chaikina g, Maria A. Surmeneva a, Roman A. Surmenev a, * a

Physical Materials Science and Composite Materials Centre, National Research Tomsk Polytechnic University, Lenin Avenue 30, 634050, Tomsk, Russian Federation Flerov Laboratory of Nuclear Reaction, Joint Institute for Nuclear Research, Joliot-Curie St.6, 141980, Dubna, Moscow Region, Russian Federation c Institute for Photon Science and Synchrotron Radiation, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344, EggensteinLeopoldshafen, Germany d €t Marburg, Hans-Meerwein-Straße 4, 35032, Marburg, Germany Fachbereich Chemie, Philipps-Universita e Laboratory for Applications of Synchrotron Radiation, Karlsruhe Institute of Technology, Kaiserstr. 12, 76131, Karlsruhe, Germany f Centre for Organismal Studies, COS, Heidelberg University, Im Neunheimer Feld 230, 69120, Heidelberg, Germany g Institute of Solid State Chemistry and Mechanochemistry of the Siberian Branch of the RAS, Kutateladze, 18, 630128, Novosibirsk, Russian Federation b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 April 2019 Received in revised form 8 June 2019 Accepted 18 June 2019 Available online 21 June 2019

We investigated the effect of varying content Sr-containing HA (SrHA) microparticles on the in vitro degradation of polycaprolactone (PCL) hybrid scaffolds. A degradation behaviour study was performed by immersing the scaffolds in phosphate buffered saline (PBS) solution at 37
C for 24 days. To evaluate the degradation rate, the following properties of the scaffolds were investigated: water uptake, pH buffer change and relative weight loss. The addition of SrHA microparticles significantly affected the PCL degradation process due to significant changes in the morphology of the hybrid scaffolds and improved wetting behaviour. The samples with a greater content of SrHA degraded faster in comparison with those with a lower content. The degradation rate of the scaffolds was revealed to increase as follows: PCL < PCL/SrHA 10 % wt < PCL/SrHA 15 % wt. Thus, the results demonstrated a higher degradation rate for the hybrid scaffolds, with a maximum weight loss of 2.41 ± 0.10 % for a PCL/SrHA 15 % wt scaffold and a minimum weight loss of 0.90 ± 0.05 % for pure PCL scaffolds implying an increased degradation rate for the hybrids. The hybrid PCL/SrHA scaffolds revealed increased wettability compared with pure PCL scaffolds that promoted the penetration of PBS into the scaffolds and increased their degradation rate in vitro. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Strontium-containing hydroxyapatite Biodegradation Scaffolds Electrospinning Hybrid Wettability

1. Introduction One of the major human health problems is the loss or damage of organs and tissues. Tissue engineering aims to restore and maintain tissue function or diseased organs by the regenerative process [1e4]. Numerous studies have been focused on the synthesis of scaffolds with structure and biological functions that match as close as possible to the natural extracellular matrix (ECM), as required for tissue engineering [1e3,5e8]. Thus, scaffolds should

* Corresponding author. E-mail address: [email protected] (R.A. Surmenev). https://doi.org/10.1016/j.polymdegradstab.2019.06.017 0141-3910/© 2019 Elsevier Ltd. All rights reserved.

promote cell adhesion, differentiation and proliferation. Along with the biocompatible, absorbable and bioactive properties of scaffolds [2,3,9,10], biodegradation plays an important role in tissue engineering [2,3,11]. The controllable rate of degradation should coincide or be slightly less than the rate of new tissue formation [8,11,12]. According to several studies, if a scaffold degrades, biodegradation by-products should not provoke a negative effect on the surrounding tissues and need to be eliminated from the body [12,13]. In this respect, different polymer/ceramic composites with the advantages of each of their components have been investigated [13e17]. Polycaprolactone (PCL) has been widely applied in different studies as a biodegradable semi-crystalline aliphatic polyester polymer, which is non-toxic and shows good

