materials

Article
Application of Ti6Al7Nb Alloy for the Manufacture
of Biomechanical Functional Structures (BFS) for
Custom-Made Bone Implants
Patrycja Szymczyk 1, *, Grzegorz Ziółkowski 1 , Adam Junka 2 ID
and Edward Chlebus 1
1 Center for Advanced Manufacturing Technologies (CAMT/FPC), Faculty of Mechanical Engineering,
Wroclaw University of Science and Technology, Łukasiewicza 5, 50-371 Wrocław, Poland;
grzegorz.ziolkowski@pwr.edu.pl (G.Z.); edward.chlebus@pwr.edu.pl (E.C.)
2 Department of Pharmaceutical Microbiology and Parasitology, Wrocław Medical University,
Borowska 211A, 50-556 Wrocław, Poland; feliks.junka@gmail.com (A.J.)
* Correspondence: patrycja.e.szymczyk@pwr.edu.pl; Tel.: +48-71-320-2074




Received: 8 May 2018; Accepted: 6 June 2018; Published: 8 June 2018


Abstract: Unlike conventional manufacturing techniques, additive manufacturing (AM) can form
objects of complex shape and geometry in an almost unrestricted manner. AM’s advantages include
higher control of local process parameters and a possibility to use two or more various materials
during manufacture. In this work, we applied one of AM technologies, selective laser melting, using
Ti6Al7Nb alloy to produce biomedical functional structures (BFS) in the form of bone implants.
Five types of BFS structures (A1, A2, A3, B, C) were manufactured for the research. The aim of
this study was to investigate such technological aspects as architecture, manufacturing methods,
process parameters, surface modification, and to compare them with such functional properties
such as accuracy, mechanical, and biological in manufactured implants. Initial in vitro studies
were performed using osteoblast cell line hFOB 1.19 (ATCC CRL-11372) (American Type Culture
Collection). The results of the presented study confirm high applicative potential of AM to produce
bone implants of high accuracy and geometric complexity, displaying desired mechanical properties.
The experimental tests, as well as geometrical accuracy analysis, showed that the square shaped
(A3) BFS structures were characterized by the lowest deviation range and smallestanisotropy of
mechanical properties. Moreover, cell culture experiments performed in this study proved that the
designed and obtained implant’s internal porosity (A3) enhances the growth of bone cells (osteoblasts)
and can obtain predesigned biomechanical characteristics comparable to those of the bone tissue.

Keywords: selective laser melting; Ti6Al7Nb alloy; biomedical functional structures; CT; osteoblasts

1. Introduction
The application of additive manufacturing (AM)—such as SLM (selective laser melting), LENS
(laser engineered net shaping), and EBM (electron beam melting)—opens up new possibilities for the
design of modern bone and cartilage implants. The novelty of AM technology is owed to its ability to
create geometrically advanced forms of implants displaying desired mechanical properties and high
biocompatibility with the surrounding living tissues. Great examples are metal and polymer scaffolds
of complex structure, containing the patient’s own stem cells seeded inside the implant together with
bioactive agents [1–5]. Other examples are implants and prostheses, customized to individual patient
anatomy and physiology, often designed on the basis of precise topographic data obtained by means
of computed tomography (CT) scans [6].
AM can produce scaffold-like, porous bone implants of high flexibility and decreased weight
and density. Another advantage of such biomechanical functional structure (BFS) geometry is an

