Materials Science and Engineering C 73 (2017) 756–766

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Materials Science and Engineering C
journal homepage: www.elsevier.com/locate/msec

Inﬂuence of different grained powders and pellets made of Niobium andTi-42Nb on human cell viability
Jana Markhoff a,⁎, Markus Weinmann b, Christian Schulze a, Rainer Bader aabUniversity Medicine Rostock, Department of Orthopedics, Biomechanics and Implant Technology Laboratory, Doberaner Strasse 142, 18057 Rostock, Germany
H.C. Starck Tantalum and Niobium GmbH, Im Schleeke 78-91, 38642 Goslar, Germanya r t i c l e

i n f oArticle history:
Received 16 August 2016
Received in revised form 15 December 2016
Accepted 20 December 2016
Available online 24 December 2016
Keywords:
NiobiumTi-42Nb
Ti6Al4V
Human osteoblasts
Human ﬁbroblasts
Powder particles

a b s t r a c t
Nowadays, biomaterials can be used to maintain or replace several functions of the human body if necessary. Titanium and its alloys, i.e. Ti6Al4V are the most common materials (70 to 80%) used for structural orthopedic implants due to their unique combination of good mechanical properties, corrosion resistance and biocompatibility.
Addition of β-stabilizers, e.g. niobium, can improve the mechanical properties of such titanium alloys further, simultaneously offering excellent biocompatibility. In this in vitro study, human osteoblasts and ﬁbroblasts were
cultured on different niobium specimens (Nb Amperit, Nb Ampertec), Nb sheets and Ti-42Nb (sintered and3D-printed by selective laser melting, SLM) and compared with forged Ti6Al4V specimens. Furthermore,human osteoblasts were incubated with particulates of the Nb and Ti-42Nb specimens in three concentrations
over four and seven days to imitate inﬂuence of wear debris. Thereby, the specimens with the roughest surfaces,
i.e. Ti-42Nb and Nb Ampertec, revealed excellent and similar results for both cell types concerning cell viability
and collagen synthesis superior to forged Ti6Al4V. Examinations with particulate debris disclosed a dose-dependent inﬂuence of all powders with Nb Ampertec showing the highest decrease of cell viability and collagen synthesis. Furthermore, interleukin synthesis was only slightly increased for all powders. In summary, Nb Ampertec(sintered Nb) and Ti-42Nb materials seem to be promising alternatives for medical applications compared tocommon materials like forged or melted Ti6Al4V.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Constraint or loss of functions in the human body can be the result oftumors, fractures, injuries as well as chronic diseases, infections or simply aging [1]. Nowadays, maintenance or replacement of those functions
can be achieved through the application of biomaterials mainly
consisting of metals, ceramics or polymers [2–4]. Titanium and its alloys
are the most common used implant materials in orthopedic surgery
with an amount of 70 to 80% [5–7]. Their application ranges from load
bearing areas like in orthodontics and orthopedics to gastroenterology
as well as cardiovascular and reconstructive aspects. They possess appreciable mechanical properties, good corrosion resistance and biocompatibility [8–12]. The most frequently used titanium-based material is
the titanium aluminum vanadium alloy Ti6Al4V (also referred to Ti
Grade 5) [13–16]. However, by now the elemental component vanadium is proved to be toxic and aluminum is suspected to cause e.g.
Alzheimer disease [7,17–19].
Titanium alloys in general crystallize in a hexagonal close-packed αphase (stable at 25 °C) which at 882 °C reversibly transforms into a βphase with body-centered cubic crystal structure. Addition of alloying⁎ Correponding author.
E-mail address: markhoffj@gmail.com (J. Markhoff).

http://dx.doi.org/10.1016/j.msec.2016.12.098
0928-4931/© 2016 Elsevier B.V. All rights reserved.

elements may stabilize this phase or cause crystallization, i.e. stabilization of α + β mixtures [20]. The β–type titanium alloys possess signiﬁcantly smaller Young's moduli compared with α- or α + β alloys. Someof the even build deformation-induced martensite structure possessing
a shape-memory effect [21]. The formation of the β–type structure may
avoid a mechanical mismatch in elasticity of bone and implant, causing
stress shielding associated with implant loosening. Speciﬁc β-stabilizers
for titanium alloys further improve the material properties [22–26]. For
example, non-toxic and non-allergenic niobium represents a relatively
new and promising implantable biomaterial, which is proved for its biocompatibility in vitro and in vivo. It has a corrosion resistance superior
to titanium resulting from a self-passivating inert (native) oxide surface
layer [27–32]. Furthermore, some niobium alloys offer a shape memory
effect (SME) and possess superelastic properties, analogous to selected
nickel alloys [7,32,33]. Nickel titanium alloys (NiTi) gained in importance in the last decades [34] being accounted for by its SME up to 8%[35], biocompatibility and mechanical properties (elastic modulus,
compressive strength) which are close to bone. Even though NiTi is corrosion and wear resistant [35–38] there are concerns including toxicity,allergenic reactions and carcinogenic potential of dissolved nickel ions[27,39,40].
Niobium and Ti-Nb alloys or niobium oxide (Nb2O5) materials represent excellent alternatives for medical applications [31,39,41,42], evenJ. Markhoff et al. / Materials Science and Engineering C 73 (2017) 756–766

