Based Scaffolds Ultrasound-Mineralized with Calcium Carbonate for

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Piezoelectric 3-D fibrous poly(3-hydroxybutyrate)-based scaffolds ultrasound-mineralized with calcium carbonate for bone tissue engineering: inorganic phase formation, osteoblast cell adhesion and proliferation Roman Viktorovich Chernozem, Maria Surmeneva, Svetlana Shkarina, Kateryna Loza, Matthias Epple, Mathias Ulbricht, Angelica Cecilia, Bärbel Krause, Tilo Baumbach, Anatolii Abalymov, Bogdan Parakhonskiy, Andre G Skirtach, and Roman A. Surmenev ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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ACS Applied Materials & Interfaces

Piezoelectric 3-D Fibrous Poly(3-hydroxybutyrate)-Based Scaffolds Ultrasound-Mineralized with Calcium Carbonate for Bone Tissue Engineering: Inorganic Phase Formation, Osteoblast Cell Adhesion and Proliferation

R.V. Chernozem1,6, M.A. Surmeneva1, S.N. Shkarina1, K. Loza2, M. Epple2, M. Ulbricht3, A. Cecilia4, B. Krause4, T. Baumbach4, 5, A.A. Abalymov6, B.V. Parakhonskiy6, A.G. Skirtach6,*, R.A. Surmenev1,*

1

Physical Materials Science and Composite Materials Centre, National Research Tomsk

Polytechnic University, 634050 Tomsk, Russia 2

Inorganic Chemistry and Centre for Nanointegration Duisburg-Essen (CeNIDE), University of

Duisburg-Essen, 45141 Essen, Germany 3

Technical Chemistry II, University of Duisburg-Essen, 45141 Essen, Germany

4

Institute for Photon Science and Synchrotron Radiation (IPS), Karlsruhe Institute of Technology,

76344 Eggenstein-Leopoldshafen, Germany 5

Laboratory for Applications of Synchrotron Radiation (LAS), Karlsruhe Institute of Technology

(KIT), 76049 Karlsruhe, Germany 6

Department of Biotechnology, Ghent University, 9000 Ghent, Belgium

* Corresponding authors: Prof. Dr. Andre Skirtach ([email protected]) and Associate Prof. Dr. Roman Surmenev ([email protected])

Keywords: scaffold, piezoelectric, calcium carbonate, bone tissue engineering, mineralization

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Abstract Elaboration of novel biocomposites providing simultaneously both biodegradability and stimulated bone tissue repair is essential for regenerative medicine. In particular, piezoelectric biocomposites are attractive due to a possibility to electrically stimulate cell response. In the present study, novel CaCO3-mineralized piezoelectric biodegradable scaffolds based on two polymers: poly[(R)3-hydroxybutyrate] (PHB) and poly[3-hydroxybutyrate-co-3-hydroxyvalerate] (PHBV) are presented. Mineralization of the scaffold surface is carried out by the in situ synthesis of CaCO3 in the vaterite and calcite polymorphs using ultrasound (U/S). Comparative characterization of PHB and PHBV scaffolds demonstrated an impact of the porosity and surface charge on the mineralization in a dynamic mechanical system, since no essential distinction was observed in wettability, structure and surface chemical compositions. A significantly higher (4.3 times) piezoelectric charge and a higher porosity (~15 %) leads to a more homogenous CaCO3 growth in 3-D fibrous structures and results in a 2 times higher relative mass increase for PHB scaffolds compared to that for PHBV. This also increases the local ion concentration incurred upon mineralization under U/S-generated dynamic mechanical conditions. The modification of the wettability for PHB and PHBV scaffolds from hydrophobic (non-mineralized fibers) to superhydrophilic (mineralized fibers) led to a pronounced apatite-forming behavior of scaffolds in a simulated body fluid. In turn, this results in the formation of a dense monolayer of welldistributed and proliferated osteoblast cells along the fibers. CaCO3-mineralized PHBV surfaces had a higher osteoblast cell adhesion and proliferation assigned a higher amount of CaCO3 on the surface compared to that on PHB scaffolds; as incurred from micro-computed tomography (μCT). Importantly, cell viability study confirmed biocompatibility of all scaffolds. Thus, hybrid biocomposites based on the piezoelectric PHB polymers represent an effective scaffold platform functionalized by an inorganic phase and stimulating the growth of bone tissue.


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1 Introduction Tissue engineering is a promising research area combining cell biology, biomaterial engineering, and medicine to fabricate new functional tissues.1 The personal medicine approach required to design novel biomaterials for the substitution of bone tissue defects with individual functional optimization and predetermined architectonics.2 In this regard, porous scaffolds are key components providing three-dimensional (3-D) structure, such as fibrous scaffolds, for cell interactions and the formation of the extracellular matrix (ECM).3 There are three methods, which allow fabrication of 3-D fiber structures: phase separation,4 self-assembly5 and electrospinning.6 In comparison to phase separation and self-assembly methods, electrospinning (ES) is an unique method, which can produce fibrous scaffolds with diameters ranging from tens of nanometers to several micrometres.7 To prepare electrospun fiber biomaterials for tissue engineering synthetic biodegradable, nontoxic and biocompatible polymers, such as polyhydroxyalkanoates (PHAs), can be used. PHA biomaterials are nowadays widely used in the packing, medicine, pharmacy, agriculture and food industry.8 PHAs are polyesters produced by microorganisms under unbalanced growth conditions9 with

many

possible

monomer

structures.10

Among

polyhydroxyalkanoates,

poly(3-

hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are biodegradable8 polymers with the piezoelectric crystalline phase,11-12 which promotes the bone growth in vivo,13-14 as well as improving cell-to-cell communication and contractility of the cardiac cells.15 Piezoelectricity is a property of non-centrosymmetric materials, whereby the electric charge is generated on the surface of materials upon mechanical stress or a mechanical deformation appeared under an external electric field.16 It is interesting to point out that the electro-mechanical coupling to human tissues is demonstrated for piezoelectric materials.17 Although the piezoelectricity is caused by a mechanical deformation of polymer scaffolds, it was shown to enhance cell migration, adhesion and secretion,18 however, hydrophobicity limits the use of these polymers in tissue engineering applications.19 A very few reliable scientific solutions to 3 ACS Paragon Plus Environment


