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Towards the development of innovative implant: NiTi alloy functionalized by multifunctional #-TCP+Ag/SiO2 coatings Mateusz Dulski, Karolina Dudek, Damian Chalon, Jerzy Kubacki, Slawomir Sulowicz, Zofia Piotrowska-Seget, Anna Mrozek-Wilczkiewicz, Robert Gawecki, and Anna Nowak ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00510 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 22, 2019

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ACS Applied Bio Materials

Development of the new type of multifunctional implant

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Towards the development of innovative implant: NiTi alloy functionalized by multifunctional β-TCP+Ag/SiO2 coatings Mateusz Dulski1,2,*, Karolina Dudek3, Damian Chalon1, Jerzy Kubacki4, Slawomir Sulowicz5, Zofia Piotrowska-Seget5, Anna Mrozek-Wilczkiewicz2,4, Robert Gawecki2,4, and Anna Nowak2,4 1 Institute of Material Science, University of Silesia, 75 Pułku Piechoty 1a, 41-500, Chorzów, Poland 2 Silesian Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland 3 Institute of Ceramics and Building Materials, Refractory Materials Division in Gliwice, Toszecka 99, 44-100 Gliwice, Poland 4 A. Chelkowski Institute of Physics, University of Silesia,75 Pułku Piechoty 1a, 41-500, Chorzów, Poland 5 Department of Microbiology, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland Corresponding author: Mateusz Dulski +48 505 676 573 *[email protected] Abstract In recent years, one of the more important and costly problems of modern medicine is the need to replace or supplement organs in order to improve the quality of human life. On this field, promising solutions seem to have been implants which base on NiTi alloys with shape memory effects. Unfortunately, this material is susceptible to the corrosion and releasing of toxic nickel to the human organism. Hence, its application as a long-term material is strongly limited. Therefore, this paper presents the new solution which should help to improve the functionality of the NiTi alloy and elongate its medical stability to use. The idea was focused on functionalization of the metallic implant surface by biocompatible, multifunctional coating without any impact on features of the substrate, i.e. the martensitic transformation responsible for shape memory effects. In this purpose prepared the colloidal suspension composed of βTCP (particle size ~450 nm) and Ag/SiO2 nanocomposite which due to the electrophoretic deposition (EPD) led to the formation of structurally atypical calcium phosphosilicate coating. Those biomaterials formed a crack-free coating, adhering well to the NiTi surface with distributed over the entire surface, low concentration of metallic and oxide silver (< 3 at. %). At the same time, the coat-forming materials had resulted in the growth of Gram-negative bacterial biofilm. Additionally, the additive of silver-silica composite enhances cell proliferation, effectively a few times higher than commonly used coat-forming materials (e.g., pure β-TCP).

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Keywords: silver-silica nanocomposites, calcium phosphates, NiTi alloy, multifunctional coatings,

electrophoresis,

morphology,

sonochemistry,

structural

modification,

physicochemical features, antimicrobial activity, cytotoxicity Introduction The NiTi alloys have been investigated since the 1970s

1–4.

However, from the medical

point of view only this alloys with a chemical composition close to equiatomic have a shape memory effect (SME) and superelasticity effects, as well as, reveal good mechanical properties, high corrosion resistance, and biocompatibility

5–7.

Crucial is to note that SME is

related to reversible martensitic transformation occurring between the B2 parent phase (cubic structure) and the B19’ martensite (monoclinic structure). The martensitic transformation (B2→B19’) is induced by reducing the temperature, whereas the reverse martensitic transformation (B19’→B2) occurs due to alloy heating. The unique features are contributed to the increased applications of NiTi alloys in a wide range of biomedical fields 5,8. However, the NiTi shape memory alloys considered as long-term implants are usually limited by the possibility of nickel ion migration into an organism due to corrosion induced by the body fluids1. The NiTi alloy is also susceptible to temperature, leading to decomposition of the B2 parent phase. Temperature decomposition of the B2 phase to equilibrium or non-equilibrium ones alters the shape memory properties and superelasticity effect. What is more, coatings should have a relatively small thickness to maintain the shape memory effect. Other problems derived from relatively low adhesion of the ceramic coating to metallic substrate or cell to layer, significantly reducing cell proliferation or loss antibacterial activities. Hence, improvement of corrosion resistance, biocompatibility and the same protect the B2 phase of NiTi shape memory alloy surface functionalization may realize by coatings’ formation, performed in a low-temperature process, e.g. electrophoretic deposition (EPD)

9–11.

Usually,

EPD enforces heat-treatment to consolidate a deposited material with the substrate. However, in literature, one can find that the combination of calcium phosphates with chitosan nanotubes

13

12

or

enhance coating durability and adhesion just after the electrophoretic deposition

process. The EPD approach gives an opportunity to manufacture uniform coatings with control thickness composed of organic polymers 17,18.

