Adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells on additively manufactured Ti6Al4V alloy scaffolds modified with calcium phosphate nanoparticles

Accepted Manuscript Title: Adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells on additively manufactured Ti6Al4V alloy scaffolds modified with calcium phosphate nanoparticles Authors: Ekaterina Chudinova, Maria Surmeneva, Alexander S. Timin, Timofey E. Karpov, Alexandra Wittmar, Mathias Ulbricht, Anna Ivanova, Kateryna Loza, Oleg Prymak, Andrey Koptyug, Matthias Epple, Roman A. Surmenev PII: DOI: Reference:

S0927-7765(18)30928-7 https://doi.org/10.1016/j.colsurfb.2018.12.047 COLSUB 9901

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

10 September 2018 3 December 2018 17 December 2018

Please cite this article as: Chudinova E, Surmeneva M, Timin AS, Karpov TE, Wittmar A, Ulbricht M, Ivanova A, Loza K, Prymak O, Koptyug A, Epple M, Surmenev RA, Adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells on additively manufactured Ti6Al4V alloy scaffolds modified with calcium phosphate nanoparticles, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.12.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

А short statistical summary of the article: - total number of words – 7152 - total number tables/figs – 1/7

Adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells

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on additively manufactured Ti6Al4V alloy scaffolds modified with calcium phosphate

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nanoparticles

Ekaterina Chudinovaa, Maria Surmenevaa,*, Alexander S. Timina,b,*, Timofey E. Karpovc,

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Koptyugf, Matthias Eppled, Roman A. Surmeneva,*

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Alexandra Wittmard, Mathias Ulbrichtd, Anna Ivanovaa, Kateryna Lozad, Oleg Prymakd, Andrey

Physical Materials Science and Composite Materials Centre, National Research

First I. P. Pavlov State Medical University of St. Petersburg, Lev Tolstoy str., 6/8,

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Tomsk Polytechnic University, Lenin Avenue, 30, 634050, Tomsk, Russian Federation

197022, St. Petersburg, Russian Federation Department of Molecular Biology, Peter The Great St. Petersburg Polytechnic

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University, Polytechnicheskaya, 29, 195251, St. Petersburg, Russian Federation

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Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE),

University of Duisburg-Essen, Universitaetsstr, 5-7, 45117 Essen, Germany e

Technical Chemistry II and Center for Nanointegration Duisburg-Essen (CeNIDE),

University of Duisburg-Essen , Universitaetsstr, 5-7, 45117 Essen, Germany f

Sports Tech Research Centre, Department of Quality Technology and Mechanical 1

Engineering, Mid Sweden University, Akademigatan 1, SE-831 25, Östersund, Sweden corresponding authors: [email protected] (Surmeneva M.A.), [email protected] (Timin A.S.), [email protected] (Surmenev R.A.)

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Graphical abstract

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Highlights 

Ti6Al4V (Ti64) scaffolds modified with calcium phosphate nanoparticles (CaPNPs) were

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investigated

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Ti64 scaffolds coated with CaPNPs exhibited improved hydrophilic surface

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Improved hydrophilicity of CaPNPs-coated scaffolds was beneficial for cells in vitro

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Ti64/CaPNPs scaffolds supported an increase in the alkaline phosphatase activity of cells

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Ti64/CaPNPs scaffolds displayed increased mineralization compared to Ti64 scaffolds

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Abstract In the present study, biocomposites based on 3D porous additively manufactured Ti6Al4V (Ti64) scaffolds modified with biocompatible calcium phosphate nanoparticles (CaPNPs) were investigated. Ti64 scaffolds were manufactured via electron beam melting technology using an Arcam machine. Electrophoretic deposition was used to modify the scaffolds with CaPNPs,

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which were synthesized by precipitation in the presence of polyethyleneimine (PEI). Dynamic light scattering revealed that the CaP/PEI nanoparticles had an average size of 46 ± 18 nm and a

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zeta potential of +22 ± 9 mV. Scanning electron microscopy (SEM) revealed that the spherical CaPNPs obtained had an average diameter of approximately 90 nm. The titanium-based scaffolds coated with CaPNPs exhibited improved hydrophilic surface properties, with a water contact

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angle below 5°. Cultivation of human mesenchymal stem cells (hMSCs) on the CaPNPs-coated

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Ti64 scaffolds indicated that the improved hydrophilicity was beneficial for the attachment and

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growth of cells in vitro. The Ti6Al4V/CaPNPs scaffold supported an increase in the alkaline

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phosphatase (ALP) activity of cells. In addition to the favourable cell proliferation and

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differentiation, Ti6Al4V/CaPNPs scaffolds displayed increased mineralization compared to noncoated Ti6Al4V scaffolds. Thus, the developed composite 3D scaffolds of Ti6Al4V

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repair.