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this challenge have been proposed, such as plasma treatment, which can damage the fibrous scaffold structure due to a polymer etching,20 or incorporation of nanoparticles21-22 (hydroxyapatite or calcium phosphate) during ES,23 which would exist in a limited amount and can be distributed in fibers irregularly.24 Another approach is based on the in situ synthesis of an inorganic material of the polymer scaffold surface in an effort to create a hybrid material,25 which can be functionalized with inorganic nanoparticles as it was shown for metal nanoparticles.26 However, a more interesting approach would be to functionalize scaffolds directly with an inorganic material that would facilitate and promote cell growth. Calcium carbonate as well as other carbonate minerals represent a possible inorganic material for scaffold functionalization and coating.27 Calcium carbonate is a bioresorbable bone filling material,28 which was reported to enhance osteoconductivity.29-30 CaCO3 can be deposited on fibrous scaffolds via in situ synthesis by mixing salts containing calcium and carbonate ions.31 32 33 It exists in 3 anhydrous modifications: more stable calcite and aragonite as well as a less stable vaterite modifications. By varying the synthesis parameters it is possible to design coatings in the porous polycrystalline vaterite form of the calcium carbonate, 34 35 36

which would provide the enhancement of degradability.37 At the same time, calcium carbonate

would be a source of calcium ions for bone reconstruction. Furthermore, its porous structure would allow to encapsulate functional molecules such as antimicrobial and anti-inflammation drugs33 or functional enzymes.38-39 In this work, we use a recently developed novel ultrasound technique for the in situ synthesis of calcium carbonate functionalized scaffolds. This ultrasound based approach to functionalize scaffolds provides both a more homogenous ion distribution with the subsequent crystal formation and a better reach throughout the whole volume of the materials.33, 40 As a result, a more uniformly mineralized polymer fibers are formed.24 Besides, we use piezoelectric 3-D fibrous substrates, which are very attactive to bone tissue engineering, since the main interaction mechanisms between the organic template and the inorganic phase are driven by the electrostatic interactions 4 ACS Paragon Plus Environment

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and molecular recognition. Recently, it has been reported for flat substrates that a mechanical deformation of a layer of piezoelectric virus particles induces voltage changes due to the piezoelectric effect by attracting polar ZnO particles formed by a homogeneous nucleation in a solution.41 However, 3-D materials are more promising compared to 2-D to be utilized for bone tissue, where ultrasound would mechanically deform piezoelectric materials generating an electrical surface potential. We hypothesized that special piezoelectrical properties of 3-D materials can have an influence on the in situ mineralization of calcium carbonate at the dynamic mechanical conditions generated by U/S, i.e. affecting the mineral phase, mass and distribution in the scaffold. To the best of our knowledge, the fabrication and investigation of simultaneously piezoelectric and biodegradable 3-D fibrous scaffolds based on PHB and PHBV polymers using CaCO3 coating have not been reported yet. Furthermore, a modification of scaffolds with CaCO3 can provide cell adhesion on the surface, particularly used for bone tissue regeneration. Therefore, the aim of the present study is to fabricate and investigate the morphology, wettability, chemical and phase composition of simultaneously biodegradable and piezoelectric biocomposites based on nonwoven 3-D scaffolds from PHB and PHBV polymer fibers uniformly coated with CaCO3 for bone tissue engineering. We demonstrate also that U/S induces a more homogenous CaCO3 coating along the fibers in the 3-D volume of a stronger piezoelectric polymer (PHB) in comparison with that for a less piezoelectric polymer (PHBV).



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2 Materials and methods 2.1 Preparation of the scaffolds The fabrication of nonwoven fibrous scaffolds was carried out by electrospinning. In the first step, the dissolution of poly[(R)3-hydroxybutyrate] (PHB; Mw = 300,000 g×mol-1, Sigma-Aldrich) and poly[3-hydroxybutyrate-co-3-hydroxyvalerate] (PHBV; Mw = 680,000 g×mol-1, Sigma-Aldrich) powders in chloroform (CHCl3) is performed. The content of hydroxyvalerate in PHBV is 12 mol%. The PHB and PHBV solutions with concentrations of 6% and 23 % (w/v) were then loaded in a plastic syringe. The syringe pump was then used to feed solutions through an extension tube capped with blunted 21-gauge needles (Fisnar). A 13 kV voltage was applied by using a high voltage power supply. A needle-collector path (13 cm), deposition time (60 min), a fixed injection flow rate (3.6 mL×h−1) and rotation speed (600 rpm) were used during ES. The prepared samples were collected on a cylindrical collector and used for further experiments. 2.2 Treatments of the scaffolds The mineralization of PHB and PHBV scaffolds was carried out using a typical process of CaCO3 particle crystallization from a mixture of Na2CO3 and CaCl2 solutions. Solutions were prepared by dissolving calcium chloride CaCl2 (1M) and sodium carbonate Na2CO3 (1M) (Sigma-Aldrich) in deionized water. PHB and PHBV scaffolds were mineralized using an ultrasonic bath (Bandelin Sonorex) using a methodology described elsewhere.24 After mineralization, the samples were washed and dried at 55 °C for 60 min. To form uniform CaCO3 coatings, the mineralization procedure was repeated three times. The mass of the samples was controlled during the treatment using an analytical balance KERN ABJ-NM/ABS-N ABS 220-4N (Germany). 2.3 Characterization of the prepared samples The SEM investigations were carried out with an ESEM Quanta 400 FEG instrument equipped with an energy-dispersive X-ray analysis (EDX analysis system Genesis 4000, SUTW-Si(Li) detector) operated in a high vacuum. Gold/palladium coatings were deposited on the samples prior 6 ACS Paragon Plus Environment