14,

carbides/nitrides, ceramics

15,16

or composites

The most perspective biomaterials for orthopedic applications are calcium phosphates

(CaPs) such as hydroxyapatite or tricalcium phosphates (β-TCP) due to their long-term stability

and

strong

bone

tissue

response

that

enhance

biocompatibility

and 2

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osteoinductivity19,20. Moreover, CaPs improve a chemical inter-layer adhesion between growing cells or tissues and calcium phosphates, enforce fibroblast responses, as well as bone formation around the implant

21.

Finally, coverage of metallic substrate by CaPs provides

better anchorage of the implant within the organism and significantly increases the duration of its long-term usage. Hence, calcium phosphates are commonly used as a potential modifier in hard tissue replacements 22, in dentistry, orthopedics, as a carrier of drugs, or primarily in the production of bioactive cell culture media (scaffolds)

23,24.

As a result, in the literature have

appeared a lot of information about CaPs-based coatings (e.g. pure HAp, β-TCP or a combination of HAp and β-TCP) engineering both on the titanium, modified titanium as well as NiTi alloys

15,17,21,22.

Unfortunately, the dissolution behavior, discontinuous and the low

adhesion strength of the coating composed of calcium phosphates have raised concerns about the stability of the implants and require the application of the novel approach to eliminate still arising problems 25. The other issue concerns the susceptibility of CaPs-based coatings to the growth of bacterial biofilm which practically always eliminates or significantly reduce the application of such products as a material for long-term usage 26,27. The biofilm infections are responsible for 80% of all human diseases

28

characterized by middle-intensity symptoms,

chronic evolution, and resistance to antibiotic treatment

29.

As an effect, it is the required

application of complex multidrug treatment strategies 30. One of the solutions seems to be embedding of inorganic antibacterial agents, e.g. silver (prepared in micrometric and nanometric size) into the ceramic matrix such as calcium phosphate or silica ones. One of the examples could be coatings built in the form of a combination of the Ag-hydroxyapatite or Ag-β-TCP deposited on titanium alloys

26,27,31–34.

However, there are no literature reports about the β-TCP-silver combination in the context of functionalization of NiTi alloys, so far. It is important to emphasize that such combinations enforce paying higher attention to the silver which in the elevated concentration and the ionic or metallic state may imply toxicity effect. It might link to rapid depletion of silver in vivo/in vitro conditions in long-term implants which from the one side may enhance temporary antimicrobial interaction but from the other one’s limit or put to depth human cells growth. Hence, an optimization of the Ag concentration in the coat-forming material becomes a critical point which has to be meticulously considered. Another problem is related to improve the adhesion, stability, and multifunctionality of the coatings. One of the potential solutions might be the usage of silica (SiO2) or silica-based structures such as silica bioglass. It is because silica and its compounds are already present in our body and are non-toxic for them. The applicational properties of silica-based systems may 3 ACS Paragon Plus Environment

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be useful, especially in the formation of bone structures enhanced the process of their limning and regeneration after fractures

35.

According to the literature, such materials improve

adhesion of ceramics to the substrates or composite system, limit dissolution effects or increase the stability of ionic form

36,37.

In turn, silica-based materials such as

polydimethylsiloxane (PDMS) are used as a preferred soft substrate for culturing different types of cells and therefore are extensively applied as a material modifier in medical implants and biomedical devices 38. Silica glass and silica-based composites are also increasingly used to development of a novel strategy of hip, knee and spine defecttreatments 39,40. A combination of positive and negative aspects of an individual biomaterial lead to the development of an entirely new concept of the bioactive coating composed of the mixture of β-TCP and silver-silica nanocomposite. The composition of different type biomaterials should enforce the stability or multifaceted the final product, improving at the same time an application character of a NiTi alloy. To realize this challenge, we developed the optimization procedure to functionalization surface of NiTi alloy by the multifunctional coatings. A quality of multifunctional coatings characterized in terms of morphology (SEM), structure (GIXRD, Raman), chemical features (Raman, SEM, XPS), a spatial distribution of individual materials (SEM+EDS, Raman) as well as biological aspect (antimicrobial activity and cytotoxicity). The impact of deposition condition on substrate features as well as coatings’ adhesion was analyzed, respectively using DSC and scratch test methods. We would like to worthwhile that our outcomes are just pilot studies which in the future will help in better understanding the processes of surface modification. Finally, we expect that our results will help in much more effective functionalization of commonly used biomaterials, improving at their features and extend the scope of their applicability, especially in cases of anastomosis of broken bones, spine disorders (treatment of scoliosis), and in facial and maxillofacial surgery. Material preparation and characterization A multifunctional composite layer composed of commercially available tricalcium phosphate (β-TCP) as well as chemically synthesized silver-silica (Ag/SiO2) nanocomposite, were deposited on NiTi shape memory alloy (Fig. 1). The X-ray measurements revealed that the initial calcium phosphate powder consisted of (87.1 ± 1.0) wt.% β-TCP and (12.9 ± 0.2) wt.% β-Ca2P2O7 calcium pyrophosphate (β-CPP). Presence of a β-CPP is the post-production impurity of the commercially available β-TCP powder which unfortunately could not be removed from the system. First of all, the silver-silica system was prepared according to the procedure outlined by Peszke et al.41. Then, calcium phosphate and Ag/SiO2 were mixed at 1:1, 5:1 and 10:1 content ratio between compounds. As a reference, the pure β-TCP system 4 ACS Paragon Plus Environment