Materials 2018, 11, 971; doi:10.3390/ma11060971 www.mdpi.com/journal/materials

Materials 2018, 11, 971 2 of 16

increased (in comparison to traditional implants) transport of tissue fluid within the implant–tissue
connections. The manufacture of BFS displaying specific chemical, biological and mechanical properties
adjusted to the patient’s actual load, bone deformation and/or displacement is an effective way to
provide and restore his mobility and life quality. However, there are presently no coherent data
concerning the implant’s optimal structure with regard to tissue overgrowth and differentiation,
probably due to considerable differences resulting from the application of various technologies
of scaffold manufacturing. Geometric matching of BFS structures with tissues is achieved by the
appropriate spatial structures’ porosity and pore sizes—two parameters essential for proper cell
migration within the implant. Several studies have shown that pore shape may have a significant
impact on cell response and the rate of tissue regeneration. These parameters are believed to increase
with the ratio of the implant’s curvature; moreover, cells grow more eagerly on concave surfaces in
comparison to convex and planar surfaces. Also, a higher cell growth was observed in implants with
a larger number of corners. Tissue formed by osteoblasts is strongly influenced by the geometrical
features of channels [7–9]. It was shown that minimal BFS porosity providing sufficient cell infiltration
and bone ingrowth should be at least 40% [10]. Other researchers estimated this parameter as 55–85%
and emphasized the fact that excessive porosity of a BFS may disturb its mechanical properties [11,12].
Another issue to overcome during the design of biocompatibile implants is selecting their optimal
pore size. According to some authors [13–17], pore sizes from 50 to 500 µm (and according to some
authors even 1200 µm) correlate with a satisfying level of bone regeneration. Importantly, it has been
shown that higher porosity results in improved bone regeneration in vivo, while lower porosity is
found to be more effective in vitro [7,18]. Additionally, it has been shown that small pore sizes are
easily clogged and this clot prevents cells from penetration inside the implant. This phenomenon
represents a major technological and manufacturing challenge, especially in the context of metal AM
technology [14,19]. In the case of non-resorbable materials, larger pores (<300 µm) enable smooth
cell migration [20]. Not only a pore size, but also global architecture has a large impact on cell
migration. Straight channels (of square cross-section) and straight connections between individual
pores facilitate ingrowth of cells into the implant and allow nutrients to be efficiently propagated
within the implant [21].
It is believed that BFS implants should be a surface for new tissue growth and temporarily
replace diseased or damaged bone. BFS should bear the loads imposed by daily routine and biological
processes. If bone replacement scaffolds with diversified structure are to be used to fill bone loss,
they should also display an appropriate level of mechanical strength. It has been established that
cortical and cancellous bone have compressive strengths in the ranges of 100–230 MPa and 2–12 MPa,
respectively. Their Young’s moduli are in the ranges of 3–30 GPa and 0.02–0.2 GPa, respectively [22].
It is of vital importance to obtain adequately low manufactured structure stiffness in comparison
to stiffness of the surrounding bone (it allows to reduce stress shielding at implant–bone contact
point) [20]. Moreover, mechanical load on bone tissue at implant–bone contact point is necessary,
because it induces bone regeneration, which in turn stimulates bone tissue growth [23]. Selection of
material meeting the aforementioned functions is extremely demanding.
Titanium alloys might be the material of choice in this case, however current SLM-based research
focuses mainly on Ti6Al4V alloy. Therefore, in this work we focused on an alternative titanium
alloy, namely Ti6Al7Nb. It is not commonly used for AM processing. However, it is gaining more
and more attention, mainly thanks to its good mechanical parameters, anticorrosive properties, and
weak solubility of niobium oxide in the tissue environment resulting in low or no toxicity to bone
cells [24–27]. Moreover, our team has longstanding expertise in analyzing and utilization of Ti6Al7Nb,
also as a potential material for implants.
It is presently known that implantation effectiveness depends not only on the implant’s mechanical
or functional properties, but also on surface characteristics [28,29]. In the case of such complex
spatial structures as BFS, their surface modification cannot be performed by means of traditional
techniques (e.g., machining or vibro-abrasive machining), but only by the application of chemical or
Materials 2018, 11, x FOR PEER REVIEW 3 of 16
Materials 2018, 11, 971 3 of 16