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functionalized with CaPNPs are promising materials for different applications related to bone

Keywords: additive manufacturing, electron beam melting, scaffold, calcium phosphate,

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nanoparticles, electrophoretic deposition, surface properties, cell adhesion, proliferation in vivo

1. Introduction 3

Biomaterials for the replacement of human body parts are experiencing rising demand, due in part to the aging world population and also to the increased number of people engaging in sports and active lifestyles, which are unfortunately connected to an increased occurrence of traumatic injury [1]. An ideal scaffold for tissue repair and replacement must fulfil three major criteria: sufficient stiffness for stability, especially in cases of load-bearing applications; biocompatibility;

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and corrosion resistance, which together support rapid integration and long-term stability in the human body [2-4].

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Many metallic materials have been certified and are widely used for bone mending and replacement including stainless steels, cobalt-based alloys (e.g., CoCrMo), and Ti and Ti-based alloys; currently, Ti6Al4V (Ti64) is the most widely used. Ti64, which is already widely used in

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technological applications such as aerospace and automotive manufacturing, finds its place in

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orthopaedic applications precisely due to its superior physical properties such as its light weight,

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high strength-to-weight ratio, and high corrosion resistance [5].

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It is often difficult to fabricate an individualized titanium scaffold using traditional methods due to the complex structure and shape that would be required. In recent years, additive

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manufacturing (AM), commonly known as 3D printing, is attracting significant attention as it

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allows for the cost-effective production of metallic components with complex geometries. AM is a family of processes joining materials together, usually layer by layer, to make objects from 3D

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computer model data, thus getting its name based on the contrast to more traditional subtractive manufacturing methodologies [3, 6]. AM began as component prototyping (‘rapid prototyping’ or ‘rapid manufacturing’[7]), and is reaching maturity quickly. Biomedicine is one of the fields

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benefitting from the advantages of AM. Electron beam melting (EBM), which belongs to the category of so-called powder bed beam-

based additive manufacturing, was made commercially available rather recently. EBM is a promising technology that allows the manufacturing of complex metallic structures layer by layer with high precision [3, 8, 9]. In the medical field, it is used to manufacture both small series of 4

implants and support equipment, as well as to manufacture patient- and case-specific implants and scaffolds designed to better fit the demands of different medical cases [4, 10, 11]. An additional advantage of AM is its ability to manufacture components combining solid and porous sections in a single process, which is quite beneficial for the manufacturing of implants. In addition, pore size and interconnectivity can also be controlled [12, 13]. Implants with porous sections better resemble

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the structure of natural bone and support cell migration, angiogenesis, and tissue infiltration. Bone ingrowth into the porous scaffold enhances the bond to the host tissues and improves the stability

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of the implants.

As manufactured, Ti- and Ti64-based implants cannot effectively bond directly to bone due to poor osseointegration and osteoinductive properties [14-16]. In order to improve this situation,

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different surface modifications of Ti and Ti64 have been employed. Calcium phosphates (CaPs)

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are the most widely used surface modifiers for metallic implants, due to their compositional

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similarity to bone mineral and excellent biocompatibility [17, 18]. In recent years, CaPs, especially

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hydroxyapatite and calcium phosphate nanoparticles (CaPNPs), have attracted significant interest and are being used in the surface coating of implants and as a drug delivery vehicle, adding a new

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dimension to their potential application [19, 20]. CaPs are more biocompatible than many other

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ceramic or inorganic materials, and their biocompatibility and variable stoichiometry, functionality, and dissolution properties make them suitable for the purposes of both drug and

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growth factor delivery.

Among the available studies on the modification of additively manufactured Ti6Al4V

scaffolds, only a few papers have focused on nanoparticle (NP) deposition [21-23], and even fewer

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have been devoted to the application of CaPNPs as a thin coating [24]. Thus, the present study focuses on the determination of the physical, chemical, and biological properties of additively manufactured Ti6Al4V scaffolds coated with CaPNPs (Ti6Al4V/CaPNPs). Human mesenchymal stem cells (hMSCs) were chosen to study cell adhesion, proliferation, and subsequent differentiation, on as-manufactured and CaPNP-coated Ti6Al4V scaffolds. 5

2. Materials and methods 2.1 Sample manufacturing An electron beam melting machine (EBM® A2, ARCAM EBM, Mölndal, Sweden) and the associated manufacturing technology, with manufacturer settings for Ti6Al4V, that were used in this work for the fabrication of porous structures has been described in detail previously [5, 25,

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26]. Standard precursor powder supplied by ARCAM EBM, with grain size distribution between 50 and 125 µm, was used in a process with layer thickness of 70 µm. The working area was

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maintained at 730 °C as suggested by the manufacturer. An array of flat coin-like samples with diameter of 7 mm and thickness of 1 mm were manufactured with the flat surface normal to the build direction. All samples were carefully blasted in the standard ARCAM powder recovery

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system, using the air flow containing the precursor powder.