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biocompatibility with organisms. The thermoplastic properties of PCL such as a melting point of 60
C enable processing in threedimensional porous scaffolds. However, in tissue engineering, the application of pure PCL is limited mainly due to a slow rate of biodegradation (3e4 years), bioactivity and its hydrophobic nature. A slow degradation rate can obstruct new tissue formation, and hydrophobicity can lead to surface erosion/degradation behaviour [18,19]. Many researchers revealed that modification of polymer scaffolds by ceramic microparticles allowed one to overcome the drawbacks of pure PCL [10,15,17,20,21]. Among ceramics, particular attention has been drawn towards hydroxyapatite (HA), since it is a bioactive, biocompatible ceramic material with a composition and structure close to that of bone [6,10,17,21]. Incorporation of strontium into the HA lattice has increased the interest of researchers due to its biological role in bone [21e23]. Strontium-containing HA (SrHA) shows increased bioactivity due to the fact that Sr enhances the activity of osteoblasts and inhibits the activity of osteoclasts, thus reducing bone resorption and stimulating bone formation. In addition, SrHA is an alkaline and hydrophilic material with higher solubility characteristics and good compressive strength similar to the mechanical properties of human bone [24e26]. While degradation studies for PCL in combination with pure HA have been previously reported [10,17,21], information about the degradation of PCL in combination with SrHA is not available. Hence, the purpose of this work was to investigate the degradation of PCL/SrHA composite scaffolds and their variability with respect to varying percentage SrHA microparticles over a period of 24 days. In this study, an electrospinning process was selected as a facile technique allowing one to control the architecture of the 3D porous scaffolds prepared close to the ECM [8,17,27,28]. Studies dealing with the degradation process mostly reveal a change in pH and the % of weight loss and absorbed water [8,13,21]. Thus, in our study, in vitro degradation of PCL/SrHA scaffolds was evaluated by monitoring the changes in morphology, weight loss, water uptake and pH under immersion in phosphate buffered saline (PBS) solution of pH 7.4 and body temperature of 37
C. 2. Materials and methods 2.1. Materials Thermoplastic research grade PCL (Mn ¼ 80 000 g mol1, m. p. 58e60
C) was purchased from the Aldrich Chemical Company Inc. (St. Louis, MO, USA). SrHA (Ca9$2Sr0$8(PO4)6(OH)2) microparticles with a diameter of 50e60 nm were supplied by the Institute of Solid State Chemistry and Mechanochemistry SB RAS (Novosibirsk, Russia). For scaffold fabrication, chloroform was employed as a solvent from the Aldrich Chemical Company Inc. (St. Louis, MO, USA). PBS was prepared by dissolving 8 g of NaCl, 200 mg of KCl, 1.44 g of Na2HPO4 and 240 mg of KH2PO4 in 800 mL of distilled water. Then, distilled water was added to a total volume of 1 L [30]. 2.2. Scaffold fabrication Functionalized hybrid 3D scaffolds based on PCL and SrHA (PCL/ SrHA) were prepared via an electrospinning technique. Samples were electrospun from a solution of PCL pellets dissolved in chloroform to yield 9 % (w/v) PCL. SrHA-containing PCL scaffolds were prepared by mixing 10 % and 15 % SrHA microparticles (w/v) into the PCL solution. All solutions were stirred and homogenized using a vibrating shaker (Multi-Reax, Heidolph, Germany) for 8 h, and then ultrasonic vibration was performed for 0.5 h before the suspension solution was loaded into a syringe. The flow rate was set to 2 mL/h. A voltage of 10 kV generated by a high voltage power supply (Hyrletron HVG 30e5/H, Eltex Electrostatic GmbH., Germany) was

applied between the syringe tip and the ground collector by connecting the cathode to the syringe needle and the anode to a metallic mandrel placed 8 cm from the tip of the needle. The resulting samples consisted of randomly arranged fibres deposited on a rotating collector operating at a speed of 600 rpm. 2.3. Characterization of pure PCL and hybrid 3D scaffolds 2.3.1. Morphological analysis by SEM A scanning electron microscope (SEM) was used to examine the fibre morphology of the scaffolds before and after incubation in PBS buffer. A JSM-7500 F scanning electron microscope (JEOL, Japan) was operated at an accelerating voltage of 10 kV under highvacuum mode. A YAG backscattered electron detector (Autrata, Czech Republic) was added to the SEM for material-specific contrast imaging by means of the effective atomic number Zeff. Prior to the analyses, the samples were coated with a layer of conductive platinum to avoid charging effects under the electron beam treatment. The average fibre diameter and diameter distribution were obtained by randomly analysing 100 fibres from an SEM image using ImageJ software. 2.4. Synchrotron mCT tomography High-resolution mCT was performed at the micro-imaging station at the Institute for Photon Science and Synchrotron Radiation of the Karlsruhe Institute of Technology (KIT, Karlsruhe, Germany) [31]. For the experiment, a monochromatic beam was used in conjunction with a sCMOS camera (sensor size 5.5 megapixels, 6.5 mm physical pixel size), a 200-mm-thick Lu3Al5O12 scintillator and a macroscope 3.6x, which allowed for a spatial resolution of approximately 1.8 mm with a field of view of 4.6  3.9 mm2. The rotation of the samples was performed with a step size of 0.24
and exposed for 1 s with 12 keV X-rays. The acquired data were reconstructed using the filtered back projection (FBP) algorithm [32]. 2.4.1. Fourier-transform infrared spectroscopy (FTIR) The molecular bonds of the scaffolds were analysed via Fourier transform infrared (FTIR) spectroscopy. FTIR spectra were obtained with a Tensor 37 FTIR spectrometer (Bruker Optics, Germany) at a temperature of 20
С. The spectra were recorded with a resolution of 4 cm1 in an attenuated total reflection (ATR) mode. The measurements were taken in the wavenumber range of 4000e400 cm1 by carrying out 100 scan iterations with further averaging. The data analysis was done using the OPUS software package [33]. 2.4.2. Powder X-ray diffraction (XRD) Powder X-ray diffraction (PXRD) was used to determine the phase composition of the prepared scaffolds. The samples were cut into slices and then characterized by a STOE STADI MP diffractometer (STOE&Cie GmbH., Germany) using Cu-Ka1 radiation (l ¼ 1.540598 nm, operating at 40 kV and 40 mA), a germanium monochromator, and a Mythen1K detector. The samples were scanned from 10
to 60
with an angle step of 0.8
and irradiation time of 10 s per step. The phases were identified using the ICDD PDF-2 database. Jana2006 software was used for processing of the diffraction data [34]. 2.5. Contact angle study The hydrophilicity of the composite scaffolds was evaluated by measuring the contact angle of the scaffold surfaces. Contact angle measurements were carried out using the “sessile drop” method

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using an OCA 15 Plus (DataPhysics Instruments GmbH, Germany) at a temperature of 22 ± 1
C. Deionized water (w) and glycerin (g) were used as liquids. Either three drops of Milli-Q water or 5 mL of glycerin were placed at different positions on the surface of each scaffold, and the results obtained were averaged.