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to the SEM study. The EDX spectra were collected with a dead time of 30 % for 60 seconds and the energy of an electron beam of 15 keV. The distribution of diameters of non-mineralized PHB and PHBV fibers was measured using ImageJ software.42 The phase composition and crystalline structure of the prepared samples were investigated by Xray diffraction using a XRD-6000 Shimadzu (Japan) with CuKα radiation (λ = 0.154 nm) in the 2θ range from 10° to 90° with a step size of 0.01 °/2θ at 40 kV and 30 mA. The database of PDF4+ was used to analyze the obtained diffractograms. To study the surface chemical composition, X-ray photoelectron spectroscopy (XPS) was performed using a Phoibos 150 analyzer, an non-monochromatic XR-50 Mg Kα X-ray source (hν=1253.6 eV) and an FG 20 flood gun for compensation of charging effects (manufacturer: SPECS, Germany). The XPS survey spectra were collected with a pass energy Ep = 50 eV and energy steps Es = 0.5 eV, the high-resolution spectra with Ep = 20 eV and Es = 0.1 eV. The base pressure of the XPS chamber was 1 × 10−8 Pa. All XPS spectra

were

analyzed

using

the

CasaXPS

(v.2.3.17PR1.1;

product

of CasaXPS Software Ltd., USA).43 The molecular structure of the samples was investigated by employing Fourier Transformed Infrared (FTIR) spectroscopy using a Bruker ALPHA-Platinum FTIR-ATR (Attenuated Total Reflectance) setup. Each sample was scanned 30 times in the range from 400 to 4000 cm-1 with the resolution of 4 cm-1. Measurements of the contact angle (CA) were carried out using an optical contact angle apparatus OCA 15 Plus (Data Physics Instruments GmbH, Germany) and the SCA20 software (Data Physics Instruments GmbH, Germany). The CA of water was estimated using the sessile drop method in air. The effective piezoelectric charge coefficient, d33, from the whole scaffold surface area of 10×10 mm2 under the frequent compression was measured using a Wide-Range d33 Tester Meter (APC International, Ltd., USA). To validate the reproducibility and stability of the piezoelectric 7 ACS Paragon Plus Environment


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effect in scaffolds, 20 samples for each polymer (PHB and PHBV) and modification (nonmineralized and mineralized) were used. The micro-computed tomography (µCT) analysis was done for mineralized PHB and PHBV samples. The tomography scans of the samples were performed with the µCT system available at a bending magnet source of the Institute for Photon Science and Synchrotron Radiation of the Karlsruhe Institute of Technology (KIT, Karlsruhe, Germany).44 The expirements were conducted by filtering the white beam spectrum (1.5-40 keV) with a 200 μm Aluminum filter. The X-ray projections were acquired with a PCO.DIMAX CMOS camera (2016×2016 pixels) equipped with an Optique Peter white beam microscope (magnification of 10x), which resulted in the effective pixel size of 1.1 μm. Conversion of X-rays was done with a 200-μm-thick Lu3Al5O12 scintillator. For each scan, 6000 projections were recorded with an exposure time of 25 ms. The distance between the sample and detector was equal to 25 mm. The projections were processed using the flat-field correction technique to suppress the beam fluctuation, noise from detector, cracks, and imperfections of the scintillator. Subsequently, a tomographic reconstruction was performed using a filtered back projection algorithm provided by the UFO framework.45 To reduce the statistical noise from the reconstructed slices, a median filter with the radius of 1.8 µm was applied to every single slice separately. Subsequently, a segmentation process was performed using the Kapur46 and Otsu47 thresholding algorithms implemented with Numpy and scikit-image packages of Python programming language48 to distinguish features of one material from another. This resulted in binary datasets, where ones are assigned to one material and zeros to another. Then, the segmented structures were analyzed with quanfima package.49 The porosity of the PHBV and PHB scaffolds was calculated for each sample. The Amira 5.4.1 software (FEI, Visualization Sciences Group) was employed for 3-D visualization.

2.4 In vitro assays 8 ACS Paragon Plus Environment

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2.4.1 Simulated body fluids Precipitation of the inorganic phase (apatite) of bone tissue is an important process for successful biomedical applications. Simulated body fluid (SBF) is used to investigate the precipitation of bioactive apatites from blood on the surface of samples. Ion concentrations of the SBF, 1.5 SBF and human blood are listed in the Table 1.50-51 Non-mineralized and CaCO3-modified scaffolds were individually immersed in 50 ml of 1.5 SBF for 7 days and stored at 37 °C. After immersion, all samples were washed in deionized water, and then dried during 60 minutes at 55 °C.