though mechanical properties require for improvement [2]. Moreover,
one of the problems of biomaterials used is the surface contamination
with residues or particles within the production process and the formation of wear debris due to mechanical strain and friction of articulating
implant components [43–45]. Particles and wear debris can accumulate
in the periprosthetic tissue being size-dependent phagocytosed and activating or inhibiting the attendant cells. For example, osteoblasts, macrophages and osteoclasts are known to interact in various fashions [43,45–47]. Those particles cause inﬂammatory reactions and moreover,lead to increased differentiation of bone resorbing osteoclasts and inhibition of bone forming osteoblasts. Finally, all these factors result in
osteolysis and aseptic implant loosening [43,45,47].
The intention of the present study is to demonstrate the superior
biocompatibility of Ti/Nb-based alloys compared with conventional metallic implant materials like Ti6Al4V. This includes biomedical issues, i.e.
preservation of cell activities (osteoblasts, ﬁbroblasts) and non-toxicity
as well as (bio-) mechanical aspects, which are work in progress andwill be published subsequently. The focus is not only on intrinsic material properties, but also on their morphologies, also considering 3Dprinted bulk materials.
Therefore, in the present in vitro study, human osteoblasts and ﬁbroblasts were cultured on two different niobium specimens (Nb Amperit,
Nb Ampertec), Nb sheets as well as Ti-42Nb specimens (sintered andmanufactured by SLM), compared with forged Ti6Al4V and referenced
to tissue culture polystyrene (TCPS) as growth control. Furthermore,human osteoblasts were incubated with four particulates of the above
mentioned groups to imitate inﬂuence of wear debris.
2. Materials & methods
Niobium powders and niobium sheet used in this study are commercial products produced and distributed by H.C. Starck Tantalum and Niobium GmbH, Goslar, Germany. Product data sheets can be obtained onrequest from www.hcstarck.com. Spherical Ti-42Nb powder was obtained by EIGA (Electrode Induction Melting Gas Atomization, EIGA)
of Ti-42Nb rods. Appropriate SEM images are displayed in Fig. 1. Chemical analysis, particle size and abbreviations of the different powders

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used in this study are provided in Table 1. Additionally, chemical analysis of niobium sheet is given.

2.1. Preparation of test specimens
All specimens prepared for cell-biological investigations were cylindrical pellets with dimensions of d × l = 10 mm × 2 mm compoundedfrom the above mentioned powders. Nb Amperit was uniaxially pressedat 25 °C using a lab toggle press applying a load of 65 bars. Nb Ampertec
was uniaxially pressed at 25 °C using a lab toggle press applying a loadof 65 bars and subsequently sintered in vacuum at 1200 °C for 10 min.
Ti-42Nb powders could not successfully be compacted by uniaxialpressing due to their spheroidal shape. Accordingly, powder consolidation was performed pressureless by sintering at 1000 °C for 10 min of a
3 mm powder bed in Al2O3 sinter rings on Nb sheets. As-obtained specimens were machined to 2 mm height. Ti-42Nb specimens were obtained by additive manufacturing, i.e. selective laser melting (SLM) of Ti42Nb (sieve fraction b 63 μm) using a TruPrint 1000 equipment(TRUMPF GmbH + Co. KG, Ditzingen, Germany) operated at
Laserzentrum Hannover, Hannover, Germany. The following laser/scanparameters were applied: laser power 50 W, scan speed 400 mm/s,
hatch 120 μm, powder bed feed rate 50 μm/layer. Surface roughness ofthe test specimens (Rz, Ra) was analyzed by means of a tactile measurement method using a Hommel-Etamic T1000 and a sampler (TKU300,
probe tip radius 5 μm,) (both: Jenoptik, Jena, Germany). Thereby, aprobe tip with deﬁned radius slides across the surface of the test specimen permeating into the surface subject to the probe tip radius. The
measuring range was 320 μm and the length of tactile measurement
was 8 mm. Three surface points per specimen (n ≥ 3) were swept(Table 2).
To illustrate surface properties of the several metallic samples ﬁeld
emission scanning electron microscopic (FESEM) images were generated. The sample surface is gold-plated (~25 nm) using a sputter coater(Leica SCD004, Wetzlar, Germany) and ﬁgured with a Merlin
VPcompact microscope (Carl Zeiss AG, Oberkochen, Germany) with a
50-fold magniﬁcation (Fig. 2).

Fig. 1. SEM images of niobium powders recorded at 100× magniﬁcation. A: AMPERIT 160 NIOBIUM METAL 14–45 μm, B: AMPERTEC NIOBIUM EB MELTED 45–75 μm, C/D: AMPERTEC MAP
Ti-42Nb powders - C: powder fraction b63 μm, D: powder fraction 103 μm - 350 μm.