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2.9. pH measurements The pH was measured using a calibrated pH meter and used to monitor the acidity alteration in the PBS buffer. It was measured prior to every solution refresh at different degradation periods. The time points were set as 4, 8, 12, 16, 20 and 24 days. After that, the PBS solution was replaced.

2.6. In vitro degradation study 3. Results and discussion In vitro degradation of the scaffolds was carried out in PBS at 37
C under pH 7.4 to imitate in vivo conditions. This PBS solution was chosen to simulate physical conditions with an osmolality and ion concentration close to that of the human body [8,15]. A study by Wu et al. has shown that due to pre-wetting, PBS solution penetrated through the pores of the scaffolds [29]. Thus, similar to the study by Revalti et al. [35], before degradation studies, scaffolds were pre-wetted by immersion in 70% ethanol for 10 min to enhance their hydrophilicity. For the in vitro degradation experiment, numerous studies employ the same methods for sample preparation towards the beginning of the experiment and sample investigation during the whole degradation time [8,10,11,13,21]. Similar to these studies, to prepare for the degradation experiment, each scaffold was cut into a square shape with dimensions of 10  10 mm2; then, the thickness and mass were measured. All the scaffolds were submerged in 10 mL of PBS in individual tubes with the screw caps tightened and incubated in a thermostatic oven at 37
C. Based on previous studies [8,12,21], the specimens were removed after 6, 12, 18 and 24 days under the same conditions. The PBS solution (pH 7.4) was refreshed every 4 days to maintain the acidity of the medium in the course of the in vitro studies. At a fixed period of soaking time, the samples were removed and rinsed thoroughly with deionized water, carefully wiped and placed in an oven at 37
C for 12 h. The dried samples were weighed for degradation analysis. The water uptake, weight loss of the samples, and pH of the solutions were determined, and the SEM observations and XRD analyses were carried out.

2.7. Water uptake The percentage weight increase was calculated from the water uptake. Water uptake was calculated at each time point by the following equation [10]:

% Water uptake ¼

Ww  Wd  100 % Wd

where Ww is the weight of the wet sample after degradation, which was recovered from PBS solution at respective time points (6, 12, 18, and 24 days), and Wd is the final weight of the sample, which was measured after drying in an oven at 37
C after degradation.

2.8. Weight loss The percent weight loss was estimated according to the following equation [10]:

% Weight loss ¼

W0  Wd  100 % W0

where W0 is the initial weight of each sample before degradation, and Wd is the final weight of the sample at a particular degradation period (6, 12, 18, and 24 days), which was measured after drying in an oven at 37
C after degradation.

3.1. Characterization of the scaffolds 3.1.1. Morphological analysis The morphology and surface structure of the scaffolds before degradation experiments were investigated using SEM in a dualsensor mode (Fig. 1). The signal from the secondary electron (SE) for morphology visualization was superimposed onto the BSEdetector signal for material contrast. A colourful appearance was adapted for each sample by varying the sensor gain. Usually electrospinning process results in different fibers alignment, which can be controlled in a various way to prepare well-aligned and cross-linked fibers. There are some reports indicating that electrospinning process parameters such as solution concentration, flow rate, voltage and distance between the syringe tip and the grounded collector affect the fibre formation with different size, which affects overall scaffold porosity [36,37]. Due to our previous study [38], the most appropriate electrospinning parameters to prepare fibrous scaffolds with randomly aligned fibres and different porosities were established and used in this study. Many researchers have shown that the addition of HA to the polymer solution causes fibre defects in the form of beading, which was explained by the influence of HA particles on the solution viscosity [5,6,39]. In our study, SEM micrographs of both pure PCL and PCL/SrHA composite scaffolds revealed that the surface had an interconnected 3D porous morphology with randomly oriented fibres. The results for an average fibre diameter are presented in Table 1. For pure PCL scaffolds, fibres are geometrically fairly uniform with an average fibre diameter of 9.20 ± 0.64 mm. The addition of SrHA microparticles into the PCL had a significant effect on the overall morphology of the scaffolds and the fibre diameter. The average fibre diameter was increased with an increase in the percentage weight of the SrHA powder in the composite scaffolds. The values for the diameter of the largest fibres were in the range of 17.35 ± 1.42 mm for PCL/SrHA 10 % wt and 20.29 ± 1.93 mm for PCL/ SrHA 15 % wt, and other fibres present in higher numbers showed a size of around 1 mm. The distribution of fibre diameters for the PCL/ SrHA composite scaffolds is broad compared with pure PCL scaffolds due to the increased concentration (viscosity) of electrospinning solvent caused by the addition of SrHA microparticles. The SEM images of the SrHA particles are shown in Fig. 2. The results show that SrHA powder consists of small crystallites, which agglomerate into large aggregated microparticles. The average crystallite diameter was 90.12 ± 3.42 nm, while the average diameter of the agglomerates was 17.44 ± 0.93 mm. At the same time, agglomerates with a diameter of up to 34.91 mm were detected. It has been reported that the fibre surface changes after the introduction of particles into polymer scaffolds [40e42]. Similar to these studies, we detected an irregular and uneven surface exhibiting a protruded granulate-like morphology. Bead-like fibres have similar values to that for the largest fibre diameters in PCL/SrHA scaffolds. Thus, the incorporation of SrHA aggregation particles hinders the formation of fibres with a uniform diameter. At a higher magnification, it is observed that some of the SrHA microparticles are not only embedded into the PCL fibres but are also located and piled up on the fibre surface of PCL/SrHA composite scaffolds.