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was also prepared. Next, each mixture was put into one of the fourth glassy flasks and diluted in 99.8 % ethanol (Avantor Performance Materials). Finally, the so-prepared suspension was magnetic-stirred through 2h and then moved into an ultrasonic bath with 55 0C for next 24h to obtain a well-dispersed colloidal suspension (Fig. 1.1). At the same time, the commercially available NiTi alloy with the chemical composition of 50.6 at.% Ni and 49.4 at.% Ti manufactured in β-phase (B2) according to the literature 42, was polished by a SiC paper with a gradation from 360 to 2000, a 1.0 µm diamond suspension, and a 0.1 μm colloidal silica suspension to obtain a mirror shine. Then, the substrates were passivated in an autoclave at 134 0C for 30 min to form the amorphous titanium oxide

43

(Fig. 1.2). Finally, β-

TCP+Ag/SiO2 suspensions were deposited from the colloidal suspension on the passivated NiTi substrate using electrophoretic deposition (EPD) 16,44. The deposition parameters such as voltage and time were determined as 40 V and 300 s, respectively. The last step was linked to the drying of the coatings at room temperature for 24 h (Fig. 1.3). The so-prepared system was characterized by several physicochemical techniques and biological tests. First, calorimetric measurements were carried out to study the influence of the deposition process on the course of martensitic transformation of the NiTi alloy by the usage of Mettler-Toledo DSC equipped with a liquid nitrogen cooling accessory and an HSS8 ceramic sensor (heat flux sensor with 120 thermocouples). The adhesion measurements between reference calcium phosphate and then multifunctional coatings to the NiTi substrate was carried out using the scratch test method on the RST S / N machine: 02-2564 from CSM Instruments. Then, the structural investigation was obtained by grazing incident X-ray diffraction (GIXRD) using X’PertPro diffractometer. More detailed structural analysis, identification of the individual phases as well as distribution of the material entirely composite coatings was performed using WITec confocal Raman microscope CRM alpha 300R equipped with an air-cooled solid-state laser (λ = 532 nm, 10 mW at the sample) and CCD detector. Next, the morphology of the samples was characterized by Scanning Electron Microscopy (SEM) TESCAN Mira 3 LMU equipped with an Energy Dispersive Spectrometer (EDS) from Oxford Instruments. The last step of samples investigation was the detailed analysis of the chemical composition of the surface. It was carried out with the use of X-ray photoelectron spectroscopy (PHI 5700/660 Physical Electronics spectrometer). Finally, biological properties of the composites such as inhibition test for model Gram-positive (Staphylococcus aureus (ATCC®6538)), Gram-negative (Escherichia coli (ATCC®25922)) strains and eukaryotic organism such as yeast (Saccharomyces cerevisiae (ATCC®18824)) was done. Cytotoxicity of multifunctional biomaterials composed of β-TCP+Ag/SiO2 coating 5 ACS Paragon Plus Environment

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deposited on NiTi alloy investigated on a model Normal Human Dermal Fibroblast (NHDF). More detailed experimental details were described in the Supporting Information. Result and dissuasion The most useful biomaterials in regenerative medicine now, are titanium (Ti), titaniumbased and NiTi shape memory alloys. However, it is worth to mention, that these materials are usually subjected to the release of Ni ions or other harmful additions due to a long-term interaction with human body fluids

1,3,27,45.

Usually, this influences negatively on the human

organism. In consequences, it is crucial to the search for new solutions to protect the patient's health, keeping the positive features of the alloy. One of the ideas is surface functionalization e.g. by application of the bioactive coating. However, a development of the individual method, choose of the material and complex characterization of the coating including analysis of the morphology, structure, physicochemical, mechanical properties, as well as biological impact, remains the critical issue. There are many reports considering surface functionalization with the use of calcium phosphates15–17,21,22,24. Unfortunately, such coatings usually are subjected to bacterial biofilm growth, featured by relatively low adhesion to the surface and discontinuous. Hence, our idea was to develop prototype-coating composed of synergistically interacted materials including commercially available calcium phosphates, and chemically synthesized silver-silica nanocomposite encapsulated in a polymer-type shell as a coat-forming composite as well as NiTi alloy as a substrate for implant biomaterial. In assumption, the multi-phase coating should have better adhesion to the substrate than other present solutions and due to crack-free and high continuously, strongly limit the release of harmful elements from the substrate to the human organisms’ environment. Also, the functionalized NiTi biomaterial should have the antimicrobial effect, enhance cell proliferation as well as improve biocompatibility, or stability of the biomaterial

25.