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Table 1
Particle size (PS) and chemical analysis of the powders used in this study. The chemical composition of the niobium sheet is provided for comparison.

AMPERIT 160 NIOBIUM METALAMPERTEC NIOBIUM EB MELTEDAMPERTEC MAP Ti-42Nba
NIOBIUM SHEETa

Abbreviation

PS (μm)Nb (%)Ti

O (ppm)C (ppm)H (ppm)N (ppm)Mg (ppm)

Nb Amperit
Nb Ampertec
Ti-42Nb
Nb Sheet

15–45
45–75
b350
n.a.

99.90%
99.90%
41.47
N99.99%n.d.
n.d.
58.26%
20 ppm

b2500
750
2212
90

b100
10
103
30

b100
b10
b10
10

b150
41
51
24

b10
b1
b10
n.d.

AMPERTEC MAP Ti-42Nb was subsequently fractionized (b63 μm, 63 μm–105 μm, 105 μm–350 μm) by sieving.

Forged Ti6Al4V pellets, which served as bench mark material tocompare with niobium (Nb) and Ti-42Nb samples, were provided by
DOT GmbH, Rostock, Germany.
2.2. Cell isolation
Cell cultivation was carried out in an incubator (Binder GmbH,
Tuttlingen, Germany) at simulated in vivo conditions at 5% CO2, 21%
O2 and 37 °C with regular medium changes. Human osteoblasts wereisolated after patient agreement from the spongiosa of patients undergoing primary total hip replacement as previously described by Lochneret al. [48]. Brieﬂy, the spongiosa was mechanically removed from thefemoral head, washed with PBS and enzymatically digested using collagenase A, culture medium without fetal calf serum (FCS) and dispase II.
The gained suspension was sown out in cell culture ﬂasks after removing spongiosa fragments. Cultivation was carried out in osteogenic cell
culture medium (MEM Dulbecco, Biochrom AG, Berlin, Germany) with
10% FCS, 1% penicillin/streptomycin, 1% amphotericin B, 1% HEPES buffer (all: Gibco®-Invitrogen, Darmstadt, Germany) and additional osteogenic additives (dexamethason (100 nM), L-ascorbic acid (50 μg/ml)
and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich,Germany). The osteogenic character of the isolated cells was proved
via alkaline phosphatase staining with fuchsin substrate chromogen(DAKO, Hamburg, Germany).
Human ﬁbroblasts were isolated from skin biopsies (breast, eyelid)
provided by a local aesthetic clinic. After excision of adipose tissue, theremaining tissue was cut in equal segments (2–3 mm edge length),which were then transferred to 6-well-plates (2–3 per well) with theepidermis upwards. After 20 min of surface drying, skin was overlaid
with 3 ml DMEM-medium (with Glutamax, 10% FCS, 1% penicillin/
streptomycin, 1% amphotericin B (all: Gibco®-Invitrogen, Darmstadt,
Germany)) and cultured for three weeks. Subsequently, cells weretransferred to tissue culture ﬂasks and cryo-preserved after further
conﬂuence.
The test pellets were seeded with osteoblasts and ﬁbroblasts in adensity of 20.000 cells (third and ﬁfth passage) per 48-Well in 500 μl
culture medium. Ti-42Nb SLM specimen were tested subsequently
and only used for human osteoblast cultivation. Cultivation on the testpellets was carried out for 96 h with an exchange of culture medium
after 48 h. The Local Ethical Committee approved the use of thehuman cells for the experiments. The characteristics of the usedhuman cells are listed in Table 3.
For incubation with niobium powders, the several powders were apportioned in amounts of 5 mg in glass jars and gamma-sterilized with
25 kGy. To produce a stock solution powders were re-suspended in

500 μl of PBS (= 10 mg/ml) and diluted with culture medium to get
three working solutions (0.1/0.2/0.5 mg/ml). These concentrations
were determined by means of appropriate pre-testings. First trials
were performed on the basis of preliminary works of the working
group regarding the inﬂuence of orthopedic wear particles on humancells [48–50]. Based on these results powder concentrations wereadapted. The wide range was used to reveal concentration-dependent
differences. Osteoblasts free of powder served as control group. Cultivation was carried out for four and seven days with exchanges of culture
medium after 48 h and after 48 h and 96 h, respectively.

2.3. Metabolic cell activity and live/dead staining
Metabolic activity of cells on the test pellets was determined via mitochondrial dehydrogenase activity (WST-1 test) (Roche, GrenzachWyhlen, Germany) after 96 h of cultivation. Activity of cells cultured
with niobium powders was proven after four and seven days, respectively. The tetrazolium salt WST (water soluble tetrazolium) is transformed to formazan by mitochondrial succinate dehydrogenase frommetabolically active cells. Thereby, adsorption is directly proportional
to the metabolic cell activity and was measured at 450 nm in a Tecan
reader (Inﬁnite F200 Pro, Männedorf, Switzerland). Live/dead staining(Live/Dead Cell Viability Assay, Invitrogen, Darmstadt, Germany) wasused to analyze qualitative cell viability. Therefore, adherent cells
were simultaneously incubated with two ﬂuorescence dyes after removing the culture medium. Vital cells were visualized green due tocalcein AM (494/517 nm), which is embedded in the cell plasma.
Dead cells get red by ethidium homodimer-1 (528/617 nm) interactingwith the cell nuclei. Microscopic pictures were taken using an inverted
microscope (Nikon TS 100, Nikon GmbH, Duesseldorf, Germany) withthe appropriate ﬁlters for ﬂuorescence images. All images were madeusing similar magniﬁcation and exposure time.