Table 1. Concentration of the ions in SBF, 1.5 SBF and human blood Fluids 1.5 SBF SBF Human blood

+

𝑁𝑁𝑁𝑁 213.0 142.0 142.0

Ions concentration (mmol/L) 𝐶𝐶𝐶𝐶 − 𝐻𝐻𝐻𝐻𝑂𝑂3− 𝐻𝐻𝐻𝐻𝑂𝑂43− 𝑀𝑀𝑀𝑀2+ 𝐶𝐶𝐶𝐶2+ 2.3 3.8 221.7 6.3 1.5 1.5 2.5 147.8 4.2 1.0 1.5 2.5 103.0 27.0 1.0

+

𝐾𝐾 7.5 5.0 5.0

𝑆𝑆𝑂𝑂42− 0.8 0.5 0.5

2.4.2 Osteoblasts cultivation Pre-osteoblastic MC3T3-E1 cells (ATCC) were cultured in MEM-alpha glutaMAX-1™ (Cat. No. 32561-

029)

supplemented

with

10%

FBS,

2

mM

glutamine,

and

100

μg/ml

penicillin/streptomycin. The media were replaced every 3 days, and the cells were maintained in a humidified incubator at 5% CO2 and 37 °C (Innova CO-170, New Brunswick Scientific). 2.4.3 Cell viability The effects of non-mineralized and mineralized scaffolds on MC3T3-E1 cells were determined in the static mechanical conditions by AlamarBlue (ThermoFisher Scientific) (Cat. No DAL1025). MC3T3-E1 cells were seeded into 96-well cell culture plates at a cell density of 104/well in the culture medium and incubated at 37 °C under 5 % CO2 with 5 × 5 mm2 samples and without samples as a control. After 24 and 72 hours, 10 μL of the fluorescent dye AlamarBlue was added to each well, and the fluorescence (540/610 nm) intensity was measured by a spectrophotometer (Infinite F200 PRO).



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2.4.4 Fluorescence microscopy To estimate the cell adhesion and proliferation on the surface of the non-mineralized and mineralized scaffolds in the static mechanical conditions, viable cells were visualized by fluorescence microscopy using a Nikon TI (Nik on, Japan) microscope with 4x and 10x objectives and appropriate filters. MC3T3-E1 cells were seeded on the surfaces of samples at a cell density of 105/sample and incubated for 1 and 3 days. Cells cultured on a plastic surface were used as a control. Afterwards, the cells were stained with Calcein AM. All optical microscopy images were post-processed using ImagJ42 performing fluorescence unmixing, linear interpolation, and image deconvolution. The number of cells was calculated from snapshots of three random zones for three replicates of the samples. 3 Results and Discussion 3.1 Surface, chemical and phase compositions of the mineralized samples Figure 1A and 1D show SEM images of electrospun fibrous PHBV and PHB fibrous scaffolds, respectively. The images demonstrate a continuous fibrous morphology. The fibers do not contain beads, independent of the polymer. The average fibrous diameter of PHBV scaffolds was 7.6±2.7 µm, while the average fibrous diameter of PHB scaffolds was 4.6±1.2 µm. The surface of uniformly coated fibers of PHBV and PHB scaffolds with CaCO3 was observed only after three U/S mineralization cycles, as shown in Figure 1B and 1E, respectively. SEM images after the second U/S mineralization cycle revealed some spots on the scaffold surface without the coating, as presented in Supporting Information (Figure S1). Moreover, the polycrystalline coating was observed on the surface of the mineralized scaffolds (Figure 1C and 1F), and the EDX analysis (Figure S2) confirmed the presence of CaCO3, insets to Figure 1C and 1F. The signals of Au and Pd correspond to a conductive gold/palladium coating deposited prior to SEM measurements, Na and Cl correspond to remaining sodium chloride after washing. It should be noted that an immersion of fibrous scaffolds in a mixture of calcium carbonate forming salts without U/S leads to a non-uniform coverage of fibers. Indeed, it has been reported that 3 U/S treatments in a mixture 10 ACS Paragon Plus Environment

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of 1M CaCl2 and 1M Na2CO3 solutions allows to attain uniform CaCO3-coated fibers in the whole volume of electrospun polycaprolactone scaffolds compared to the immersion, when CaCO3 particles mostly are observed in gaps between the fibers.24

Figure 1. SEM images of (A-C) PHBV and (D-F) PHB scaffolds: (A,D) a top view of nonmineralized; (B, E) a top view and (C,F) magnified top view of mineralized. Insets in (A,B,D,E) show optical images of water CA for corresponding scaffolds. Insets in (C,F) show quantified EDX spectra of corresponding mineralized scaffolds, where “Others” are referred to as Au, Pd, Na and Cl.