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Fig. 1. Combined SEM/BSE micrographs of a pure PCL scaffold and the hybrid scaffolds PCL/SrHA 10 % wt and PCL/SrHA 15 % wt before degradation experiments. Images with the subscripts (a), (c), (e) were recorded with low magnification (120), and images with the subscripts (b), (d), (f) were obtained at a high magnification (2000). White arrows show SrHA particles, which are embedded into and located on the PCL fibres.

Table 1 The main parameters for the electrospun scaffolds. Scaffold

Average fibre diameter, mm

Average particle size, mm

PCL PCL/SrHА 10 wt % PCL/SrHА 15 wt %

9.20 ± 0.64 17.35 ± 1.42 20.29 ± 1.93

e 5.91 ± 1.62 6.26 ± 1.49

3.1.2. Fourier-transform infrared spectroscopy (FTIR) FTIR spectroscopy was performed to confirm the molecular composition of the synthesized scaffolds. The FTIR spectra obtained from pure PCL and PCL with 10 and 15 % wt SrHA contents are shown in Fig. 3 and summarized in Table 2. In the spectra for the pure PCL scaffolds, a strong and sharp peak at 1721 cm1 attributed to the carbonyl stretching mode is observed. The two peaks at 2944 cm1 and 2865 cm1 correspond to the asymmetric and symmetric stretching of the CH2 group, and the two peaks at 1293 cm1 and 1162 cm1 correspond to the stretching of the CeO and CeC groups in the crystalline and amorphous phase, respectively. Furthermore, the band at 1239 cm1 originates from asymmetric stretching of the COC group, and the band at 1193 cm1 is related to the stretching of the OeCeO

group [43]. After the addition of SrHA microparticles into the PCL polymer scaffold, the patterns for the PCL absorption bonds did not change. New bands, arising from one hydroxyl group OH liberation and two deformation vibrations of the PO3 4 group, appeared at 633 cm1, 600 cm1 and 572 cm1, respectively. All of these new peaks are attributed to SrHA [45e47]. The peak for the PO3 4 vibration, which is at 960 cm1 in SrHA, is overlapped with the peak of the CeO stretching vibration band of PCL. We should note that the same spectrum was obtained for both composite scaffolds, such as PCL/SrHA 10 % wt and SrHA 15 % wt. Thus, the FTIR results confirmed that composite scaffolds of PCL/SrHA were prepared. 3.1.3. Powder X-ray diffraction (PXRD) The powder XRD patterns for pure PCL and PCL/SrHA composite scaffolds were collected and are shown in Fig. 4, and the results are summarized in Table 3. In the XRD patterns for semi-crystalline PCL, the most intense reflections were observed at 2q ¼ 21.36
, 21.98
, and 23.68
, corresponding to the (110), (111) and (200) planes, respectively [48]. Also, a broad amorphous halo in the low-angle region (2q  30
) was found. This confirms the presence of the amorphous phase of the PCL polymer. After the incorporation of SrHA, the patterns for

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Fig. 2. SEM micrographs of the SrHA powder. The image with the subscript (a) was recorded with low magnification (500), and the image with the subscript (b) was obtained at a high magnification (50000). White arrows show crystallites and agglomerated aggregates.

Fig. 3. FTIR spectra for a pure PCL scaffold (a), PCL/SrHA 10 % wt (b) and PCL/SrHA 15 % wt (c) hybrid scaffolds.

Table 2 Characteristic IR bands for the PCL/SrHA hybrid scaffolds. Wavenumber, cm1 This work

Literature data [43,44]

2944 2865 1721 1293 1239 1193 1162 633 598 572

2949 2865 1727 1293 1240 1190 1157 630 601 571

the composite scaffolds showed reflections belonging to both PCL and SrHA. The most intensive reflections due to the SrHA microparticles were found at 2q ¼ 31.6
, 32.2
and 32.8
, which were assigned to (211), (112), and (300) [49]. As we showed earlier using the Le Bail refinement method against the PCL/SrHA 10 % wt diffraction data [50], the PCL phase belongs to the orthorhombic crystal system with a ¼ 7.516 (3), b ¼ 4.9894 (10), c ¼ 17.201 (5) Å, V ¼ 645.0 (4) Å3 at 293 K. The SrHA phase belongs to the tetragonal crystal family with a ¼ 9.4547 (5), c ¼ 6.9157 (6) Å, and V ¼ 535.38 (6) Å3 at 293 K. Both findings are in

Vibration assignment

Abbreviation

asymmetric CH2 stretching symmetric CH2 stretching carbonyl stretching CeO and CeC stretching in the crystalline phase asymmetric COC stretching OCeO stretching CeO and CeC stretching in the amorphous phase hydroxyl group OH liberation bending vibrations of the PO3 4 group bending vibrations of the PO3 4 group

nas (CH2) ns (CH2) n(C]O) ncr nas (COC) n(OC-O) nam nL (OH) n4(PO43) n4(PO43)

perfect agreement with the literature data [47,48]. 3.1.4. Water contact angle study The contact angle for the scaffold surface was measured, and the results are shown in Table 3. Some reports indicate that the PCL polymer is hydrophobic [18,19], while the SrHA microparticles are hydrophilic [24,26]. Our measurements showed that pure PCL and PCL/SrHA hybrid scaffolds are hydrophobic, since the water contact angle is more than 90
. However, in the case of PCL/SrHA 10 % wt and PCL/SrHA 15 % wt composite scaffolds, the hydrophobicity of

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Fig. 4. XRD patterns for the pure PCL scaffold (a), PCL/SrHA 10 % wt (b) and PCL/SrHA 15 % wt (c) hybrid scaffolds.