In this purpose, a combination of calorimetry, diffraction,

microscopy, and spectroscopy outcomes was applied to characterize the physical and physicochemical features of the entire biomaterial. Finally, cytotoxicity, antimicrobial efficacy, and cell proliferation were analyzed to check the usefulness of the prototypecoatings. The features of the NiTi shape memory alloy after surface modification by the use of EPD were checked, respectively by DSC, while adhesion of created coatings to the metallic substrate by a scratch test. DSC data revealed no impact of surface functionalization on onestep martensitic transformation. This effect was observed in cases of pure calcium phosphate as well as composite materials (β-TCP+Ag/SiO2) (Fig. 2). Some modification appeared 6 ACS Paragon Plus Environment


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during the adhesion studies. Here, we found that calcium phosphate coatings adhered rather weak because a relatively low force (~10.7 kN) was needed to detach the coating from the substrate. In turn, much higher force (~37.3 kN) were applied to detach multi-phase coatings from the NiTi alloy’s substrate (see Supporting Information). The quality of the functionalized NiTi biomaterial was checked as the first using X-ray diffraction (XRD). Here, grazing incident XRD returned signal from NiTi with B2 structure and coat-forming materials i.e. a crystalline calcium phosphates (β-TCP, β-CCP) and metallic silver with typical cubic structure (Fig. 3 and Table 1). Presence of a β-CPP is probably the post-production impurity of the commercially available β-TCP powder which unfortunately could not be removed from the system. Silica due to lack of visible diffraction lines had amorphous character (detailed results are described in Supporting Information). The more precise, surface investigation (< 2 – 5 nm) confirmed presence in the surface layer: calcium phosphates

46,47,

oxidation states

41

non-stochiometric SiO2-x

48

and silver in ionic

49,50,

metallic

51,52

and

(Fig. 4 and see Supporting Information). In turn, SEM and Raman as bulk

techniques showed co-deposition of agglomerated particles in the form of heterogeneously distributed and irregular shape structures (Figs. 5, 6). Moreover, Raman imaging and postprocessing manipulations including K-means cluster and integrated intensity analysis distinguished five different images illustrating phase differentiation within the coating. A green and light blue cluster corresponded to calcium phosphates while yellow, dark blue and white to atypical amorphous phases, probably due to a combination of calcium phosphosilicate system with silver-polymeric structures (Fig. 6). Similar outcomes revealed elemental distribution from SEM+EDS analysis (Fig. 5). According to such complicity, the determination of the morphology, as well as estimation of the particle size of different coatforming materials, requires atypical approach in the form of so-called cross-section analysis through granules of variable chemical composition (A to B, or C to D in Fig. 7). Here, the real size of particles estimated by application of the fitting procedure using Voigt function on designed cross-section profile (Fig. 7b). As a result of the mathematical approach reported by Dudek et al.44, the particle size of green and yellow cluster estimated as close to 590 nm and 450 nm in diameter (Fig. 7c). The data from the green cluster correlates with calcium phosphates as in previously reported data

44

while the yellow cluster data corresponded

probably to the atypical submicron-sized structures formed due to electrophoresis. To explain the structural properties of the coat-forming materials as well as the origin of atypical submicron-sized structures, it is worth to get back into the stage of sample preparation. First of all, calcium phosphate and silver-silica nanocomposite 41 has been mixed 7 ACS Paragon Plus Environment

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in a glass flask, fill over with the aqua solution of ethanol and sonicated in an ultrasonic bath. In such suspension, the material has been probably subjected to so-called “sonochemical effect” arises from acoustic cavitation linked to the formation of bubbles with the temperature inside even up to 5000 ºC

53,54.

The bubbles usually are growing during the process and very

fast collapsing, provide formation of microjets throwing the particle nucleus to the surface of the material. It may provide (i) partial melting of the organic materials or ii) silica or calcium phosphate surface due to the rapid local growth of temperature resulting from the collision with the silver nanoparticles. In this context, silver probably plays the role of a catalyst inducing structural modification of the materials. Hence, the calcium phosphate or silica present in colloidal suspension might be from the molecular point of view subjected to (iii) amorphization, or (iv) depolymerization of its primary crystal structure and provide the appearance of unsaturated chemical bonds. The same effect can be observed may be observed on the substrate, especially titanium dioxide passivated the NiTi. As a result of partially surface melting of various inorganic phases (SiO2, CaP, TiO2) in linkage to its structural modification, the material probably has undergone coagulation and form more durable multiphase composites transferring into the increase of the coating adhesion after drying of the biomaterial. Those results seem to be unobvious, drawing higher attention to the way of the preparation method of a colloidal solution, and a substrate, as well as the selection of suitable deposition parameters. Given the above information, more precise characterization features of the multi-phase coatings should have to be done. Hence, as the first point, crucial is to look at the impact of sonochemical effect on structural modification of calcium phosphates (Fig. 6). Here, bands typically ascribed to the ν1(PO4)3- modes are slightly broadened relative to literature-reported positions, (Fig. 6). Additionally, the band splitting resulted from differences in nonequivalent position of monophosphate ions is lower than expected through theory

55.