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2.2. Preparation of calcium phosphate nanoparticles (CaPNPs) and their deposition

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CaPNPs with polyethyleneimine (PEI) as stabilizer were used for the bioactive coating. Aqueous solutions of [CH3CH(OH)COO]2Ca·5H2O (calcium lactate, 6 mM; Merck Chemicals,

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Darmstadt, Germany) and (NH4)2HPO4 (3.6 mM; Aldrich, Steinheim, Germany) were adjusted to pH 9 with NH3(aq) and then mixed in a tubular reactor (50 mm length, 2.54 mm inner diameter, Y-

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connector) with a flow rate of 25 mL min−1 each. Simultaneously, a 2 g L–1 PEI solution (25 000

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g mol-1, branched; Aldrich, Steinheim, Germany) was added through another Y-connector at a flow rate of 12.5 mL min−1 to inhibit particle growth and to colloidally stabilize the nanoparticles. The particles were collected by centrifugation (4 000 rpm, 30 min), and re-dispersed in ethanol.

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The calcium concentration in the deposition solution was 49 μg mL-1, as determined by

atomic absorption spectroscopy (AAS). A 3 mL stainless steel beaker was used for the electrophoretic deposition. The distance between the Ti6Al4V sample and counter electrode was maintained at 3 mm. Deposition of CaPNPs from the ethanol suspension was carried out in ethanol

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at a constant voltage of 50 V for 30 min. After that, the scaffolds were dried at 50 °C. A schematic

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of the synthesis and electrophoretic deposition process is illustrated in Fig. 1.

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Fig. 1. CaPNP synthesis and electrophoretic deposition.

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2.3. Characterization of the surface morphology The morphology and elemental composition of the scaffolds were examined and

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characterized by scanning electron microscopy (gold–palladium [80:20]-sputtered samples; ESEM Quanta 400, Thermo Scientific) coupled with energy-dispersive X-ray spectroscopy (EDX;

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detector: S-UTW-Si(Li)). The hydrodynamic diameter and zeta potential of the NPs were measured through dynamic

light scattering (DLS) using a Malvern ZetasizerNano ZS system. To estimate the number of deposited NPs, the calcium content was determined by atomic absorption spectroscopy (AAS) (MSeries; Thermo Electron Corporation) with a detection limit of 1 μg L-1. For this measurement, the

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CaPNPs on the scaffolds were dissolved with HCl. Ultraviolet (UV) spectroscopy was used to determine the PO43- content of the CaPNPs dissolved from the scaffolds. To determine the internal structure and phase composition of the studied samples, a Bruker D8 ADVANCE X-ray powder diffractometer with Cu Kα radiation (λ=1.54 Å; 40 kV and 40 mA) was used. Diffraction patterns were obtained in the 2θ range of 5–90° with a step size of 0.01° and

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a counting time of 0.6 s at each step. Qualitative phase analysis was carried out with a DIFFRAC.SUITE EVA V1.2 from Bruker, using the pattern of titanium (#44-1294) in the

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database of the International Center for Diffraction Data (ICDD)) as reference.

Contact angle (CA) analyses were performed with an optical contact angle apparatus (OCA 15 Plus; Data Physics Instruments GmbH, Germany) using the SCA20 software (Data Physics

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Instruments GmbH, Germany). The static CA was recorded using a sessile drop method in air at

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room temperature. A minimum of 10 droplets (2 µL) of water, diiodomethane, or ethylene glycol

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were examined for each sample, and the mean values of CA and surface free energy were

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calculated. The surface free energy and its dispersive and polar components were estimated using

2.4. Cell culture

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the Owens-Wendt-Rabel-Kaelble (ORWK) method.