2.4. Enzyme-linked immunosorbent assays
Protein syntheses were quantiﬁed by means of enzyme-linked immunosorbent assays (ELISAs). Pro-collagen type I (Metra C1CP EIA Kit,
Quidel, Buende, Germany) was chosen for human osteoblasts and ﬁbroblasts. Therefore, protein content in the culture supernatants was determined according to the manufactures instructions after 96 h ofcultivation with test pellets. CICP synthesis of osteoblasts cultured
with niobium powders was measured after four and seven days,
respectively.

Table 2Dimensions and roughness valuesa of the test pellets.
Test pellet

Dimensions d × l [mm]

Ø Rz [μm]Ø Ra [μm]Ti6Al4V
Nb Amperit
Nb Ampertec
Nb sheetTi-42Nb sinteredTi-42Nb SLM

10 × 2
10 × 2
10 × 2
10 × 1
10 × 2
10 × 215.71 ± 1.27
7.04 ± 3.08
17.50 ± 8.95
18.14 ± 3.23
41.65 ± 28.39
46.83 ± 5.70

2.38 ± 0.15
1.28 ± 0.88
2.49 ± 1.08
4.39 ± 0.38
7.38 ± 5.86
9.06 ± 1.28

aRz - arithmetical mean deviation of the proﬁle|Ra – average roughness of all individual measured values.

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Fig. 2. FESEM images of the tested pellets. A – Ti6Al4V, B – Nb Amperit, C – Nb Ampertec, D – Nb sheet, E – Ti-42Nb sintered; F - Ti-42Nb SLM; magniﬁcation: 50×; bar: 100 μm.

2.5. Cytokine analysis
To verify a possible immune-stimulatory effect of the different niobium powders, a cytokine multiplex (interleukin (IL)-6, IL-8, monocyte
chemotactic protein (MCP-1)) (Bio-Rad, Munich, Germany) was done
according to the manufactures instructions. In brief, a multi-cytokinedetection in the culture supernatants was conducted by means of microsphere beads interlinking several cytokines via speciﬁc monoclonal
antibodies and ﬂuorescence dyes. The appropriate cytokine concentration was calculated based on a standard curve. Concentration of MCP1 was mainly out of range.
2.6. Statistical analysis
The statistical signiﬁcance was calculated with Mann-Whitney-U
test using IBM® SPSS® Statistics Version 20 (IBM Corp., New York,USA). Data were shown as box plots or mean + SEM. Boxes denote interquartile ranges, horizontal lines within the boxes denote medians,
and whiskers denote minimum and maximum values. Values ofp b 0.05 were set to be signiﬁcant.
3. Results
Human osteoblasts and ﬁbroblasts were cultured on different niobium pellets for 96 h to verify their biocompatibility. The specimens werecompared to Ti6Al4V pellets and referenced to TCPS as growth control.
Metabolic activity of human osteoblasts was signiﬁcantly decreased on
Nb Amperit (− 52%) and Nb sheet (− 80%) (Fig. 3, left). Ti6Al4V(− 29%), Nb Ampertec (− 15%) and Ti-42Nb SLM (− 8%) caused aslightly reduced activity compared with the reference. Against this, cultivation on Ti-42Nb sintered (+17%) signiﬁcantly increased metabolic
activity of human osteoblasts towards control.
Similar results were obtained if cultivating human ﬁbroblasts on
those surfaces. While Ti6Al4V (− 68%) and Nb Amperit specimen(−52%) as well as Nb sheet (−92%) led to signiﬁcant loss of metabolic

Table 3Characteristics of the human donorsa used for the cell experiments.
Test material

DonorsGenderAverage age

Pellets (A-E in Fig. 2)
Ti-42Nb SLM
Pellets (A-E in Fig. 2)
Powders (A-D in Fig. 1)

Osteoblasts (n = 7)
Osteoblasts (n = 4)
Fibroblasts (n = 4)
Osteoblasts (n = 8)

5♂/2♀
4♂/–♀
–♂/2♀ (2 n/a)
4♂/4♀

65.29 ± 14.47
61.25 ± 6.34
40 ± 7.07 (2 n/a)
69.25 ± 8.46

an = number of different donors.