traditional techniques (e.g., machining or vibro-abrasive machining), but only by the application of
chemical or electrochemical
electrochemical methods.
methods. In our In work
previous our previous work implants,
on Ti6Al7Nb on Ti6Al7Nb implants,that
we observed we the
observed
use of that
the
the use
latter of the strongly
method latter method strongly
correlated withcorrelated with
a decreased a decreased
ability ability of Staphylococcus
of Staphylococcus aureus and
aureus and Pseudomonas
Pseudomonas
aeruginosa aeruginosa
(common bone(common bone
pathogens) to pathogens) to adhere
adhere to cleansed to cleansed
scaffold scaffold
surfaces. surfaces.
Probably Probably
nitrogen and
fluorine, elements of known bactericidal ability and used as cleansing agents, were responsible forwere
nitrogen and fluorine, elements of known bactericidal ability and used as cleansing agents, this
responsible for
phenomenon this phenomenon [30,31].
[30,31].
Bearingin
Bearing inmind
mindthetheneed
needfor
fordeveloping
developingnewnewand
andfunctional
functionalimplants,
implants,thetheaim
aimof
ofpresent
presentstudy
study
was to design, manufacture and investigate the properties of Ti6Al7Nb alloy structures
was to design, manufacture and investigate the properties of Ti6Al7Nb alloy structures with regard with regard
to their
to their functional
functional and
and biological
biological features
features and
and to
toevaluate
evaluate their
theirpotential
potential application
application as
as modern
modern
custom-made bone implants.
custom-made bone implants.

2.2.Materials
Materialsand
andMethods
Methods

2.1.
2.1.Specimen
SpecimenManufacturing
Manufacturing
AAcomputer-aided
computer-aideddesign
design(CAD)
(CAD)waswascreated
createdusing
usingthe
theMagics
Magicssoftware
software(15.0,
(15.0,Materialise
Materialise HQ,
HQ,
Leuven,
Leuven,Belgium).
Belgium). A A model
model of
of aa porous
porous unit
unit cell
cell built
built of
ofstruts
strutswas
wasdesigned
designed in inthe
theform
formofofaacube
cube
(Figure
(Figure1a).
1a).Strut
Strutdiameter
diameterwas
wassetsetat
at150 μm,while
150 µm, whilethethedistance
distance between
between strut
strut axes
axes waswas 600 μm [26].
600 µm [26].
The
Thecubic
cubicmodel
modelof ofthe
thespecimen
specimenusedusedin
inthe
thestudy
studywas wascomposed
composedof of512
512unit
unitcells
cells(8
(8 in
in each
each direction)
direction)
that
thatformed
formedaa4.954.95×× 4.95
4.95 ×
× 4.95 mm cube (Figure
(Figure 1b,c).
1b,c). The
The porosity
porosity of
of the
thedesigned
designedstructure
structurewas
was
85%. The designed geometry of BFS specimens is shown
85%. The designed geometry of BFS specimens is shown in Figure 1. in Figure 1.

Figure 1.1. (a)

Figure (a) Unit
Unit cell
cell model
modelofofthe
thedesigned
designedstructure
structureofof
thethe
specimens forfor
specimens technical research,
technical (b)
research,
designed geometry, (c) technological model, (d) SLM 50 device.
(b) designed geometry, (c) technological model, (d) SLM 50 device.