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Human mesenchymal stem cells (hMSCs) were obtained from the bone marrow of healthy

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donors who gave informed consent. Cells were isolated using a direct plating procedure. 1 mL of whole bone marrow, heparinized, was re-suspended in alpha-MEM (Lonza, Switzerland) supplemented with 100 IU mL-1 penicillin, 0.1 mg mL-1 streptomycin (Biolot, Russia), 10% foetal

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bovine serum (FBS; HyClone, USA), and 2 mM UltraGlutamine I (Lonza, Switzerland). The hMSCs were cultured in DMEM under standard cell culture conditions (i.e., 37 °C, 5/95 % CO2/air, humidified sterile environment) to above 85 % confluency. Subsequently, hMSCs were detached using trypsin (Sigma-Aldrich, UK) and used for the second stage (passage P2) for cultivation with the scaffolds. 8

2.5. Cell adhesion Adhesion of hMSCs on the surface of the samples was evaluated after 24 h of incubation. To evaluate cell morphology, hMSCs were seeded at 6.0·104 cells per well on the untreated and CaPNP-coated Ti6Al4V in a 24-well plate (n = 3). After 24 h, hMSCs were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. After 2 rounds of washing, the cells were

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permeabilized with 100 µL of a solution containing 0.5 mg/mL saponin and 5 mg/mL glycine in PBS for 5 min. The hMSCs were washed two more times with PBS and phalloidin conjugated

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with Alexa Fluor 488 to stain the cytoskeleton, and 4',6-diamidino-2-phenylindole (DAPI) diluted in PBS was added to the cells for 20 min to stain the nuclei. Then, the scaffolds and the hosted cells were visualized with a Cytell Cell Imaging System (GE Healthcare Life Sciences). The

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images of the cell nuclei were examined by the FiJi cell counter java application

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(http://imagej.net/Cell_Counter) to determine the number of cell nuclei. Cells were quantified by

2.6. Cell viability analysis

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manually outlining 10 cells per image per group in FiJi. In total, 10 images were evaluated.

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Cell viability studies were performed with a fluorescence-based approach using resazurin. Resazurin is non-toxic and almost non-fluorescent, however, it turns into fluorescent resofurin

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upon irreversible reduction in a redox reaction. For this analysis, 6.0·104 hMSCs were seeded on

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untreated and CaPNPs-coated Ti6Al4V samples placed in a 24-well plate. Each well (surface area 1 cm2) contained 1000 µL of cell growth medium. Cells were then incubated for 24 h. After incubation, the growth medium was discarded and a new medium containing 10% alamarBlue was

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added to the cells for 4 h. Next, the supernatant from each sample was transferred into a 96-well plate and analysed with a microplate reader (excitation and emission wavelengths 560 and 575 nm). The maximum of the fluorescence emission, which correlates with cell viability, was used for the evaluation. Each measurement was performed in triplicate to obtain the mean value and standard deviation. The mean fluorescence value was then plotted against incubation time. 9

2.7. Cell proliferation studies The hMSCs were seeded onto the samples placed in a 24-well plate at a density of 3.5·104 cells/well. The growth medium was changed every second day. As controls, only cells and cells seeded on scaffolds without capsules were used. Cell proliferation was analysed over 21 days. At each time point in the analysis, cells were washed twice with PBS and fixed with 4%

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paraformaldehyde for 15 min. Cells were then permeabilized with 100 µL of a solution containing 0.5 mg mL-1 saponin and 5 mg mL-1 glycine in PBS for 5 min. The hMSCs were washed two more

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times with PBS and phalloidin conjugated with Alexa Fluor 488 to stain the cytoskeleton, and DAPI diluted in PBS was added to the cells for 20 min to stain the nuclei. Then, the scaffolds with cells were visualized with the Cytell Cell Imaging System (GE Healthcare Life Sciences). Images

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of the cells showing the nuclei were examined by the FiJi cell counter java application to count

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the number of cell nuclei. The cells were quantified by manually outlining 10 cells per image per

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group in FiJi. In total, 10 images were evaluated. All measurements were performed in triplicate. 2.8. In vitro osteogenic differentiation and mineralization analysis

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In order to study osteogenic differentiation of hMSCs on scaffolds, cells were seeded onto

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the samples placed in a 24-well plate at a concentration of 3.5·104 cells/well. The growth medium was replaced the next day with an osteogenic medium consisting of DMEM supplemented with

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0.1 μM dexamethasone (Sigma-Aldrich, UK), 10 mM β-glycerol phosphate (Sigma-Aldrich, UK), and 0.2 mM ascorbic acid (Sigma-Aldrich, UK), which was changed twice a week. Cells were then placed in an incubator for 21 days, and the medium was replaced every third day. All samples were

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prepared in triplicate. 2.8.1. Staining with Alizarin Red Analysis of the calcium deposits caused by the differentiated and mineralized osteoblasts was carried out using alizarin red staining. After 14 and 21 days of incubation, cells were washed with PBS and fixed with 10% formalin for 30 min. The fixed cells were then washed 3 times with 10