This modification may result from the local disordering of the ideal network and

formation of unsaturated oxygen linkage, especially within phosphate units. Such a hypothesis was proved by the XPS data (Fig. 4). Moreover, SEM+EDS microimages showed that silver was distributed in the vicinity of the CaP-built elements suggesting the impact of silver nanoparticles on structural modification of calcium phosphate network (Fig. 5). It turns, the origin of the submicron-sized structures observed on the Raman images may originate from the system combined of silver-silica nanocomposite and calcium phosphates. To explain this effect more precisely, again it is crucial to get back into the preparation of the colloidal suspension. Here, placing the silver-silica nanocomposite in ethanol leads to the releasing of mobile ionic form of Ag which in addition to silver nanoparticles may 8 ACS Paragon Plus Environment


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additionally interact both with silica and calcium phosphate particles. This is because microjets of the sonochemical process provide high-speed throw silver nucleus which due to collisions with the calcium phosphates and silica may provide formation of amorphous calcium phosphosilicate system. As mentioned before, it is resulted from locally melting of the inorganic materials, especially at the contact zone between nanometer-size particles41. This effect is enhanced in case of the low-diameter in size structures. Hence, we expect that introduction of the d = 12 nm silver nanoparticles and d = 50 nm silica one into the solution with CaP’s should support the significant reinforcement of the sonochemical effect. What is more, the diameter of CaP particles equal to 590 nm probably favored the embedding of the silica into the calcium phosphate particles. From a structural point of view, this process may promote strong depolymerization of crystal network and formation of Si-O and P-O bonds in so-called Qn subunits of SiO4 or PO4 tetrahedra (n = number of bridging oxygen per tetrahedron). It may be observed on XRD data in the form of a slight background raised at angles about 35 - 450 (Fig. 3). Even better, this effect appeared during the Raman data analysis due to the widening of the bands (black spectra in Fig. 6). Hence, it is worth to look more closely on such results. The band fitting procedure returned very low intense bands in the region 1200 - 1000 cm-1 (1) and strong ones in the region 900 - 500 cm-1 (2). The intensity of bands from the region (1) pointed to the low impact of Q3 units with terminal oxygen atoms within SiO4 and one non-bridging oxygen (NBO) as well as fully polymerized Q4 subunits 56,57.

At the same time, the bands are overlapping with the vibration of PO32- and PO2- as well

as with the P-O-Si linkages 58. The significant vanishing of bands’ intensity in this region is resulted in local increasing the concentration of silver and correlate with amorphous or disordered calcium phosphosilicate system reported previously in the literature

59.

Strong

depolymerization is highlighted through the Raman signal linked to the Si-O-Si within Q1 subunits

60,

P-O-P and O-P-O within Q2, and Q1 subunits, as well as P-O-Si and P-O-Ca

linkages

57,58.

The spectrum of disordered calcium phosphosilicate phase is affected by

relatively intense band centered at about 235 cm-1 correlating to vibration within Ag-O linkage 41,52. Moreover, the intensity of bands in the region 900 - 500 cm-1 and around 235 cm1,

as well as yellow and dark blue clusters, indicate mutual correlation in spatial distribution

between amorphous calcium phosphosilicate and silver (Fig. 6 and Supporting Information). Similar observation proved SEM+EDS chemical maps and indirectly outcomes from XPS analysis (Figs. 4, 5 and Supporting Information). Another important thing connected to structural analysis was the appearance of bands inbetween 1600 - 1300 cm-1 and below 700 cm-1, especially well visible on the spectrum 9 ACS Paragon Plus Environment

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originated from the white area on Raman cluster image (Fig. 6). It is worth to note that those bands are not linked to the typical modes assign to main units built the calcium phosphates or silica. Therefore, to explain their presence, it is worth to look at the paper describing the silver-polymeric nanocomposite and the impact of local temperature growth on thermal degradation of polymer chains

53.

In this paper, similar like in case of our data, the broad

Raman bands in the 1600 - 1300 cm-1 region assigned to the vibration of disordered and graphitic carbon with intensity enhanced due to the impact of silver nanoparticles (Fig. 6). The position of a band at ~1200 cm-1 linked to the appearance of sp2-sp3 bonds in the system or to the C-C and C=C stretching vibrations of polyene-like structures

53.