Wettability is one of the key parameters for cell attachment and proliferation.52 It was reported that hydrophilic surfaces have a better affinity for cells in comparison to hydrophobic surfaces.5354

Besides, the hydrophilicity of the surface affects the cellular response, e.g. cell adhesion and

proliferation.55 The surface of the non-mineralized scaffolds is hydrophobic, i.e. the average water CA for PHBV and PHB was 136±4° and 133±1°, respectively, as shown in insets to Figure 1A and 1D. It can be seen that after mineralization the surface of scaffolds becomes superhydrophilic, since the water droplets completely spread immediately after the deposition on the surface of each 11 ACS Paragon Plus Environment


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sample. It was reported that non-mineralized electrospun fibrous PHBV56 and PHB57 scaffolds possess CA values of over 100°, i.e. characteristic of a hydrophobic surface. We note that the hydrophilization of electrospun scaffolds by the deposition of biomimetic coatings (e.g. calcium phosphates) was reported elsewhere.58 The results obtained in current study are found to be in a good agreement with literature. The surface chemical composition of the prepared samples was subsequently studied by XPS. XPS spectra of the non-mineralized and mineralized scaffolds are presented in Figure 2A. The spectra of PHB and PHBV scaffolds show characteristic peaks of carbon and oxygen. The XPS peaks attributed to Ca, Na, and Cl appeared after the mineralization of the scaffolds, where Na and Cl can be attributed to sodium chloride observed in EDX. To investigate the surface compounds, high-resolution XPS spectra of the Ca 2p, C 1s and O 1s regions were measured and are presented in Figure 2B, 2C and 2D, respectively. The C 1s intensity (Figure 2C) of non-mineralized PHB and PHBV scaffolds can be attributed to 3 components with the binding energies 284.8 eV (aliphatic carbon), 286.4 eV (ether), and 288.8 eV (ester groups). 20 59 60 After the mineralization, an additional peak in the C 1s region of both PHB and PHBV scaffolds appeared at 289.6 eV, attributed to the C-O bond in carbonate ions (𝐶𝐶𝑂𝑂32− ).

59 61 62

For mineralized PHB and PHBV scaffolds, high-resolution XPS spectra of Ca 2p showed

the expected double-peak structure with a multiplet splitting of 3.5 eV (Figure 2B).59 The Ca 2p3/2 peaks were observed at 347.1 eV corresponding to Ca bonded to the oxygen atoms from the carbonates groups (𝐶𝐶𝑂𝑂32− ).59 61 63 Finally, the O 1s regions for non-mineralized PHB and PHBV

scaffolds were fitted with two components (Figure 2D) at 531.8 and 533.2 eV, attributed to ether (C-O), and ester groups (O=C-O), respectively.

59 61 63 64

For the O 1s region of mineralized

scaffolds an additional peak appeared at 531.5 eV (Figure 2D), most likely corresponding to the carbonate group (𝐶𝐶𝑂𝑂32− ).59, 61-62


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Figure 2. (A) Survey XPS spectra measured from 1000 to 20 eV and high-resolution XPS spectra for each observed regions of (B) Ca 2p, (C) C 1s and (D) O 1s for PHBV and PHB scaffolds before and after mineralization.



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Figure 3 presents recorded XRD patterns of the scaffolds before and after the mineralization. No significant differences between the crystal structure of non-mineralized PHB (# 49-2212 PDF 4+) and PHBV65 electrospun scaffolds were observed. After the mineralization, the analysis of the XRD patterns revealed the presence of calcium carbonate corresponding to rhombohedral calcite (# 01-072-1652 PDF 4+), hexagonal vaterite (# 01-072-0506 PDF 4+) and trace amount of the byproduct sodium chloride (# 00-05-0628 PDF 4+). For the quantitative phase analysis, the Rietveld refinement was performed. The ratio of vaterite and calcite phases was 82:18% for PHB scaffold and 89:11% for PHBV scaffold, respectively. Thus, XRD results confirm the formation of calcite and vaterite phases of calcium carbonate onto fibrous surface. In turn, the formation of vaterite is predominant for both PHB and PHBV scaffolds under used experimental conditions.

Figure 3. XRD patterns of PHBV and PHB scaffolds before (bottom row) and after (top row) mineralization with CaCO3: C – calcite, V – vaterite, H – halite.


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3.2 Tomography, increase in mass and piezoelectric response of the prepared scaffolds. 3-D reconstructions of the selected regions of the mineralized PHB and PHBV scaffolds are presented in Figure 4A and 4B, respectively. μCT analysis of the mineralized scaffolds reveals that the distribution of CaCO3 particles is not homogeneous. A clear difference between the distribution of CaCO3 particles for PHB and PHBV scaffolds was observed (Figure 4A and 4B, side view), i.e. CaCO3 particles were grown deeper in the volume (μCT depth profile – 385 μm) of PHB scaffolds than those in PHBV scaffolds. Moreover, after each U/S mineralization a relative increase in mass was significantly higher (~2 times) for PHB scaffolds compared to that for PHBV scaffolds (Figure 4C). Stitz et al showed that due to electrostatic interactions, the ZnO coating has more crystalline structure during biomimetic mineralization onto 2-D piezoelectric template surface. Therefore, the electrostatic interactions play a key role for biomimetic mineralization.41 Furthermore, in the present study surfaces of both PHB and PHBV scaffolds were initially hydrophobic (Figure 1) and had similar chemical compositions (Figure 2). Three possible mechanisms have been suggested to explain a more homogeneous distribution of CaCO3 particles and a higher relative increase in the mass for PHB scaffolds in comparison with PHBV; (i) different scaffolds porosity; (ii) difference in the polymer piezoelectric performance stimulated by mechanical deformation via ultrasound, and, therefore, a different surfaces charge; or (iii) a combination of both (i) and (ii) mechanisms. Despite of the smaller pores possessing higher capillary pressure, a larger porosity (by 15%) (Figure 4D) for PHB could provide higher liquid transport dynamics, i.e. increasing local ions concentration with the consequent mineralization. The measured piezoelectric charge coefficient of PHB scaffolds (d33 = 3.0 ± 0.5 pC/N) was 4 times higher than that for PHBV scaffolds (d33 = 0.7 ± 0.5 pC/N) (Figure 4E). The corresponding piezoelectric coefficient, d33, for PHB scaffold was reported to be 2.0 ± 0.3 pC/N,12 while that for PHBV was reported to be 0.7 - 1.0 pC/N11 at the 17 % content of HV. The surface electric potential should be higher with an increasing piezoelectric coefficient.12 Also, U/S treatment generates the surface electric charge 15 ACS Paragon Plus Environment