MilliQ water. Afterwards, 1 mL of 40 mM alizarin red staining reagent was added to each well and left for 30 min at room temperature. The dye was then removed, the cells were washed 5 times with MilliQ water, and pictures were taken of the stained calcium deposits. Scaffolds were then transferred into 1.5 mL centrifugation tubes, to each of which 300 µL of 10% acetic acid was added, and the tubes were shaken for 30 min at room temperature. Next, samples were vortexed

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and heated to 85 °C for 10 min. Scaffolds were then centrifuged at 20 000 g for 5 min and supernatants were transferred to new tubes. Finally, 75 µL of 10% NH3(aq) was added to adjust the

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pH to between 4.1-4.5. The absorbance of all samples was then measured at 405 nm. The mineralization efficiency in the samples was then represented as the absorbance at 405 nm normalized to the DNA content. Picogreen was used to estimate the amount of DNA in the

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samples, following the standard protocol. Briefly, the lysate solution was collected and mixed with

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the Picogreen dye solution, and a microplate reader set at 485 nm excitation and 528 nm emission

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wavelengths was used to measure the fluorescence intensity. DNA content was calculated using a

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calibration plot prepared from serial dilutions of a solution with known DNA concentration.

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2.8.2. Alkaline phosphatase (ALP) activity

The ALP activity of the cells was measured using p-nitrophenyl phosphate (pNPP, Sigma)

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at certain time points during incubation for 21 days [27]. The cells were lysed with 0.1% Triton

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X-100 and freeze-thawed from -80 to 37 °C over 10 min. Upon thawing, 50 µL of the lysate was added into the wells of a 96-well plate, and an equal amount of pNPP (50 µL) was added. After 1 h incubation, the absorbance at 405 nm was recorded using the microplate reader. ALP activity

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was normalized to the total DNA content. DNA was then quantified using the Picogreen dsDNA quantification kit (Molecular Probes). Briefly, the lysate solution was collected and mixed with the Picogreen dye solution. The microplate reader set to 485 nm excitation and 528 nm emission wavelengths was used to measure the fluorescence intensity, and DNA content was calculated from a calibration plot prepared from serial dilutions of a solution of known DNA concentration. 11

3. Results and discussion 3.1. Morphology, composition and wettability of calcium phosphate nanoparticles Prior to electrophoretic deposition, the synthesized NPs were characterized by various methods. SEM showed that the CaPNPs functionalized with PEI had a near-spherical shape with average core diameter of 90 nm (Fig. 2A). The hydrodynamic diameter of the NPs was 46 ± 18

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nm, as measured by dynamic light scattering (DLS; by number). The zeta potential (ζ) was +22 ±

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9 mV.

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Fig. 2. (A) SEM image of the PEI-functionalized CaPNPs and (B) DLS measurement results for the nanoparticles dispersed in ethanol.

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Fig. 3 presents the surface morphologies of the non-coated and CaPNP-coated Ti6Al4V scaffolds. The upper images exhibit the typical surface topography of EBM-manufactured Ti6Al4V samples at the sub-millimetre level carrying traces of the 70 μm-thick manufacturing

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layers and partially fused powder grains. At the sub-micrometre range, the surface is rather homogeneous and smooth. After electrophoretic deposition, the surface of the scaffolds was completely coated with CaPNPs.

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Ti6Al4V+CaPNP s

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Ti6Al4V

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Ti6Al4V+CaPNP s

Fig. 3. SEM images of the EBM-manufactured non-coated and CaPNPs-coated Ti6Al4V alloy scaffolds (top left and right, respectively), the corresponding EDX spectra of Ti6Al4V and Ti6Al4V/CaPNPs (middle), and EDX element mapping data of the deposited NPs (bottom). 13

As shown in Fig. 3 (top right), the CaPNP coating is composed of grain agglomerates with some porosity. According to Boccaccini et al. [28], similar particles of hydroxyapatite electrochemically deposited at a relatively low electrochemical potential cannot properly migrate or produce a fully homogenous coating. In refs. [29, 30] it is noticed that the higher number of agglomerates observed after deposition of CaPNPs at 60 V than at 20 V. The authors reveal that

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the observed effect occurs due to the faster kinetics of the migration process, and thus a reduced time taken for the NPs to locate and occupy the most suitable position for the formation of a

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uniform coating.

EDX analysis of the scaffold surface confirmed the presence of the CaPNPs layer. The following elements such as Ca, O, and P were detected besides the elements from the substrate

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(Ti, Al, V). A mean Ca/P ratio was calculated to be of 1.41.

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Fig. 4 presents the XRD patterns of the non-coated and CaPNP-coated Ti6Al4V samples. In

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all cases, only peaks for Ti (#35-0816ICDD PDF2+) were observed.