Such results well

correlate with data obtained due to the analysis of C 1s line from XPS surface analysis (Fig. 4 and Supplementary data). In turn, the bands around 660 and 235 cm-1 are associated with the PVP-stabilized silver nanostructures 53 as in case of previously mentioned disordered calcium phosphosilica structures. Unfortunately, such results are nonintuitive and to explain its origin, it is worth to look at the synthesis procedure of the Ag/SiO2 nanocomposite. Here, in the assumption of the fabrication procedure, a silver-silica system was encapsulated through the carbon polymeric structure to prevent the metallic state of silver from the oxidation

41.

The

polymeric substructures equipped with silver nanoparticles present in colloidal suspension may imply the formation of free carbon and ensure, similar as for the chitosan, specific properties of a solution such as good film-forming ability or superior adhesion to metallic surfaces 18. An interesting observation is also the fact that the electrochemical deposition enforced the formation of relatively thin, crack-free and continues layer with the thickness depending on the phosphate-like structures. Moreover, the coating seems to be thicker in areas around calcium phosphate agglomerates. Such observation has been partially reflected through SEM and XPS data (Figs. 4, 5 and Supplementary data). The coating thickness in polymeric-rich areas estimated as not higher than 650 nm due to the depth penetration of Raman (Figs. 5, 6). Moreover, lack of titanium signal during the XPS observation testified about the uniformity of the coating probably as a result of the fulfillment of free spaces between calcium phosphate granules (green and light blue images in Fig. 6) by the carbon-like material (Figs. 4, 5 and Supplementary data). Reference β-TCP coating and multifunctional coatings prepared at the different ratio between β-TCP and Ag/SiO2 (1:1, 5:1, 10:1) were investigated using microbial toxicity test on bacterial strains of E. coli, S. aureus, and yeast S. cerevisiae, what verify this hypothesis. The antimicrobial test illustrated that multifunctional coating regardless of the mutual 10 ACS Paragon Plus Environment


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concentration between calcium phosphates and silver-silica system pointed out significantly higher toxicity to Gram-negative bacteria in comparison with pure reference β-TCP coating (Fig. 8). It may indicate that coating with silver doped may inhibit organization of biofilm created by Gram-negative bacteria. At the same time, all of the analyzed coatings demonstrated relatively low impact to S. aureus, and yeast. The strongest antimicrobial activity was found for coating composed of 1:1 and 5:1 β-TCP and Ag/SiO2 ratios. Similar bacterial behavior has been reported earlier in the case of coatings composed of silvertricalcium phosphate structures61. The explanation of antibacterial effect, notably reduction in E. coli viability can be explained by the strong inhibition of bacterial growth by silver presents in the implant 62. In this hypothesis, the release of silver correlates with the variable kind of interaction on bacteria, e.g., silver ions may interact with the bacterial outer membrane, causing the formation of irregularly shaped pits to induce changes in membrane permeability and release lipopolysaccharide from the bacteria

63.

Another mechanism is

associated with passing Ag+ ions through the cell membrane into the interior of the cell, interacting with a ribosomal subunit protein linked to thiol groups. Silver ions may also interact with sulfhydryl groups of the respiratory enzymes or proteins blocking of breath process and as a consequences lack ATP and the death of the bacterium64. On the other hand silver can react with the oxygen dissolved in the water generating reactive form of oxygen: O*, hydroxyl radicals (OH) or superoxide anions (O2–) degrade intracellular and extracellular structures, interfere with the electron transport system of the bacteria, and damage deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)

65.

Bacteria-Ag interactions may

explain the strong inhibition of E. coli growth by silver in our study, especially for coatings prepared with β-TCP+Ag/SiO2 at 1:1 and 5:1 ratio (Fig. 8). One of the explanation might be an elevated concentration of silver (> 2 at.% according to XPS data, Table 2) or relatively weak interaction between calcium phosphates and/or silica enforcing higher rate of Ag-ion released from the surface of silver nanoparticles as well as from the coat-forming material. In turn, the lower inhibition zone for 10:1 β-TCP to Ag/SiO2 ratio corresponding to lower antimicrobial effect was related to lower concentration of Ag in relation to previously mentioned coatings where according to XPS data silver was in the content below 2 at.%. Similar results previously appeared at the paper of Matsumoto et al. 34 At the same time, MTS assays have shown no cytotoxicity after 72 h of incubation on every functionalized NiTi alloys. Here, the reference calcium phosphate coating composed mainly of β-TCP at only a small amount of β-CPP (~12 %) showed the global response of the cell without the inhibition of the proliferation of the tested cells (Fig. 9). In turn, multi-phase 11 ACS Paragon Plus Environment

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coatings with the various mutual ratio between CaP and Ag/SiO2 composite pointed out much better cell response and higher cell proliferation (Fig. 9). According to XRD data similar to a reference sample, the main component of the coating is linked to tricalcium phosphate with an only small content of the second one calcium phosphate. It is, however, difficult to explain on the first glance the higher proliferation in relation to the reference sample. Thus, it is worthwhile to look not only on the result from XRD but primarily at the XPS data, illustrating a strong correlation between the percentage atomic values of elements built the surface of the coatings (Table 2). A different amount of silver-silica composite in relation to the calcium phosphates implied discrepancies in the percentage of atomic values between β-TCP and Ag/SiO2. The higher amount of calcium phosphate in relation to the silica before functionalization of the NiTi surface (10:1→5:1→1:1) implied the higher atomic concentration of calcium phosphate-built elements in accordance to composite-built elements after deposition (Table 2). Those data well-agree with the theoretical predictions, illustrating that soluble Si, Ca, P ions possess the good capability to produce intracellular and extracellular effects at the interface between the bioactive material and the cellular environment

66,67.