activity, Nb Ampertec (−6%) and Ti-42Nb sintered (−13%) only slightly deviated from the reference. Accordingly, the metabolic activity ofhuman ﬁbroblast on these specimen was much higher than on theother samples (Fig. 3, right). These results are to a certain extend
sustained by live/dead staining (Fig. 4). Thereby, results depended on
the amount of metabolic cells as well as the speciﬁc level of cell activity.
Furthermore, protein synthesis of matrix relevant pro-collagen type
1 (CICP) was determined using ELISA. CICP synthesis was signiﬁcantly
reduced in supernatants of human osteoblasts cultured on the metallic
surfaces. Nb Amperit (−92%) and Nb sheet (−78%) resulted in the lowest synthetic activity, followed by Ti6Al4V (− 58%), Ti-42Nb sintered(− 45%), Ti-42Nb SLM (− 39%) and Nb Ampertec (− 26%) comparedto TCPS (Fig. 5, white). Accordingly, Ti-42Nb sintered as well as Ti42Nb SLM and Nb Ampertec revealed a signiﬁcantly higher metabolic
activity towards CICP compared with the other metals. Cultivation ofhuman ﬁbroblasts on Ti6Al4V and niobium surfaces, i.e. Nb Amperit(−69%) and Nb sheet (−54%) resulted in a distinct decrease of metabolic activity towards control (Fig. 5, gray). Ti6Al4V (−3%) and control
medium displayed a similar performance in the Pro-collagen type I synthesis whereas CICP synthesis was noticeably increased on Nb Ampertec(+12%) and Ti-42Nb sintered (+9%)
In addition, human osteoblasts were cultured in the presence of different Nb powders using three concentrations (0.1/0.2/0.5 mg/ml) overfour and seven days. The metabolic activity of the cells was signiﬁcantly
reduced compared with the control in all experiments and mostly concentration-dependent. To receive information of the inﬂuence of surface
area of the powders on the metabolic activity, two sieve fractions of Ti42Nb powder namely a ﬁne-grained powder with x b 63 μm and acoarse powder with 103 μm b x b 350 μm were investigated.
Each experiment indicated a considerable impact of the powders on
the cell metabolism, i.e. a decrease of activity after four and seven daysof cultivation (Fig. 6). While the cell activity of Nb Amperit powder was
reduced from day four to seven there was no time-dependent inﬂuence
for Nb Ampertec detectable. However, the latter had the strongest impact on the cell metabolism and reduced the cell activity to b 40% compared with the reference, for concentrations of 0.2 and 0.5 mg Nb/ml. In
contrast, Ti-42Nb powders provoked higher activities after seven days
than after four days. Interestingly, the coarse Ti-42Nb powder(103 μm b x b 350 μm) caused a slight concentration-dependent increase of metabolic activity at day four, which became signiﬁcant at
day seven. Table 4 depicts the signiﬁcant differences between all tested
powders at day 4 and 7, respectively.
To investigate the cell-powder interaction, light microscopic pictures were recorded using an inverted microscope (Nikon TS 100,
Nikon GmbH, Duesseldorf, Germany). Those indicate up-take of small
particulates (Fig. 7, right picture) and partially powder agglomeration.

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Fig. 3. Metabolic activity of human osteoblasts (left, n ≥ 4) and human ﬁbroblasts (right, n = 4) on different test pellets (Ti6Al4V, Nb Amperit, Nb Ampertec, Nb sheet, Ti-42Nb sintered, Ti42Nb SLM) after 96 h cultivation. Boxes denote interquartile ranges, horizontal lines within the boxes denote medians, and whiskers denote minimum and maximum values. For statisticalanalysis Mann-Whitney-U test was conducted. Data were compared to the growth control (100%, *p) and against each other (°p). *p b 0.05, **p b 0.01, ***p ≤ 0.001.

Fig. 4. Live/dead staining of human osteoblasts and ﬁbroblasts on different test pellets (Ti6Al4V, Nb Amperit, Nb Ampertec, Nb sheet, Ti-42Nb sintered, Ti-42Nb SLM) after 96 h cultivation.
Living cells are displayed in green, dead ones in red. Magniﬁcation: 40×, Scale bar: 50 μm.

After cultivation with several niobium powders live/dead staining was
conducted which approved the results of the investigations on the metabolic activity (Fig. 8).
In addition, pro-collagen type I synthesis was analyzed. Data of collagen synthesis were relativized to metabolic activity and referred topowder-free control (Fig. 9). Again, concentration-dependent inﬂuenceof niobium powders was clearly visible. While Nb Amperit powder

resulted in a signiﬁcant increase of collagen synthesis higher than control at nearly any point, Nb Ampertec and ﬁne-grained Ti-42Nb revealeda decreasing pro-collagen type I synthesis with raising powder concentrations. Ti-42Nb powder (103 μm b x b 350 μm) only led to a negligible
decrease of synthesis compared to control. Altogether, the pro-collagentype I syntheses increased with increasing time for either of the substrates in either concentration (Table 5).