The specimens were fabricated using ReaLizer SLM 50 (Realizer GmbH, Borchen, Germany)
The specimens were fabricated using ReaLizer SLM 50 (Realizer GmbH, Borchen, Germany)
(Figure 1d) from Ti6Al7Nb alloy of 20–63 μm particle diameter (TLS Technik GmbH & Co
(Figure 1d) from Ti6Al7Nb alloy of 20–63 µm particle diameter (TLS Technik GmbH & Co Spezialpulver
Spezialpulver KG,Bitterfield, Germany). During the fabrication, an argon inert atmosphere was used
KG, Bitterfield, Germany). During the fabrication, an argon inert atmosphere was used and oxygen
and oxygen volume was in the range of 0.2–0.4 ppm. Manufacturing process parameters are shown
volume was in the range of 0.2–0.4 ppm. Manufacturing process parameters are shown in Table 1.
in Table 1.
Table 1. Process parameters used to manufacture test specimens
Table 1. Process parameters used to manufacture test specimens
Process Parameter
ProcessLaser Power
Laser Spot Size ScanScan
Velocity Layer
LayerThickness OverlapScanning
Scanning Strategy
unit Parameter W
Spot Size mm/s
Overlap
%
Power µm Velocity µm
Thickness Strategy -
value 25 100 200 50 30 XY
unit W μm mm/s μm % -
value 25 100 200 50 30 XY
The use of such manufacturing process parameters ensures sufficient material melting and allows
to achieve material
The use density
of such close to or equal
manufacturing to 100%
process [26]. Three
parameters variants
ensures of BFSmaterial
sufficient specimens featuring
melting and
diversified internal structures were manufactured (Figure 2). Several proposals were
allows to achieve material density close to or equal to 100% [26]. Three variants of BFS specimens prepared of
geometric
featuring strut structures
diversified that structures
internal could servewereas the scaffold for bone
manufactured implants.
(Figure Theproposals
2). Several application of
were
varying orientation of individual struts within the cubic sample aimed to determine the
prepared of geometric strut structures that could serve as the scaffold for bone implants. The impact of the
architecture
application of
of the BFS sample
varying relative
orientation to the forcestruts
of individual direction with
within therespect
cubic to mechanical
sample aimedproperties.
to determine
the impact of the architecture of the BFS sample relative to the force direction with respect to
mechanical properties.
Materials 2018, 11, 971 4 of 16
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FigureFigure
2. BFS2. specimen
BFS specimen types:
types: (a) type
(a) type A—specimen
A—specimen structure
structure without
without rotation
rotation with
with perpendicular
perpendicular
Figure 2. BFS specimen types: (a) type A—specimen◦ structure without rotation with perpendicular
strutsstruts
in eachin each
plane;plane; (b) type
(b) type B—structure
B—structure rotated
rotated 45 45° with
with respect
respect to one
to one axis;
axis; (c)(c) type
type C—structure
C—structure
struts in◦each plane; (b) type B—structure rotated 45° with respect to one axis; (c) type C—structure
rotated
rotated 45 with45° with respect
respect to two
to two axes.axes.
rotated 45° with respect to two axes.
In addition, some specimens (type A) were manufactured in three different orientations with
InIn
addition,
addition,some
somespecimens
specimens(type
(type A) A) were
were manufactured
manufactured in in three
threedifferent
differentorientations
orientationswith
with
respect to the build direction (Figure 3) to assess the impact of the manufacturing process on the
respect to to
respect thethe
build direction
build (Figure
direction 3) to3)assess
(Figure the impact
to assess of theofmanufacturing
the impact process
the manufacturing on theon
process quality
the
quality of the manufactured structures.
of quality
the manufactured structures.
of the manufactured structures.