It has also been reported that the presence of such ions stimulates gene

expression, enhance bone metabolism through the signal transduction, as well as cell differentiation or even osteogenesis. On the other side, higher cell proliferation in case of multifunctional coatings in relation to the reference coating can be associated with the presence of low percentage atomic concentration (< 3 at.%) of silver. It was also found that the lower content of silver, the higher cell proliferation (Fig. 9). It means that the concentration of silver in the coat-forming material cannot be higher than 2 % at. because at higher concentration probably a greater role may play the cytotoxicity effect. Those results correlate quite well with previous data for Ag-hydroxyapatite coatings deposited on a titanium substrate 68,69. As reported in studies provided on Ag-modified sol-gel coating on titanium, the good cell proliferation on coatings including a low content of silver usually explains through the bio-stimulative action of silver correlated to the production of dsDNA 70. Conclusions The article was based on the development of a suitable procedure to functionalize of the metallic implant surface by a biocompatible, multifunctional coating composed of calcium phosphate and silver-silica systems without impact on features of the substrate, i.e. the martensitic transformation responsible for shape memory effect. As a result of electrophoretic deposition (EPD) process and the various mutual ratio between β-TCP and Ag/SiO2, structurally atypical amorphous calcium phosphosilicate coatings were obtained. All of such 12 ACS Paragon Plus Environment


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biomaterials imply the formation of a crack-free, as well as adhering well coating to the NiTi surface with a low concentration of metallic and oxide silver (< 3 at.%). Stronger adhesion obtained for the multiphasic system in contrast to pure calcium phosphate system might have been explained by partial melting of the surface of coat-forming materials with titanium dioxide and formation of unsaturated bonds enforcing formation of more durable composites. Here, silver nanoparticles played the role of a catalyst implied such structural modification. Another explanation is related to the presence of polymer structure which similar to chitosan may enhance the adhesion effect of the coating. Multi-phase coatings revealed positive antimicrobial effect inhibiting the growth of Gram-negative bacteria. Also, the additive of silver-silica composite additionally enhances cell proliferation, effectively a few times higher than commonly used coat-forming materials (e.g., pure β-TCP). Due to our investigations, the best biological impact had coatings prepared with 5:1 β-TCP to Ag/SiO2 ratios with a silver concentration of about 1.9 at.%. At this concentration, we found the best balance between antimicrobial features and cell proliferation. As a result, we expect that our prototype coatings will be a good alternative for currently used metallic biomaterials and functionalized biomaterials with implantology properties. Supporting Information Due to the character of the paper, a detail characterization of the multifunctional coatings, taking into account the physicochemical properties of the material, were summarized in a few individual paragraphs. Here, it can be found more detail structural interpretation of the coatforming materials by the usage of XRD and Raman data, as well as chemical interpretation using SEM+EDS and XPS data. Acknowledgments The authors are thankful for the ﬁnancial support from the National Center of Science based on

the

decision

2014/13/D/NZ7/00322

(A.M.W.),

2017/26/D/ST8/01117

(M.D.),

2017/25/N/ST8/01479 (K.D.), and 2017/26/D/NZ9/00448 (S.S.).