Fig. 5. Pro-collagen type I synthesis of human osteoblasts (white, n ≥ 4) and human ﬁbroblasts (gray, n ≥ 2) on different test pellets (Ti6Al4V, Nb Amperit, Nb Ampertec, Nb sheet,Ti-42Nb
sintered, Ti-42Nb SLM) after 96 h cultivation. Boxes denote interquartile ranges, horizontal lines within the boxes denote medians, and whiskers denote minimum and maximum values.
For statistical analysis Mann-Whitney-U test was conducted. Data were compared to the growth control (100%, *p) and against each other (°p). *p b 0.05, **p b 0.01, ***p ≤ 0.001.

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Fig. 6. Metabolic activity of human osteoblasts cultivated with different Nb and Ti-42Nb powders in three concentrations (0.1, 0.2 and 0.5 mg/ml) for four and seven days. Data are mean ±SEM (n = 8). For statistical analysis Mann-Whitney-U test was conducted. Data were compared to the growth controls (day 4 = *p/day 7 = °p) and between day 4 and day 7 (^p).
Comparison of powder concentrations per niobium/Ti-42Nb powder is marked with +p (4 days) and #p (7 days); *p b 0.05, **p b 0.01, ***p ≤ 0.001.

To evaluate an inﬂammatory response of human osteoblasts to four
different niobium powders, interleukin syntheses were examined. IL-6
production was slightly increased after cultivation with Nb Amperit,
Nb Ampertec and ﬁne-grained Ti-42Nb powder at day four compared
to control and raised further to day seven (Fig. 10). Dose-dependency
is not that consistent between the powders. Coarse-grained Ti-42Nb
caused a slight dose-dependent increased IL-6 synthesis being signiﬁcantly higher than control at day four and decreasing with further cultivation until day seven.
Measurement of IL-8 synthesis revealed similar results compared toIL-6 synthesis for the several niobium powders. However, IL-8 was synthesized in a lower amount and was most widely equal to the control
group (Fig. 11). There was no consistent trend perceptible between
the several groups concerning dose- and time-dependency.

4. Discussion
In the present in vitro study, the biocompatibility of ﬁve different niobium-based metallic samples (Nb Amperit, Nb Ampertec, Nb sheet, Ti42Nb sintered, Ti-42Nb SLM) towards human osteoblasts and ﬁbroblasts is investigated and compared with Ti6Al4V pellets. In a previous
work niobium exhibited better performance than titanium, using celllines and testing proliferation, metabolic activity and maintenance ofcell morphology [51]. Woo et al. [52] also used sintered Ti-42Nb,
which was obtained by high energy ball milling, and demonstrated
that mechanical properties and biocompatibility were superior to
Ti6Al4V. An improved biocompatibility of coated Ti-Nb alloys has also
been stated compared with pure Ti [53]. Moreover, shape memory effect and superelasticity are proved for Ti-Nb alloys [25,53]. Park et al.

Table 4
Appropriate signiﬁcances to Fig. 5 comparing the different test powders. For statistical analysis Mann-Whitney-U test was conducted. Values of p b 0.05 were set to be signiﬁcant. Significances for day 4 are above those of day 7, respectively.

n.s. - not signiﬁcant.

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Fig. 7. Light microscopic pictures of control cells without powder (left, scale bar: 100 μm) and incorporation of powder particles by human osteoblasts at day four (Nb Ampertec (0.5 mg/ml), scale bar: 10 μm).

Fig. 8. Live/dead staining of human osteoblasts cultured with different Nb and Ti-42Nb powders in three concentrations (0.1, 0.2 and 0.5 mg/ml) after four and seven days. A – Nb Amperit,
B - Nb Ampertec, C – Ti-42Nb powder (63 μm b x), D - Ti-42Nb powder (103 μm b x b 350 μm). Living cells are displayed in green, dead cells are displayed in red. Magniﬁcation: 40×, Scalebar: 50 μm.

Fig. 9. Pro-collagen type I synthesis of human osteoblasts cultivated with different Nb and Ti-42Nb powders in three concentrations (0.1, 0.2 and 0.5 mg/ml) for four and seven days. Data
are mean ± SEM (n = 8). For statistical analysis Mann-Whitney-U test was conducted. Data were compared to the growth controls (day 4 = *p/day 7 = °p) and day 4 towards day 7 (^p).
Comparison of concentrations is marked with +p (4 days) and #p (7 days); *p b 0.05, **p b 0.01, ***p ≤ 0.001.

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Table 5
Appropriate signiﬁcances to Fig. 8 comparing the several test powders against each other. For statistical analysis Mann-Whitney-U test was conducted. Values of p b 0.05 were set to besigniﬁcant. Signiﬁcances for day 4 are above those of day 7, respectively.

n.s. - not signiﬁcant.