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on the surface of piezoelectric materials due to mechanical deformations initiated by acoustic waves in liquids.66-67

68

Such deformations of piezoelectric materials in water can lead to the

formation of reactive oxygen species, which provide antibacterial properties and pollution cleanning.68,69 Probably, the difference between piezoelectric charge coefficients for PHB and PHBV polymers leads to generation of different electric potentials on their surfaces, which can have an influence on the ions distribution during U/S mineralization, as illustrated in Figure 4F. This would provide a deeper growth providing a more homogeneous distribution of CaCO3 in the coating. Recently, it was reported that electrical stimuli enhance the bone tissue formation effect upon a mechanical deformation of a piezoelectric polymer in vivo in comparison to a nonpiezoelectric polymer.13 Besides, an increase of the calcite phase was also observed in the XRD analysis (Figure 3) for PHB scaffolds compared to PHBV scaffolds can also contribute to a relative increase in the mass (Figure 4C). This can be attributed to the fact that the density of porous vaterite particles is significantly lower than that for calcite. It is know that the density of solid CaCO3 is 2.7 g/cm3, while vaterite particles have a porous structure compared to calcite. Volodkin et al reported that the volume occupied by the solid CaCO3 and internal pores of vaterite are ~ 59% and 41%, respectively, 34 although the porosity can be controlled by temperature from ~ 19 nm to ~ 44 nm.70 Therefore, the density of vaterite particles is deduced from above data to be 1.6 g/cm3.34 At the same time, it was published that an increase of the local ions concentration accelerates the recrystallization process from vaterite to calcite.36 Due to electrostatic interactions, the ion concentration will be higher for PHB scaffolds, which exhibit 4.3 times higher piezoelectric properties compared to those for PHBV (Figure 4E). That would also lead to a faster recrystallization from vaterite to calcite.


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Figure 4. μCT images of the mineralized (A) PHB and (B) PHBV scaffolds with fibers and without fibers to show CaCO3 particles distribution; (C) a relative increase in the mass of scaffolds after each U/S treatment; (D) calculated porosity of the scaffolds; (E) the piezoelectric charge coefficient of scaffolds before (black and orange) and after (blue and red) 3 U/S treatment; (F) scheme of a scaffold cross-section view demonstrating an influence of the surface charge on mineralization. 17 ACS Paragon Plus Environment


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3.3 Simulated body fluid assays The surface properties of biomaterials determine the outcome of interactions between biomedical devices and the surrounding biological environment.71 To replace bone tissue defects, the apatiteforming behavior on the prepared scaffolds is essential. In this study, non-mineralized and mineralized scaffolds were soaked into 1.5 SBF for 7 days to evaluate the influence of the apatiteforming behavior by different compositions of prepared biocomposites. Due to hydrophobic surface of polymeric electrospun scaffolds (Figure 1A and 1D), no apatite-formation was observed. Only some particles were observed in the gaps between fibers on the surface of nonmineralized scaffolds as shown in supporting information (Figure S3). In contrast, after the immersion of the mineralized scaffolds in 1.5 SBF for 7 days, uniformly coated fibers were observed as shown in Figure 5A and 5D. Moreover, the formation of a coating on the surface of calcium carbonate is distinguishable (Figure 5B and 5E). The analysis of EDX spectra (Figure S4) confirmed the presence of phosphorus on the surface of the mineralized scaffolds after 7 days immersion in 1.5 SBF as shown in insets of Figure 5B and 5E. Furthermore, the analysis of the FTIR spectra confirmed the presence of phosphate: both for PHB and PHBV scaffolds the vibrations of phosphate group were observed at 598 cm-1 of 𝜈𝜈4, while only for PHBV an additional

peak of 𝜈𝜈3 vibration appeared at 1038 cm-1.71 It can be explained by the reflective mode during the measurements and the presence of a higher amount of CaCO3 on the top of the PHBV scaffolds, as observed from μCT images (Figure 4A and 4B). In addition, FTIR spectra resolved the bands of O-H ions at 665 and 1634 cm-1 corresponding to apatites,72-73 which are the inorganic phase of bone tissue. The formation of compounds on the surface of the mineralized scaffolds after 7 days of immersion in 1.5 SBF were further investigated by XPS. The survey XPS spectra revealed the presence of phosphorus for both mineralized PHB and PHBV scaffolds (Figure 6A). For more detailed information about the surface chemical compounds after SBF soaking, high-resolution XPS spectra of the C 1s, O 1s, Ca 2p, P 2p and O 1s regions were measured as presented in Figure 6B18 ACS Paragon Plus Environment

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E, respectively. For both polymers, the C 1s region was fitted with four components at the binding energies 284.8 eV (aliphatic carbon), 286.4 eV (ether), 288.8 eV (ester groups) and 289.6 eV attributed to C-O bindings in carbonate ions (𝐶𝐶𝑂𝑂32− ) from calcium carbonate after mineralization (Figure 6B).20, 59, 61-62 The Ca 2p3/2 were registered at the binding energy of 347.1 eV (Figure 6C), which can be attributed to calcium carbonate59,