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List of Figures Fig. 1 Schematic representation of manufacturing procedure to develop the multifunctional composite coatings composed of a β-TCP+Ag/SiO2 on the TiO2/NiTi alloy. 1.1: preparation of the colloidal suspension, 1.2: passivation of the NiTi, 1.3: coating formation. Fig. 2 Exemplary DSC cooling (red) / heating (black) curves measured for NiTi sample coated by multifunctional coating composed of calcium phosphate and silver-silica composite, where: As - start temperature of the reverse martensitic transformation, Af - finish temperature of the reverse martensitic transformation, Ms - start temperature of the forward martensitic transformation (B2→R), Mf - finish temperature of the forward martensitic transformation (B19’→B2). Fig. 3 GIXRD patterns (α = 0.20) registered for β-TCP/Ag/SiO2/TiO2/NiTi biocomposite with different mutual concentration between tricalcium phosphate and silver-silica nanocomposite (1:1, 5:1, 10:1) deposited at 40 V for 300 s. Fig. 4 The exemplary Si 2p, O 1s, Ca 2p, P 2p, Ag3d, and C 1s core-levels for hybrid βTCP+Ag/SiO2 biocomposite coating (5:1 concentration ratio) deposited on TiO2/NiTi substrate. The core levels were fitted using Voigt function, while the background was subtracted by Shirley baseline for each peak. Fig. 5 a) Exemplary SEM image of β-TCP+Ag/SiO2 (5:1 concentration ratio) composite coating manufactured at 40 V, 300 s correlated with chemical composition imaging of all elements (Ag, Si, O, Ca, P, Ni, Ti) performed through the EDS analysis (b-m). EDS imaging data were divided into three main diagrams taken into account areas correlated with b) Ag, Si, O, c) Ca, P, O as well as d) Ni, Ti, O. The spatial distribution of an element of silver-silica, calcium phosphate, and NiTi substrate were presented in the form of a combination view (b-d) as well as an individual one (e-m). Fig. 6 Chemical phase differentiation performed by K-means cluster analysis in a fragment of the hybrid coating. Exemplary image map was performed for coating with 5:1 of β-TCP: Ag/SiO2 atomic ratio in 1600 µm2 area. a) The root cluster divided into five sub-clusters correlating to various chemical and structurally-different phases: b) blue, violet, yellow and white images come from depolymerized silica and phosphate network with strong impact of silver oxide and organic phase signal of PVP

53,

while green and light blue maps are

associated to tricalcium phosphate and calcium pyrophosphates, respectively. c) Raman 20 ACS Paragon Plus Environment


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spectra of individual phases built the multifunctional hybrid coating visualized in the 1800 250 cm-1 range with region-highlighted names (CaP: calcium phosphate, CaP∙SiO2: calcium phosphate silica, AC: amorphous carbon) with fitting analysis obtained using Voigt function with preservation of a minimum of components. Color-highlighted stars correspond to appropriate phases. Fig. 7 a) Chemical phase differentiation performed due to cluster analysis for hybrid coating, b) estimation of the particle size of β-TCP and silver-polymeric phases performed on the basis of cross-section through the green (a-b) and yellow granule (c-d) and fitted by Voigt function, and c) particle size distribution estimated for ten different β-TCP and silver-polymeric phases and fitted using logarithmic function to find maximum value of the particle distribution. Fig. 8 a) Antimicrobial activity of β-TCP+Ag/SiO2 coatings on the passivated NiTi alloy against Escherichia coli strain: photography of the inhibition zone test. b) Mean area of the inhibitory zone. Different letters (a-c) indicate significant differences between the area of inhibition (Tuckey’s test, α=0.05). Fig. 9 Evaluation of cell proliferation using MTS assay for materials prepared in the form of coatings with the variable ratio between β-TCP and Ag/SiO2 as well as coatings consist of pure β-TCP as a reference. The data are expressed as the mean values (MV) ± standard deviation (SD) of three independent experiments. No significant differences were observed between the groups (P > 0.05). List of Tables Table 1. Lattice parameters of silver (Ag), NiTi, tricalcium phosphate (β-TCP), and calcium pyrophosphate (CPP) for coatings prepared in a different ratio of calcium phosphate and silver-silica nanocomposites, obtained based on the Rietveld refinement. Reliability factors: Rexp (expected value) and Rwt (weighted value) refinement using the Rietveld method are within the range 8.61 - 9.61 % and 11.12 – 11.57 %, respectively. Table 2. Results of the surface (XPS) chemical analyses from 2 - 3 nm as well as the and Si/O and Ca/P ratios. All errors (± 0.1 eV) were estimated based on the statistical approach.


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Figures

Fig 1.

Fig. 2.



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Fig. 3.

Fig. 4.


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Fig. 5.

Fig. 6.



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Fig. 7.

Fig. 8.


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Fig. 9. Tables β-TCP+Ag/SiO2/(TiO2/NiTi) 1:1

5:1

10:1

Space group

Ag

a0 [Å]

4.084(1)

4.085(1)

4.085(2)

Fm3m

NiTi

a0 [Å]

3.014(8)

3.015(4)

3.014(2)

Pm3m

a0 [Å]

10.420(1)

10.421(7)

10.422(1)

c0 [Å]

37.354(1)

37.355(4)

37.356(2)

a0 [Å]

6.683(9)

6.681(8)

6.680(8)

c0 [Å]

24.146(9)

24.148(9)

24.146(3)

β-TCP CPP

R3c P41

Table 1. XPS surface composition [at.%] β-TCP+Ag/SiO2

Si

O

Ca

P

Ag

C

Si/O

Ca/P

1:1

27.1

42.8

1.2

0.8

2.8

25.3

0.63

1.50

5:1

22.8

48.6

1.6

1.1

2.3

23.6

0.47

1.45

10:1

23.7

47.2

3.2

2.1

1.9

21.9

0.51

1.52

Table 2.


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