[22] cultured ﬁbroblast cell lines on several pure metals and Ti alloys
containing e.g. Nb, V or Al. WST-1 test showed good biocompatibilityof pure Nb and the highest cell viability for Ti-10Nb. Those results areapproved by de Andrade et al. [8] and Cremasco et al. [6] culturing rat
osteoblasts and a ﬁbroblast cell line on Ti-35Nb, respectively. Do Prado
et al. [23] analyzed expression of relevant genes encoding for cell adhesion, differentiation and matrix synthesis in human osteoblasts. Thereby, Ti-35Nb revealed similar results to titanium. Bai et al. [31] usedﬁbroblast and osteoblast cell lines to examine and prove cell proliferation and differentiation on Ti-45Nb. Biocompatibility of Ti–50Nb, Ti30Nb, Ti-26Nb as well as Ti-15Nb, Ti-10Nb and Ti-5Nb with osteoblastand ﬁbroblast cell lines was also attested [24,32,54–56]. Hofstetter et
al. [57] also used human osteoblasts suggesting that Nb surfaces are

more suitable than titanium. Likewise alloys containing Ti, Nb and a further element like e.g. tantalum or aluminum are proved to be suitable
for medical applications [7,14,18,58–60]. Besides in vitro biocompatibility, niobium is also veriﬁed for in vivo use [27,61,62]. Nevertheless, a cell
speciﬁc behavior on materials with different surface roughness is also
stated. For instance, proliferation and differentiation of osteoblasts isproved to be roughness-dependent [63–66]. It is controversiallydiscussed whether osteoblasts prefer rough surfaces [63,67] [10,68,69]. Our present study strongly supports these thesis, since the specimens with the roughest surfaces (both Ti-42Nb and Nb Ampertec) revealed the best results concerning metabolic activity and collagensynthesis. As described by Lohmann et al. [70], cell response of osteoblasts to surface roughness depends on their maturation state. In

Fig. 10. IL-6 synthesis of human osteoblasts cultivated with different Nb and Ti-42Nb powders in three concentrations (0.1, 0.2 and 0.5 mg/ml) for four and seven days. Data are mean ±SEM (n = 8). For statistical analysis Mann-Whitney-U test was conducted. Data were compared to the growth controls (day 4 = *p/day 7 = °p) and day 4 towards day 7 (^p).). §p tags
difference between the several niobium powders at day 7; *p b 0.05, **p b 0.01, ***p ≤ 0.001.

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J. Markhoff et al. / Materials Science and Engineering C 73 (2017) 756–766

Fig. 11. IL-8 synthesis of human osteoblasts cultivated with different Nb and Ti-42Nb powders in three concentrations (0.1, 0.2 and 0.5 mg/ml) for four and seven days. Data are mean ±SEM (n = 8). For statistical analysis Mann-Whitney-U test was conducted. Data were compared to the growth controls (day 4 = *p/day 7 = °p) and day 4 towards day 7 (^p). $p tags
difference between the several niobium powders at day 7; *p b 0.05, **p b 0.01, ***p ≤ 0.001.