Figure 3. Specimens build orientation: (a) type A1—specimen surface parallel to build platform; (b)
Figure3. 3.A2—specimen
Specimens build
Specimens build orientation: (a) type A1—specimen surface parallel to build platform; (b)
Figuretype builtorientation:
on edge; (c) (a)
typetype A1—specimen
A3—specimen built surface parallel
on corner. to build platform;
type A2—specimen built on edge; (c) type A3—specimen built on corner.
(b) type A2—specimen built on edge; (c) type A3—specimen built on corner.
A comprehensive analysis of the samples will determine the relationships between their
A comprehensive analysis of the samples will determine the relationships between their
geometry
A and mechanical
comprehensive analysis properties, as well
of the samples willas will assess
determine thethe extent to which
relationships betweenthe their
applied process
geometry
geometry and mechanical properties, as well as will assess the extent to which the applied process
and parameters
mechanical affect the
properties, biological
as well and material
asmaterial
will assess characteristics
the extent to of the manufactured structures (BFS).
parameters affect the biological and characteristics of which the appliedstructures
the manufactured process parameters
(BFS).
affect the biological and material characteristics of the manufactured structures (BFS).
2.2. Evaluation of Mechanical Properties
2.2. Evaluation of Mechanical Properties
2.2. Evaluation of Mechanical Properties
The manufactured cubic specimens were subjected to static compression test performed with
The manufactured cubic specimens were subjected to static compression test performed with
INSTRON 3384
The manufactured (Instron,
cubic Norwood,
specimens MA, USA). The teststo were carried out at atestspeed of 0.5 mm/min.
INSTRON 3384 (Instron, Norwood, MA, were
USA).subjected
The tests were static compression
carried out at a speed performed
of 0.5 mm/min.with
Young’s
INSTRON modulus
3384 (Instron, was calculated
Norwood, as
MA, the slope
USA). The of the
tests initial
were linear
carried range
out at of
a the
speedstress–strain
of 0.5 curve.
mm/min.
Young’s modulus was calculated as the slope of the initial linear range of the stress–strain curve.
Compression
Young’s modulus tests calculated
was were carried the out slope
until of
thetheoccurrence of the firstofdecrease in stress, curve.
which
Compression tests were carried as out until initial of
the occurrence linear
the range the stress–strain
first decrease in stress, which
corresponded to the first breakdown of segments (pillars) supporting the subsequent levels of BFS.
Compression
correspondedteststo thewere
first carried
breakdownout of
until the occurrence
segments of the first
(pillars) supporting the decrease
subsequent inlevels
stress,of which
BFS.
The maximum substitute stress σmax was calculated by dividing the highest load sustained by the
corresponded to the first breakdown of segments (pillars) supporting
The maximum substitute stress σmax was calculated by dividing the highest load sustained the subsequent levelsbyofthe
BFS.
specimen before fracture by the initial cross-sectional area. Samples were analyzed in relation to the
The maximum
specimen substitute
before fracture stress max was
by theσinitial calculated by
cross-sectional dividing
area. Samplesthe werehighest
analyzedloadinsustained
relation tobythe
the
direction of force (Figure 4).
specimen
directionbefore fracture
of force (Figureby 4).the initial cross-sectional area. Samples were analyzed in relation to the
direction of force (Figure 4).
Materials 2018, 11, 971 5 of 16
Materials 2018, 11, x FOR PEER REVIEW 5 of 16

Figure
Figure 4.4.BFS
BFSmodels
modelsfor
foranalysis
analysis and
and manufactured
manufactured sample
sampleorientation
orientationrelative
relativetotothe
thebuild
buildplatform
platform
and marked loading directions for individual samples: (a) A1, (b) A2, (c) A3, (d) type B, (e) typeC.C.
and marked loading directions for individual samples: (a) A1, (b) A2, (c) A3, (d) type B, (e) type