63-64

or apatite-based compounds, such as

hydroxyapatite (HA) or amorphous calcium phosphate (ACP).62, 64, 74 The P 2p region showed the binding energy of P 2p3/2 at 133.1 eV (Figure 6D), which is linked to the phosphorus bonded to the oxygens in the (PO4)3- groups, in the apatite-like structures.62, 64, 74 The O 1s region was fitted with two binding energies as follows (Figure 6E): at 513.5 eV, which can be attributed to oxygen bonded to carbon or phosphor in CaCO3 or apatite-like structure, respectively; and at 533.3 eV was established to ester groups (O=C-O).20, 59-60

Figure 5. SEM images of (A,B) CaCO3-mineralized PHBV and (D,E) PHB scaffolds after 7 days immersion in 1.5 SBF. FTIR spectra of the (C) PHBV and (F) PHB scaffolds after mineralization and subsequent 7 days immersion in 1.5 SBF. Insets of quantified EDX spectra of (B) CaCO3mineralized PHBV and (E) PHB scaffolds after 7 days immersion in 1.5 SBF, where “Others” are referred to Au, Pd, Na and Cl.



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Thus, the analysis of SEM and EDX results reveales the formation of phosphorus-contained coating onto the surface of mineralized PHB and PHBV scaffolds after the immersion in 1.5 SBF for 7 days. At the same time, FTIR and XPS analysis confirmed successful growth of apatite-like structures, such as HA or ACP. Therefore, U/S calcium carbonate mineralization of the electrospun scaffolds allows to achieve a very pronounced apatite-forming behavior during the interaction of the biocomposite surface with the surrounding media compared to the non-mineralized fibrous surface.

Figure 6. (A) Survey XPS spectra measured from 1000 to 20 eV and high-resolution XPS spectra for each observed regions of (B) C 1s, (C) Ca 2p, (D) P 2p and (E) O 1s for mineralized scaffolds after 7 days soaking in 1.5 SBF. 20 ACS Paragon Plus Environment

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3.4 Adhesion and proliferation of osteoblasts onto the surface of hybrid scaffolds In the next step, the osteoblastic cell growth is investigated by fluorescence microscopy assays to explore proliferation and adhesion properties of fabricated samples. Fluorescence microscopy images showed a more spread morphology of cells onto mineralized surfaces (Figure 7A) compared to control or non-mineralized scaffolds surface. Moreover, the surface of the mineralized scaffolds showed well-distributed cells that proliferate along the fibers creating a network of cell clusters (Figure S5). After 3 days of incubation on the mineralized samples, cells formed a dense monolayer, thereby, covering the entire surface of the scaffolds, despite the fact that the samples has a 3-D structure (Figure 7A). In contrast, non-attached and somewhat clustered cells on fibers of non-mineralized scaffolds were observed. This difference of MC3T3-E1 cells behavior can be explained by a significantly higher hydrophilic surface of the scaffolds (Figure 1) and/or the presence of the inorganic phase of the bone tissue after the mineralization. This is consistent with previously reported good adhesion and spread morphology of cells on calcium carbonate mineralized fibrous scaffolds.24 The results of the AlamarBlue test (Figure 7B) revealed no statistical difference of cell viability of the samples compared to control, i.e. all scaffolds completely lack the toxicity. In addition, mineralized scaffolds provide a significant (p < 0.05) increase of the cell density on the surface (Figure 7C) compared to the non-mineralized scaffolds. It can be noted that after one day of incubation, the number of cells was significantly lower for non-mineralized scaffolds compared to control, whereas, mineralized scaffolds led to similar or even higher cell adhesion. Comparing the mineralization on the 1st and 3rd days of the incubation, it can be seen that the mineralization of the surface of scaffolds significantly improves the cells proliferation (Figure 7C). The highest cell density after the 1 and 3 days of incubation on the surface of mineralized PHBV scaffolds can be explained by a higher amount of CaCO3 observed in μCT analysis (Figure 4). All samples are thus biocompatible and the mineralized scaffolds provide significantly better osteoblasts adhesion and proliferation, which are key parameters for bone tissue engineering. 21 ACS Paragon Plus Environment


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Figure 7. (A) Optical images of MC3T3-E1 cells cultivated on the surface of the samples during 1 and 3 days (calcein, green colour). The scale bar is 50 μm. The scale bar inside the red inserts is 10 μm. (B) Cell viability measured at different times using an AlamarBlue test on MC3T3-E1 cells incubated with samples in the culture medium. (C) The number of cells adhered to the surface of the samples for 1 and 3 days. An asterisk (*) indicates significant differences from the control cell group. An ampersand (&) denotes significant differences between mineralized samples. A caret (^) indicates a significant difference in cells density between modified samples. A dollar sign ($) indicates a significant difference in cells density between modified and mineralized scaffolds based on the same polymer. The statistical analysis was performed by ANOVA followed by the Tukey test (p < 0.05).