contrary, preference for smooth surfaces is well displayed for ﬁbroblasts[67,68,71]. The presented data do not endorse these studies since behavior of human osteoblasts and ﬁbroblasts was nearly similar indicating the best results for both Ti-42Nb surfaces and Nb Ampertec. Those
surfaces differed strongly in their roughness values. Thus, the results
might also be inﬂuenced by the surface material. Furthermore, Nb
Ampertec and Nb sheet showed nearly identical coefﬁcients of roughness, while Nb Ampertec resulted in great metabolic activity and procollagen type I synthesis for both types of cells. Hence, the adverse results for Nb sheets might be ascribed to production residues sincethose specimen were only stamped from on big sheet of metal without
any further post-processing except ultrasonic bath and sterilization.
Furthermore, niobium powders (Nb Amperit, Nb Ampertec, andspherical Ti-42Nb in two particle sizes) were used for osteoblast exposition to imitate inﬂuence of particulate debris on potential osteolysis. The
time- and dose-dependent inﬂuence of powders on the metabolic activity of human osteoblasts (Fig. 6) is supported by the live/dead ﬂuorescence staining quite well (Fig. 8). A decrease of cell density andincrease of dead cells is perceptible mainly for Nb Ampertec and Ti42Nb powder (63 μm b x) with higher concentrations and cultivation
time reﬂecting the results of metabolic activity. Nb Amperit und Ti42Nb powder result in a slight decrease of cell density. Nevertheless,human cells are more sensitive than cell lines, but reacting in a donorspeciﬁc manner. Accordingly, metabolic activity is not reﬂected bylive/dead staining, generally. For comparison it should be taken into account that pure powders (Amperit, Ampertec) and “alloy powders” (Ti42Nb) had been used offering different chemical compositions. Apartfrom that particle size is one of the main factors inﬂuencing cell reaction.
Particles of Nb Ampertec are larger than Nb Amperit ones, which might
be a reason for the decreased metabolic cell activity. Since Ti-42Nb powders revealed an opposite result, here the chemical particle composition
might be with higher inﬂuence than the particle size. As discernible inFig. 1, titanium free niobium powders show a different particle morphology compared to Ti-42Nb ones. The particles are more edged than
the spherical particles of the Ti-42Nb powders, which is proven to be
an important factor for biological response. Dependency of cellular responses on the shape of particles is stated for at least polymer wear debris showing a decreased aggressive potential of spherical particles
compared to other morphologies [72,73]. For a more detailed evaluation
further investigations on this topic are required. A previous work investigating wear debris of Nb alloys like e.g. Ti-6Al-7Nb compared with
common Ti6Al4V indicated a lower inﬂammatory response of monocytes cultured with Nb-containing particulates [27]. Kuroda et al. [74]examined inﬂuence of Nb and Ti wear debris in different concentrations
over four and seven days on osteoblast cell lines and proved a dose-dependent inﬂuence on cell viability being depressed and lowest with thehighest particle concentration. Thereby, viability of cells was still slightly higher using Nb debris. Similar experiments were done by Sakai et al.
[46], who approved these results, but suggested that rather particulates
wielded a slight effect on cells, but not the extracts. It is still proven thatthe effects of particulates on cells are caused by the direct contact of particulates with the cell membrane and phagocytosis of particles by cells[46]. Nevertheless, the recovery of viability is possible if the exposurelevel is below the “threshold particulate concentration value” [46]. Furthermore, it is well known, that cell viability is inﬂuenced by particulatesize, concentration, composition and dissolved ions. Even contact withbody ﬂuid (blood, urine, saliva, serum) or culture medium results in corrosion processes of metals releasing ions to their environment [3,75],
while intensity of ion dissolution is depending on medium properties(pH, temperature, chemical composition) [11,75,76] and the dissolution
rate and roughness of the metal [77,78]. Caicedo et al. [79] published acomprehensive study concerning metal ion-induced cell effects. Additionally, in our present study random supernatants of the test specimens were analyzed after 72 h via Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) for testing purposes using an
ICP Optical Emission Spectrometers (Varian/Agilent 715-ES,
Waldbronn, Germany). Sample digestion and dilution were done with
HNO3 and with H2O2 and H2O, respectively. Due to technical incidents
a high dilution of samples was necessary, so all tested supernatants
were under the detectable limit. However, Obata et al. [80] demonstrated a concentration-dependent inﬂuence of Nb ions, which induced differentiation and mineralization of osteogenic cells rather than initial celladhesion and proliferation. Niobium surfaces are well known to exhibit
good resistance against corrosion in simulated body ﬂuid and excellent
biocompatibility [41].
There are no previous in vitro studies regarding the inﬂuence of niobium and Ti-42Nb powders on human cells examining cell viability,
collagen and interleukin synthesis. Our results indicate a slight releaseof IL-6 after particulate exposure, while IL-8 synthesis is nearly unaffected. Therefore, further work concerning inﬂammatory inﬂuence should
be done. In addition, gene expression analysis and measurement ofother matrix relevant markers should be taken into account. Moreover,comparative analysis in terms of the inﬂuence of particle sizes to cell behavior are obligatory.
In summary, there are only few previous studies covering the in vitro
biocompatibility of niobium and especially Ti-42Nb. None of them aredealing with spherical Ti-42Nb powders which are required for production of patient-speciﬁc implants using 3D-printing, e.g. SLM or EBM. In
the present study, those materials show promising results withhuman osteoblasts and ﬁbroblasts and might be an alternative for medical applications to common metallic implant materials like Ti6Al4V
alloys.

J. Markhoff et al. / Materials Science and Engineering C 73 (2017) 756–766

5. Conclusions
Specimens and powders based on niobium (Nb Amperit, Nb
Ampertec) and spherical Ti-42Nb (sintered, SLM) were determined incell cultures of human osteoblasts and ﬁbroblasts. Thereby, the roughest
surfaces, both Ti-42Nb surfaces and Nb Ampertec specimen, revealed
excellent results concerning cell viability and collagen synthesis compared to commonly used implant material Ti6Al4V. Nevertheless, thecell speciﬁc preferences for surface roughness known from literature
could not be ascertained. Both cell types acted in a similar manner. Examinations with particulate debris disclosed a dose-dependent andpartly time-dependent inﬂuence of all four powders, thereby Nb
Ampertec showed the highest decrease of cell viability and collagensynthesis. Furthermore, interleukin synthesis was only slightly increased for all powders. In summary, Nb Ampertec and Ti-42Nb seemto be promising alternatives for medical applications compared to commonly applied implant materials such as Ti6Al4V.
Conﬂicts of interest
The authors declare no conﬂict of interest.
Acknowledgments
The authors thank Dr. Jürgen Weber (Aesthetics clinic Rostock) for
allocation of tissues for ﬁbroblast isolation. We would like to thank theelectron microscopy center, University Medicine Rostock for enabling
FESEM images. We thank DOT GmbH Rostock for providing theTi6Al4V test pellets. Furthermore Dr. Anika Jonitz-Heincke, Doris
Hansmann and Mario Jackszis are gratefully acknowledged for technicalsupport and Ronny Hagemann (Laser Zentrum Hannover e.V., LZH) for
selective laser melting (SLM) of the Ti-42Nb test specimens. We thank
PD Dr. Wolfgang Baumann (Leibniz Institute for Catalysis, Rostock) for
performing ICP-OES measurements.
This research did not receive any speciﬁc grant from funding agencies in the public, commercial, or not-for-proﬁt sectors.
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