Table 3 Water contact angles on the scaffold samples. Scaffold

Contact angle (
)

qw

qg

PCL PCL/SrHA 10 % wt PCL/SrHA 15 % wt

132.1 ± 4.9 119.2 ± 2.1 112.6 ± 4.2

125.2 ± 3.2 115.5 ± 2.1 110.2 ± 3.4

the surfaces decreased compared with the pure PCL scaffolds. Thus, the water contact angle was decreased from 132
to 119
and 112
for PCL/SrHA 10 % wt and PCL/SrHA 15 % wt composite scaffolds, respectively. It was observed that the contact angle was decreased with an increasing amount of SrHA microparticles. These results are in good agreement with those obtained for composite scaffolds based on polyurethane and HA, where the addition of HA increased the hydrophilicity of the polymer matrix [6]. Thus, the addition of hydrophilic SrHA microparticles into the PCL matrix improved the hydrophilicity of the hybrid scaffolds since SrHA contains hydroxyl groups [21]. The obtained FTIR data confirmed the presence of OH groups (Fig. 4). The surface roughness of the fibres is another factor that can affect the hydrophobic nature of the PCL matrix [51,52]. SEM results revealed that the surface roughness of the fibres changed with the addition of SrHA particles (Fig. 1). 3.2. In vitro degradation study 3.2.1. Morphological changes observed during in vitro degradation SEM results revealed the morphology of the scaffold surface after immersion in PBS for 24 days in Fig. 5. After 6 days of degradation in PBS at 37
C, no significant morphological differences were found for the PCL, PCL/SrHA 10 % wt and PCL/SrHA 15 % wt compared with that before degradation except for the weight loss and water uptake properties. The changes in morphology after 12 days of degradation are consistent with a change in the pore size in the scaffold fibres. The pore size was further increased over the whole degradation time. After degradation for 18 days, some micro-cracks appeared on the surface of the PCL/SrHA 10 % wt and PCL/SrHA 15 % wt scaffolds (Fig. 5 (h,k)). We assume that these cracks are formed in the rough fibre surface sites, where SrHA microparticles are embedded within the polymer structure. This hypothesis was confirmed by micro-

computed tomography analysis (Fig. 6 (b,c)), where it can be clearly seen that most of the largest SrHA microparticles are embedded into the polymer material, forming a fibrous, porous structure. After 24 days, a higher amount of micro-cracks and pores with bigger sizes were observed. The average fibre diameter of samples at each degradation time point based on SEM data was calculated and compared with the original samples before the beginning of the degradation experiments. No significant changes were determined for each sample, but one can observe that the pore morphology changes on the external surface of the composite PCL/SrHA scaffolds (Fig. 5 (f,l)). This fact is consistent with the study reported by Wu et al., where the fibre diameter was not significantly varied although the pore size was changed with degradation time [29]. In this study, the average pore size and pore size distribution were obtained manually by randomly analysing 300 pores from a SEM image using ImageJ software. The results are presented in Fig. 7 and Table 4. After 24 days of degradation, the average pore size of each scaffold was increased to a value of 1.00 ± 0.05 mm for PCL, 1.25 ± 0.06 mm for PCL/SrHA 10 % wt and 1.53 ± 0.06 mm for PCL/SrHA 15 % wt. A unimodal pore size distribution was observed. The PCL/SrHA 15 % wt scaffolds are characterized by the presence of some pores with a size of 3.9 mm. This can be associated with the degradation of the pore wall, which leads to the formation of larger pores [29]. The change in pore morphology of the PCL/SrHA 10 % wt scaffold was similar to that of the PCL/SrHA 15 % wt scaffold. The morphology of the PCL scaffold reveals no visual changes for the whole biodegradation period. However, the PCL/SrHA 10 % wt and PCL/SrHA 15 % wt fibres in the scaffolds were observed to have a rougher surface morphology with additional micropores after immersion in PBS buffer. Presumably, the SrHA microparticles, due to their hydrophilic nature, promoted penetration of the PBS buffer media into the PCL polymer scaffolds. 3.2.2. Weight loss of scaffolds during in vitro degradation Measurements of the changes in the quantitative weight play an important role in the evaluation of the efficiency of the scaffold degradation behaviour in vitro. The percentage of weight loss for the hybrid scaffolds PCL, PCL/SrHA 10 % wt and PCL/SrHA 15 % wt versus incubation time is calculated and shown in Fig. 8. The overall rate of weight loss was relatively slow for the whole period of incubation in PBS. While a maximum weight loss was observed for the PCL/SrHA 15 % wt scaffold at 2.41 ± 0.10 % after 24

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Fig. 5. Combined SEM/BSE micrographs of PCL, PCL/SrHA 10 % wt and PCL/SrHA 15 % wt scaffolds after degradation in PBS. White arrows show the micro-cracks and enhanced pores formed on the fibre surface during the degradation process. In Fig. S3 (supplementary), additional images with different magnifications are presented.