Fig. 4. XRD patterns of the non-coated and CaPNP-coated Ti6Al4V prepared by additive manufacturing.

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Due to the fact that reflections from CaPNPs could not be clearly detected on the diffractograms, we were unable to make any conclusions on the structure of the deposited NPs. As for the CaPNPs, it was theorized that they may exist in the structure of either amorphous calcium phosphate or nanocrystalline hydroxyapatite. Welzel et al. [31] and Urch et al. [32] studied CaPNPs synthesized under similar conditions and identified broad diffraction peaks which

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corresponded to hydroxyapatite (Ca5(PO4)3OH) of low crystallinity. Hu et al. synthesized crystalline hydroxyapatite and amorphous calcium phosphate NPs with the same size distribution

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by changing the reaction time [33]. In this case, the preparation of crystalline hydroxyapatite NPs was performed under similar conditions to those employed for the amorphous CaPNPs, but the reaction time was extended to 24 h. Assuming that the systematized CaPNPs have the

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hydroxyapatite structure, the thickness of the deposited layer would be estimable. The

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concentration of calcium in the deposited layer was determined to be 940 μg cm−2 after dissolution

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in 0.5 M HCl. Taking the area of the sample to be 0.385 cm2 (as for the smooth flat disk of the

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same diameter) and propagating the assumption that the NPs possess hydroxyapatite structure, with density of 3.160 g сm-3. The thickness of the layer of the deposited NPs was calculated as

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follows: Thickness = Mass/(Density×Substrate area). According to the formula, it was approximately 7.5 μm. When a close packing of the spheres of equal size with a packing density

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of 74 % was assumed, the effective layer thickness was calculated to be 10 μm. Due to surface

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roughness, the actual surface area should be larger, and thus the estimated layer thickness is an estimate of the upper limit; the actual thickness is expected to be somewhat lower. As evaluated by SEM, average particle diameter was 90 nm. Thus, the coating was calculated to consist of

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approximately 125 layers of NPs. However, it should be noted that the calculated thickness is only an approximation, with a probable error of at least 10% because the sample surface is quite rough and the deposited layer might be not uniform. Some polymer remained on the surface of the particles. The thickness of the polymer layer is generally difficult to determine, but it should be of the order of the size of the molecules or a 15

few nanometres according to a study by Wallat et al. [34]. Further, it has been shown that PEI can affect cell membranes due to its positive surface charge [35, 36]. According to the water CA measurements, the deposited CaPNP assemblies exhibited a highly hydrophilic surface with water CA < 5°, while the uncoated scaffold surfaces exhibited a much higher water CA of 95.9 ± 0.9° and a moderate surface energy which was dominated by

Table 1. Summary of the wettability results

Ti6Al4V/+CaPNPs

Water

95.9 ± 0.9

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Diiodomethane

41.6 ± 0.3

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Ethylene glycol

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Contact angle (CA), °

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Parameters

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dispersive interactions (Table 1).

61.7 ± 0.4

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38.4 ± 4.8 0.39 ± 0.03

– –

38.0 ± 4.0

–

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Surface energy (SE), mN m-1 Polar contribution to SE, mN m-1

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Dispersive contribution to SE, mN m-1

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Drelich et al. [37] obtained similar results for porous, highly hydrophilic, and

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biocompatible CaP coatings. Similar results for a superhydrophilic surface of the deposited CaP coatings were presented elsewhere [38-41]. It was not possible to calculate free surface energy because liquid droplets immediately spread over the surface, but de Gennes [42] reported a high

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hydrophilicity of a similar surface, which was due to the contributions of strong polar chemical bonds toward the free surface energy in compounds with PO43- and OH- groups. It was concluded that the topography of the non-coated scaffold surface does not affect the surface hydrophilicity because the substrate has a hydrophobic surface (Table 1). However, it is worth noting that the electrophoretically deposited CaPNP coating is porous, with a significantly increased surface area (Fig. 3, top right). It is known that surface porosity causes liquids to wet the spherical elements 16

of the surface [43-46] (partially fused Ti6Al4V powder particles) and spread significantly faster if hydrophilic CaPNPs are present, which results in a decrease in the water CA. Moreover, as noted in [47-49], according to the Wenzel equation, a hydrophilic surface with micro-roughness enhances hydrophilicity due to an increase in the intrinsic surface area. It has been shown that the hydrophilic nature of implant surfaces significantly improves cell differentiation and growth factor

3.3. Adhesion, viability, and cell growth of hMSCs on scaffolds

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production [40, 50, 51].