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In summary, CaCO3-mineralization represents an essential step in functionalization of piezoelectric scaffolds based on PHB and PHBV polymers. In perspective, further surface functionalization with enzyme can be used to regulate cell proliferation,75-76 which can be deposited in the form of brushes77 or polymeric coatings.78

4 Conclusions Herein, novel piezoelectric PHB and PHBV fibrous scaffolds were assembled by electrospinning and subsequently mineralized with CaCO3 in vaterite and calcite phases using ultrasound. Characterization of hybrid biocomposites by SEM, XPS and XRD techniques reveals uniformly coated fibers of three-dimensional PHB and PHBV scaffolds. The influence of the porosity and local electric charge on CaCO3 formation at dynamic mechanical conditions is determined, since there is no difference in the wettability, phase and chemical compositions of non-mineralized and mineralized PHB and PHBV polymer scaffolds. Based on a significantly higher piezoelectric charge coefficient (d33 = 3.0 ± 0.5 pC/N) of PHB compared to that of PHBV scaffolds (d33 = 0.7 ± 0.5 pC/N), it is proposed that different surface potential affects ions distribution during the U/S mineralization by increasing the local ion concentration and providing a deeper ion penetration. At the same time, an increased porosity for PHB scaffolds (by 15 %), as incurred from μCT measurements, allows for solutions to penetrate deeper. This results in a more homogenous CaCO3 distribution in the whole volume of PHB scaffolds compared to that for PHBV, which is confirmed by a deeper CaCO3 growth observed from μCT analysis and resulting in ~ a 2 times relative increase in the mass of the scaffolds. The U/S mineralization of PHB and PHBV scaffolds with biocompatible CaCO3 transforms their surfaces from hydrophobic (for non-mineralized) to hydrophilic (for CaCO3-mineralized), and this significantly enhances the osteoblast cell adhesion and proliferation, and improves apatite-forming behavior. Although all prepared samples exhibit no toxicity and generally good cell growth rates, mineralized PHBV scaffolds facilitate a statistically significant higher osteoblast cell growth compared to that grown on PHB – this is 23 ACS Paragon Plus Environment


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assigned to a higher amount of CaCO3 on the surface as confirmed by the μCT analysis. Thus, biodegradable PHB-based piezoelectric scaffolds can be very effective to replace defected bone tissue and stimulate formation of inorganic bone phase under dynamic mechanical conditions.

Acknowledgments This research was mainly carried out at Tomsk Polytechnic University within the framework of Tomsk Polytechnic University Competitiveness Enhancement Program grant. The authors are thankful to Maksim Syrtanov for the assistance with XRD measurements. RC acknowledges support of the German-Russian Interdisciplinary Science Center (G-RISC). The authors also acknowledge the support from the Special Research Fund (BOF) of Ghent University (BOF16/FJD/029). Anatolii Abalymov thanks the Russian government funding program “Global education”. BVP thanks FWO (1524618N) for support. AGS acknowledges support of the Special Research Fund (BOF) of Ghent University (01IO3618, BAS094-18, BOF14/IOP/003) and FWOVlaanderen (G043219, G0D7115N). BVP is a FWO post-doctoral fellow. We thank the Deutscher Akademischer Austauschdienst (DAAD) for funding in the Leonhard-Euler program.

Supporting information Other experimental results describing the surface and the element composition of the samples after mineralization and SBF assays using SEM and EDX. Images of cell morphology are also available in Supporting Information. Notes The authors declare no competing financial interests.[


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Figure 1. SEM images of (A-C) PHBV and (D-F) PHB scaffolds: (A,D) a top view of non-mineralized; (B, E) a top view and (C,F) magnified top view of mineralized. Insets in (A,B,D,E) show optical images of water CA for corresponding scaffolds. Insets in (C,F) show quantified EDX spectra of corresponding mineralized scaffolds, where “Others” are referred to as Au, Pd, Na and Cl. 197x115mm (96 x 96 DPI)


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Figure 2. (A) Survey XPS spectra; and high-resolution XPS spectra of the (B) Ca 2p, (C) C 1s and (D) O 1s regions for PHBV and PHB scaffolds before and after mineralization.



Figure 3. XRD patterns of PHBV and PHB scaffolds before (bottom row) and after (top row) mineralization with CaCO3: C – calcite, V – vaterite, H – halite. 304x215mm (300 x 300 DPI)


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Figure 4. μCT images of the mineralized (A) PHB and (B) PHBV scaffolds with fibers and without fibers to show CaCO3 particles distribution; (C) calculated porosity of the scaffolds; (D) the piezoelectric charge coefficient of scaffolds before (black and orange) and after (blue and red) 3 US treatment; (E) a relative increase in the mass of scaffolds after each US treatment; (F) scheme of a scaffold cross-section view demonstrating an influence of the surface charge on mineralization.



Figure 5. SEM images and insets of quantified EDX of (A,B) CaCO3-mineralized PHBV and (D,E) PHB scaffolds after 7 days immersion in 1.5 SBF, where “Others” are referred to Au, Pd, Na and Cl. FTIR spectra of the (C) PHBV and (F) PHB scaffolds after mineralization and subsequent 7 days immersion in 1.5 SBF


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Figure 6. Survey XPS spectra and high-resolution XPS spectra of C 1s, O 1s, Ca 2p and P 2p regions for mineralized scaffolds after 7 days soaking in 1.5 SBF. 186x168mm (96 x 96 DPI)



Figure 7. (A) Optical images of MC3T3-E1 cells cultivated on the surface of the samples during 1 and 3 days (calcein, green colour). The scale bar is 50 μm. The scale bar inside the red inserts is 10 μm. (B) Cell viability measured at different times using an AlamarBlue test on MC3T3-E1 cells incubated with samples in the culture medium. (C) The number of cells adhered to the surface of the samples for 1 and 3 days. An asterisk (*) indicates significant differences from the control cell group. An ampersand (&) denotes significant differences between mineralized samples. A caret (^) indicates a significant difference in cells density between modified samples. A dollar sign ($) indicates a significant difference in cells density between modified and mineralized scaffolds based on the same polymer. The statistical analysis was performed by ANOVA followed by the Tukey test (p < 0.05).


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Based Scaffolds Ultrasound-Mineralized with Calcium Carbonate for

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