days, the PCL scaffold lost an average of 0.90 ± 0.05 % over the same period. The weight loss for the PCL scaffold revealed a gradual and uniform decrease throughout the 24-day period. These results correspond well with the results reported elsewhere [53]. In contrast, the degradation of the hybrid scaffolds was found to be non-uniform and displayed two weight loss rates throughout the same period. From 0 to 6 days, a rapid mass loss was observed compared to a gradual mass loss close to saturation thereafter. In a study by Diaz et al., a PCL/HA scaffold, produced by the lyophilization technique, showed the same characteristic weight loss curve after 4 weeks of degradation but revealed a smaller weight loss [10]. In a study of PCL/HA, the same authors fabricated samples via a melt-moulding/porogen leaching technique and showed that samples reached a weight loss of approximately 1.5 ± 0.1 % for PCL and 3.7 ± 0.1 % for PCL/HA after 6 months of degradation [17]. Differences in weight loss can be caused by different fibre structures and morphologies, which depend on the methods used for scaffold synthesis. Regarding the degradation behaviour of HA, Nagano et al. reported that a HA coating could undergo complete coating resorption [54]. The degradation of HA is accompanied by the release of calcium and phosphate ions that leads to enhanced osteoconduction [55]. Ducheyene and Cuckler stated that the greater the dissolution rate of ceramic materials that are rapidly replaced by bone, which achieves mechanical interlocking with the porous surface of the materials, the greater is the effect of boneingrowth fixation [56].

These results indicate that the PCL/SrHA 15 % wt exhibited a faster degradation rate in PBS buffer compared to PCL/SrHA 10 % wt. Also, the PCL scaffold revealed the lowest weight loss for the pure PCL scaffold in the whole set of samples. This fact was affected by the combination of the high molecular weight and hydrophobic nature of the polymer. Diaz et al. states that polymers with a high molecular weight have increased chain length, thus taking longer to require a greater number of ester bonds to be cleaved; consequently, degradation takes longer [10]. Many researchers report that semi-crystalline PCL consists of crystalline and amorphous regions. Crystalline regions are highly ordered by polymer chains and do not allow fast water penetration. Thus, in the initial stage, hydrolysis proceeds in the amorphous regions, resulting in cleavage of the hydrolytic chain [19,57,58]. The studies [19,57e60] on PCL degradation revealed that the hydrolytic degradation takes place due to the diffusion-reaction phenomena, which involve water absorption, ester bond cleavage, diffusion, and solubilisation of the formed molecules. Thus, the degradation mechanism of the polymer can proceed via prevailing the specific pathways of the surface or bulk degradation. The surface degradation occurs when the aqueous medium cleaves the polymer backbone only at the surface. Because of hydrolytic chain cleavage, the oligomers and monomers are formed and diffused into the surrounding media. In this pathway, the rate of the polymer bonds cleavage is faster than the rate of the water infiltration into the polymer bulk. Thus, the water molecules do not have the possibility to penetrate inside the

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Fig. 6. X-ray mCT-based 3D rendering (left column) and transverse sections through sample height (right column) for a pure PCL scaffold (a) and PCL/SrHA 10 % wt (b) and PCL/SrHA 15 % wt (c) hybrid scaffolds. White arrows show SrHA microparticles embedded within the polymer fibre structure.

polymer matrix. Consequently, the polymer scaffolds are slowly degraded over time from the surface to their bulk, thus leaving the molecular weight of the bulk polymer intact. In contrast, the bulk degradation involves penetration of the medium into the whole polymer volume. In this pathway, the rate of water infiltration into the polymer bulk is faster than the degradation rate of the polymer chains. As a result, the medium has the opportunity to penetrate into the entire polymer matrix, which enables the polymer chains cleavage. Formed in such a way PCL degradation byproducts are hydroxyl and carboxyl end groups. The formation of these groups results in the production of carboxylic acid, which acts as a catalyst for hydrolysis. The homogeneous polymer chains cleavage, e. i. Homogeneous molecular weight reduction, defines the bulk degradation of the polymer. The disturbance of this diffusionreaction balance can lead to internal autocatalysis, which can occur when the hydrolysis by-products can't easily diffuse into the media from the polymer matrix and act as the catalyst for this reaction. They provide the increase of the concentration of carboxylic acid, resulting in the concentration gradient of carboxylic acid from the centre to the surface of the scaffolds. As a result, the exponential trend of the degradation rate at the core of the material can be observed. Consequently, degradation becomes faster internally than at the surface, which leads to the bimodal molecular weight distribution, i.e. higher molecular weight of the scaffold surface, and lower molecular weight of the degraded interior of the scaffold. Some studies revealed degradation pathways of PCL proceeded via