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The viability, proliferation, and adhesion of cells to the surface of biomaterials are key factors in tissue engineering applications. For a tissue engineering scaffold, surface properties and

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structure are major factors in regulating cell behaviour and growth [52]. The biological

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performances of the non-coated and CaPNP-coated Ti6Al4V scaffolds were evaluated via testing

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the adhesion of hMSCs as well as their growth and viability. First of all, the effect of the CaPNP

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coating on the adhesion of hMSCs was investigated. Figs. 5 A and B left illustrate the hMSC test results, which revealed good adhesion of hMSCs on the scaffolds after 24 h incubation. It can be

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seen that cell adhesion to the Ti6Al4V scaffold functionalized with CaPNPs was higher than that to the non-coated Ti6Al4V scaffold, indicating that the CaPNP coating enhanced adhesion. It has

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been shown that cell adhesion depends on numerous characteristics of the underlying material,

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including its surface profile (roughness, pore size) and wettability, that affect the adsorption of the external cellular matrix (ECM) components and proteins, originating from the serum components of the cultural medium as well as produced by hMSCs. It was previously demonstrated that cell

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adhesion is very sensitive to surface wettability [53]. As shown previously, the deposition of CaPNPs improves the wettability of the scaffold surface and makes it more hydrophilic, which can result in increased adhesion of hMSCs. The viability of the hMSCs on the tested scaffolds was also investigated after 24 h incubation. The cytotoxicity was evaluated using an alamarBlue cell viability assay. As shown in Fig. 5 B right, the survival rates of the cells after 24 h of incubation 17

were above 85% for all tested scaffolds. These results clearly demonstrate that the CaPNP coating on the scaffolds did not have any influence on cell viability. Viability is a very important criterion for cell growth, since the initial cell adhesion after 24 h has an impact on cell proliferation due to integrin-mediated signalling. In contrast, low cell viability or poor adhesion would lead to apoptosis, preventing cell growth [54]. These results indicate that the Ti6Al4V scaffold

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functionalized with CaPNPs is biocompatible, supporting enhanced cell adhesion.

Fig. 5. Fluorescent images of hMSCs adhered to non-coated and CaPNPs-coated Ti6Al4V scaffolds (A: left and right, respectively). Quantitative analysis of hMSC adhesion on the non18

coated and CaPNPs-coated Ti6Al4V scaffolds after 24 h incubation (B left). Cell viability analysis of hMSCs adhered to scaffolds after 24 h of incubation (B right). Values are shown as mean ± SD, n = 3; *P < 0.05. To investigate the influence of the CaPNPs coating on cell proliferation, hMSCs were cultured on the surface of both (non-coated and CaPNPs-coated Ti6Al4V) scaffold samples over

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21 days. To visually observe the spreading behaviour of the hMSCs on the scaffolds, the cells were stained with DAPI for the cell nuclei and Alexa Fluor 488-phalloidin for F-actin. The cells were

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then visualized using a Cytell Cell Imaging System. The fluorescent images showed that, on the first day of incubation, the cells were sparsely distributed on the surfaces of both scaffolds. After 21 days, it was shown that the cells had proliferated well on the scaffolds and formed a confluent

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monolayer of cells (Figs. 6 A top and middle). The comparison of cell proliferation over the 21

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days of incubation (Fig. 6 B) demonstrates the differences in hMSC cell density between the non-

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coated and CaPNPs-coated scaffolds. The results indicate that the CaPNP coating was beneficial

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for cell proliferation, which was attributed primarily to the enhanced wettability of the CaPNPscoated surface. After 7 and 21 days of culturing, the number of cells on the CaPNPs-coated

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Ti6Al4V scaffold was higher than that on the non-coated Ti6Al4V scaffold, indicating that the

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growth.

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enhanced hydrophilicity of the CaPNPs-coated Ti6Al4V exerted a positive influence on cell

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Fig. 6. Proliferation of hMSCs adhered on non-coated and CaPNPs-coated Ti6Al4V scaffolds: (A: top and middle, respectively) fluorescent images of cells on the scaffolds after 1, 7, and 21

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days of incubation; (B) density of the hMSCs cells adhered to the scaffolds after 1, 7, and 21 days of incubation. Values are shown as mean ± SD, n = 3; *P < 0.05.

3.4. Alizarin Red S (ARS) staining and ALP activity The potential for differentiation to a variety of connective tissue cell types, including osteoblasts, adipocytes, and chondrocytes, can be considered one of the main functional 20

characteristics of hMSCs [53]. Osteoblasts can be induced to produce extracellular calcium deposits; this process is called mineralization, which is one of the key factors for bone regeneration. The composition and surface properties of the scaffold, including wettability, play a significant role in the generation of nucleation spots for mineralization with calcium phosphate [55]. The hMSCs were cultured on both non-coated and CaPNPs-coated Ti6Al4V scaffolds in

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order to evaluate the effect of the CaPNP coating on the capacity to promote mineralization. Calcium deposition was evaluated after hMSCs were cultured on scaffolds for 14 and 21 days

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(Fig. 7A left) The CaPNPs-coated scaffold exhibited enhanced mineralization compared to the non-coated scaffold, suggesting that the CaPNPs coating may promote mineralization. ALP activity in hMSCs was measured after 14 and 21 days of incubation on the scaffolds

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and used as an early stage marker to investigate osteogenic differentiation. As shown in Fig. 7A

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right, all samples showed increased ALP activity after 14 and 21 days when compared with the

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negative control (hMSCs on uncoated scaffolds cultured in growth media). The cells on the

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CaPNPs-coated scaffolds exhibited 1.2-fold higher ALP activity than those on the non-coated scaffold after 21 days of incubation. This result is in accordance with the experimental data from

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the biomineralization assays. Images of the samples stained by ARS are presented in Fig. 7B. The

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scaffolds with cells incubated in osteogenic media showed positive staining, whereas the negative controls did not show positive staining.

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These findings clearly demonstrate that the cells on the Ti6Al4V/CaPNPs scaffolds exhibited enhanced osteogenic maturation, which can be explained as follows. As previously demonstrated, the incorporation of the CaPNPs coating improved the surface hydrophilicity of

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Ti6Al4V in the composite scaffolds. It has been reported that the wettability of the surface depends on both surface energy and the roughness of the surface [56]. Numerous studies have demonstrated that enhanced surface roughness and hydrophilicity could improve the biocompatibility of scaffold materials [47-49]. Highly biocompatible scaffolds support optimal cell spreading and cell morphology. The cell-substrate adherence and induction of appropriate spread morphology may 21

promote the osteogenic lineage commitment and enhanced biomineralization in Ti6Al4V/CaPNPs

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scaffolds.

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Fig. 7. Evaluation of osteogenic differentiation of hMSCs on non-coated and CaPNPs-coated Ti6Al4V scaffolds: (A left) production of the mineralized matrix determined by quantifying the

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amount of Alizarin Red S that stained the mineralized matrix; (A right) ALP activity in hMSCs grown on both scaffolds after 14 and 21 days; (B) representative digital photographs of the ARSstained scaffold surfaces after 21 days, in the presence and absence of osteogenic supplements.

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Results are presented as average values ± standard deviation, *p < 0.05. GM, growth medium; OM, osteogenic medium.

Conclusions

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In this paper, electrophoretic deposition of near-spherical CaPNPs on the surface of additively manufactured Ti6Al4V scaffolds is reported. The wet-synthesized CaPNPs were electrophoretically deposited on the scaffolds and exhibited a homogeneous distribution on the surface. XRD patterns showed peaks corresponding to metallic Ti and no peaks which could be assigned to crystalline CaP. According to SEM, EDX, and AAS analyses, CaPNPs were uniformly

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deposited on the surface of the scaffolds. Modification of the Ti6Al4V scaffolds resulted in a significant decrease in the water CA, making the surface of the CaPNPs-coated scaffold highly

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hydrophilic with a water CA below 5. Results of the in vitro study showed that deposition of CaPNPs on Ti6Al4V scaffolds improved both adhesion and growth of hMSCs. Further, the osteogenic differentiation behaviour of hMSCs, as measured via ALP activity, was significantly

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different on the CaPNPs-coated scaffold than on the non-coated counterpart. Mineralization after

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cell culturing for 21 days was higher on the CaPNPs-coated scaffold than on the non-coated

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Ti6Al4V scaffold. Overall, the immobilization of CaPNPs on the surface of additively

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manufactured Ti6Al4V-based scaffolds is a promising approach for improving the biocompatibility of this material and enhancing the osteogenic potential of cells, both of which are

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highly important factors in bone tissue engineering applications.

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5. Acknowledgements and conflict of interest statement

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This work was supported by the grants from the Russian Science Foundation (No. 15-1300043; sample fabrication, modification, investigation, and analysis) and Russian Foundation for Basic Research (No. 18-015-00100; parts 3.2 and 3.3 of Results and Discussion). Ms. Chudinova

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thanks the German Academic Exchange Service (DAAD) for support within the Leonhard-Euler program, and the German-Russian Interdisciplinary Science Center (G-RISC) funded by the German Federal Foreign Office via the German Academic Exchange Service (DAAD).

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