the surface degradation mechanism at pH 7.4 [58e60]. However, Lam et al. provided the results that PCL degrade via the surface degradation mechanism at the initial stages, and then the degradation is changed to the bulk one under the long-term simulated condition [19,57]. Changes in weight loss have been observed in other studies, where the PCL scaffold was blended with the bioceramic materials tricalcium phosphate (TCP) [19,58] and HA [10,21]. These articles showed that samples with bioceramic fillers revealed an increased weight loss. The results of our study reveal that the addition of SrHA microparticles to the PCL scaffold increased the weight loss rate degradation. According to the results of the study [13] in respect with the polymer weight loss, much faster degradation process of the scaffolds during the early stages was revealed. The FTIR- and XRD-patterns for the scaffolds obtained in this study before and after degradation experiments in vitro are presented in the supplementary file (Figs. S1 and S2). No significant changes were found in the FTIR spectra recorded for the scaffolds before and after degradation tests. It can be observed that the number of the diffraction peaks in the XRD patterns remains unchanged and all the peaks belong to the standard PCL and SrHA materials, however, the intensity of all the diffraction peaks decreased significantly after the degradation experiments. It is reported that after six months of immersion the intensity corresponding to PCL peaks decreased, while the intensity of the HA peaks increased [17]. The HA content in the polymer scaffold was increased after 6 months of degradation in PBS solution (pH ¼ 7.4). This fact is consistent with the results reported elsewhere [13], where the composition of PLGA and TCP was estimated separately as soluble and insoluble fractions in chloroform during the degradation period. In the period at every 2 week interval the hybrid scaffold was treated in chloroform to dissolve polymeric part of the composite. The obtained insoluble white TCP powder was vacuum dried for more than 24 h and then weighted. Thus, the weight proportion change of TCP component was measured in the PLGA/TCP composite scaffold after every degradation time point. The TCP content in the hybrid polymer scaffolds was slightly increased from 30.0 ± 0.4 to 31.8 ± 2.2 % at 4 weeks after degradation. After immersion in PBS buffer at 22 weeks at the weight loss of PLGA/TCP composite scaffold of 26.5 ± 3.4 % the content of TCP in the scaffold was 35.5 ± 3.7 %. It is reported that the weight of TCP in the residual scaffold is slowly decreased due to the fact that TCP is a water soluble inorganic salt and it is slowly released from the composite scaffold during its degradation. According to the results of the above-mentioned studies, it can be concluded that in our case the degradation of PCL and PCL/SrHA scaffolds mainly takes place due to hydrolysis of ester PCL bonds, which allow the release of SrHA microparticles from the polymer scaffolds [13,17]. The SrHA microparticles are observed not only on the fibre surface but they are also embedded into the polymer fibres (Figs. 1 and 6), thereby increasing their surface area. These microparticles facilitate the increase of the water diffusion into the PCL polymer scaffolds internal structure due to their hydrophilic nature. Consequently, the weight loss measurements demonstrated significant role of the SrHA microparticles as an ‘accelerator’ for the penetration of PBS buffer into the PCL fibrous scaffolds, which corresponds well with the results reported elsewhere [10]. 3.2.3. Water uptake Fig. 9 shows the percentage water uptake of the scaffolds immersed in PBS buffer over various time points up to 24 days of incubation, as a function of degradation time. It was observed that the water uptake in PBS buffer increased from 0 to 1.82 ± 0.11 % for the PCL scaffold within 24 days, whereas the PCL/SrHA 10 % wt and PCL/SrHA 15 % wt scaffolds absorbed

E.V. Melnik et al. / Polymer Degradation and Stability 167 (2019) 21e32

29

Fig. 7. SEM analysis of scaffolds before and after degradation. Graphs show the percentage of pore distribution for PCL, PCL/SrHA 10 % wt and PCL/SrHA 15 % wt scaffolds.

4.90 ± 0.19 and 5.70 ± 0.28 % of water, respectively. At day 6 in PBS buffer, the water uptake significantly increased to 3.33 ± 0.13 % and 4.12 ± 0.12 % for the PCL/SrHA 10 % wt and PCL/SrHA 15 % wt, respectively. The water uptake changes synchronized with the weight loss of the scaffolds is shown in Fig. 8. Maximum water

uptake of each scaffold type was reached at the time of the highest rate of weight loss. Many researchers have shown that when the scaffolds are capable of water uptake, their pore size is increased in diameter, which corresponds well with the SEM results shown in Fig. 5. This allows PBS buffer both to attach to the surface of the

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E.V. Melnik et al. / Polymer Degradation and Stability 167 (2019) 21e32

Table 4 Average pore size for the scaffolds. Scaffold sample

Average pore size, mm Degradation time, days

PCL PCL/SrHА 10 wt % PCL/SrHА 15 wt %

0

6

12

18

24

0.76 ± 0.02 0.84 ± 0.02 1.01 ± 0.03

0.82 ± 0.03 0.9 ± 0.03 1.05 ± 0.12

0.82 ± 0.04 1.08 ± 0.03 1.15 ± 0.03

0.95 ± 0.04 1.12 ± 0.05 1.25 ± 0.05

1.00 ± 0.05 1.25 ± 0.06 1.53 ± 0.06

Fig. 8. Weight loss of the scaffolds versus degradation time in vitro.

measured and compared with that of pure PCL scaffolds, as shown in Fig. 10. The PBS solution was refreshed every 4 days for each studied sample (pH ¼ 7.4). The pH of the PBS buffer solution for the PCL scaffold continuously decreased and reached 7.36 after 24 days. This means that the acidity increased. Many studies describe that this fact arises as a result of the dissolution of acidic degradation PCL products, namely, oligomers, in the PBS buffer medium [19,48,49]. In the case of the PCL/SrHA 10 % wt and PCL/SrHA 15 % wt composite scaffolds, the pH was higher than that of the pure PCL scaffold. Thus, after 24 days of degradation, the pH of the PCL/SrHA 10 % wt scaffold was 7.392 ± 0.003, and the pH of the PCL/SrHA 15 % wt scaffold remained relatively constant at pH 7.404 ± 0.003 throughout the experiment. Xiao et al. stated that the reduction in the acidity was due to the alkaline nature of SrHA. The pH of the buffer solution is reduced due to the polymer degradation involving acidic byproducts. This behaviour could compensate for the elevated pH due to the release of alkaline ions, which mainly come from the partial dissolution of SrHA [21]. Consequently, pH measurements have demonstrated the significant role of SrHA microparticles in the reduction of the acidity of the degradation products of PCL [13]. 4. Conclusions

Fig. 9. Water uptake of the scaffolds during degradation in vitro.

scaffolds and also to penetrate inside the interpolymer network during the in vitro degradation study. Since PCL/SrHA 15 % wt hybrid scaffolds show the maximum percentage of water uptake, it is considered that this type of scaffold has the largest surface to volume ratio compared with the PCL and PCL/SrHA 10 % wt scaffolds. The addition of a higher percentage weight of SrHA particles into a PCL polymer matrix can increase the free volume for liquid absorption due to the hydrophilic nature of bioactive particles [24,26].

3.2.4. pH measurements The change in pH of the PBS buffer solution with degradation time was determined to check for the release of acid degradation products from the polymer scaffolds. The pH measurement is a major factor in the degradation experiment because a decrease in pH means the accumulation of acidic degradation products, which can induce bacteria-free inflammation in the surroundings of an implant. The pH change of the PBS buffer solution for PCL/SrHA 10 % wt and PCL/SrHA 15 % wt scaffolds immersed for 24 days was

Pure PCL and PCL/SrHA hybrid scaffolds were fabricated by an electrospinning process. SEM results for PCL/SrHA composite scaffolds confirmed the incorporation of SrHA microparticles into the PCL polymer matrix. The in vitro degradation study confirmed that the addition of SrHA microparticles affected the degradation rate of PCL. The degradation rate of the scaffolds was revealed to increase as follows: PCL < PCL/SrHA 10 % wt < PCL/SrHA 15 % wt. This increase in the degradation rate of the hybrid scaffolds correlated well with the results obtained from weight loss, water uptake and pH measurements. After being soaked in PBS solution for 24 days, the PCL scaffolds showed a weight loss of 0.90 ± 0.05 %; however, the samples with 15 % wt SrHA content revealed an

Fig. 10. Change in PBS solution used for in vitro degradation of porous scaffolds with a varying amount of SrHA composition at 37
C.

E.V. Melnik et al. / Polymer Degradation and Stability 167 (2019) 21e32

increased weight loss of 2.41 ± 0.10 %. The water uptake changes correlated well with the weight loss for the scaffolds of 1.82 ± 0.11 % and 5.70 ± 0.28 % for the PCL and PCL/SrHA 15 % wt scaffolds after 24 days, respectively. The pH decreased for the PCL scaffold to 7.362 ± 0.004, while the pH remained constant at 7.404 ± 0.003 for the hybrid scaffolds. The SrHA microparticles significantly affected water penetration into the scaffolds, which resulted in an increased degradation rate for the composite scaffolds, as they are more hydrophilic than the pure PCL scaffold. Thus, the results of this study allowed a more in-depth understanding of the details for the effect of inorganic fillers such as SrHA microparticles within polymer fibres on scaffold degradation behaviour in vitro. Acknowledgements The research was conducted at the Tomsk Polytechnic University within the framework of a Tomsk Polytechnic University Competitiveness Enhancement Program grant. We thank Prof. Dr. Florian Kraus and Dr. Hendrik Martin Reinhardt (Marburg, Germany) for their kind support. We also acknowledge the financial support from the German ministry of education and research BMBF (project n. 05K16VH1) and the ERA-Net project INTELBIOCOMP n. 01DJ15025B. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.polymdegradstab.2019.06.017. Conflicts of interest There are no conflicts to declare. 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. References [1] M.M. Stevens, Biomaterials for bone tissue engineering, Mater. Today 11 (5) (2008) 18e25. [2] B. Dhandayuthapani, Y. Yoshida, T. Maekawa, D.S. Kumar, Polymeric scaffolds in tissue engineering application: a review, Int. J. Polym. Sci. 2011 (2011) 19. Article ID 290602. [3] E. Eisenbarth, Biomaterials for tissue engineering, Adv. Eng. Mater. 12 (2007) 1051e1060. [4] K. Nakayama, In vitro biofabrication of tissue and organs, in: G. Forgacs, W. Sun (Eds.), Biofabrication: Micro-and Nano-Fabrication, Printing, Patterning, and Assemblies, Elsiever Inc., Waltham, 2013, pp. 1e16. [5] P. Tyagi, S.A. Catledge, A. Stanishevsky, V. Thomas, Y.K. Vohra, Nanomechanical properties of electrospun composite scaffolds based on polycaprolactone and hydroxyapatite, J. Nanosci. Nanotechnol. 9 (8) (2009) 4839e4845. [6] H.Y. Mi, S. Palumbo, X. Jing, L.S. Turng, W.J. Li, X.F. Peng, Thermoplastic polyurethane/hydroxyapatite electrospun scaffolds for bone tissue engineering: effects of polymer properties and particle size, J. Biomed. Mater. Res. B Appl. Biomater. 102 (7) (2014) 1434e1444. [7] B.P. Chan, K.W. Leong, Scaffolding in tissue engineering: general approaches and tissue-specific considerations, Eur. Spine J. 17 (4) (2008) 467e479. [8] F.H. Zulkifli, F.S. Hussain, M.S. Rasad, M.M. Yusoff, In vitro degradation study of novel HEC/PVA/collagen nanofibrous scaffold for skin tissue engineering applications, Polym. Degrad. Stabil. 110 (2014) 473e481. [9] J.R. Porter, T.T. Ruckh, K.C. Popat, Bone tissue engineering: a review in bone biomimetics and drug delivery strategies, Biotechnol. Prog. 25 (6) (2009) 1539e1560. [10] E. Díaz, I. Sandonis, M.B. Valle, In vitro degradation of poly (caprolactone)/nHA composites, J. Nanomater. (2014) 185. [11] Y. Lei, B. Rai, K.H. Ho, S.H. Teoh, In vitro degradation of novel bioactive polycaprolactone e 20 % tricalcium phosphate composite scaffolds for bone engineering, Mater. Sci. Eng. C 7 (2) (2007) 293e298.

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