2.3. Geometric Analysis Using CT

2.3. Geometric Analysis Using CT
Technical computed tomography was used to record the external and internal geometry of the
Technical computed tomography was used to record the external and internal geometry of the
fabricated structures. The analysis was performed using Zeiss METROTOM 1500 CT system (Carl
fabricated structures. The analysis was performed using Zeiss METROTOM 1500 CT system (Carl Zeiss
Zeiss GmbH, Oberkohen, Germany) to enable coordinate measurements of complex geometric
GmbH, Oberkohen, Germany) to enable coordinate measurements of complex geometric structures.
structures. All CT measurements were performed at resolution of 21.3 μm. Based on CT
Allreconstruction,
CT measurements were of
an analysis performed
geometry at resolution
deviations wasofperformed
21.3 µm. usingBasedGOMon CT reconstruction,
Inspect V7.5 (GOMan
analysis of geometry deviations was performed using GOM Inspect V7.5 (GOM
GmbH, Braunschweig, Germany), while wall thickness analysis of the struts was performed using GmbH, Braunschweig,
Germany),
VG Studio while
Maxwall thicknessGraphics,
2.0 (Volume analysis of the struts was
Heidelberg, performed
Germany). using VG Studio
Additionally, porosity Max 2.0 (Volume
analysis was
Graphics, Heidelberg, Germany). Additionally,
performed with Avizo 8.0 (FEI, Hillsboro, OR, USA). porosity analysis was performed with Avizo 8.0 (FEI,
Hillsboro, OR, USA).
2.4. Biological Evaluation
2.4. Biological Evaluation
To confirm that BFSs can be effectively used to replace lost bone tissue and particularly to
To confirm
determine that BFSs
the extent can
of cell be effectively
adhesion used toinitial
to the surface, replace lost bone
in vitro tissue
studies wereand particularly
performed usingto
determine
osteoblast cell line hFOB 1.19 (ATCC CRL-11372) (American Type Culture Collection, VA, USA). using
the extent of cell adhesion to the surface, initial in vitro studies were performed For
osteoblast
the purposescell line hFOB 1.19 (ATCC
of microbiological CRL-11372)
testing, (American
A3 type samples Type Culture
of cylindrical form Collection, VA, USA).
have been produced
For theexternal
with purposes of microbiological
dimensions of ø 6.2 × 6testing, A3 type
mm. Because of samples
complex of cylindrical
internal formofhave
geometry been produced
the manufactured
with external dimensions of ø 6.2 × 6 mm. Because of complex internal
BFS structures, to improve surface quality and to remove powder grains remaining on thegeometry of the manufactured
surface of
BFS
thestructures,
struts theirto improve
surface has surface quality and
been chemically to remove
etched powder
in accordance grains
with remainingpresented
the guidelines on the surface
in ourof
other
the paper
struts their[30]. As inhas
surface thebeen
above-mentioned work,inwe
chemically etched divided the
accordance samples
with into two groups:
the guidelines presentednon-CP
in our
samples—submitted to preliminary cleaning using ultrasonic cleaner containing
other paper [30]. As in the above-mentioned work, we divided the samples into two groups: non-CP 99.7% isopropyl
alcohol; CP samples—pre-cleaned
samples—submitted to preliminary in 99.7% isopropyl
cleaning alcohol cleaner
using ultrasonic and then subjected 99.7%
containing to a chemical
isopropyl
polishing
alcohol; CPinsamples—pre-cleaned
ultrasonic bath with thein mixture
99.7%ofisopropyl
5 mL HF (50%)
alcohol andand
15 mL
thenHNO3 (50%) to
subjected in 200 mL of
a chemical
ultrapure water.
polishing in ultrasonic bath with the mixture of 5 mL HF (50%) and 15 mL HNO3 (50%) in 200 mL of
ultrapure water.
2.4.1. In Vitro Cytotoxicity of BFS Structures against Osteoblast Cell Line
2.4.1. In Vitro
The Cytotoxicity
extracts of BFS Structures
were prepared accordingagainst Osteoblast
ISO 10993 Cellbiological
standard: Line evaluation of medical
devices; Part 5: Tests for in vitro cytotoxicity; Part 12: Biological evaluation
The extracts were prepared according ISO 10993 standard: biological evaluation of medical devices,ofsample
medical
preparation and reference materials (ISO 10993-5:2009 and ISO/IEC 17025:2005). BFS structures
devices; Part 5: Tests for in vitro cytotoxicity; Part 12: Biological evaluation of medical devices, sample were
sterilized at 120 °C. Next, sterile specimens were placed in 1mL of sterile F12 medium
preparation and reference materials (ISO 10993-5:2009 and ISO/IEC 17025:2005). BFS structures were (for osteoblasts
culture) atand
sterilized 120left
◦ C.for 24 sterile
Next, h/37 °C/5%CO
specimens 2. After incubation, the conditioned medium was used in
were placed in 1mL of sterile F12 medium (for osteoblasts
cytotoxicity evaluation. Briefly, ◦ the conditioned media were introduced to osteoblast cell cultures and
culture) and left for 24 h/37 C/5% CO2 . After incubation, the conditioned medium was used in
incubated for 24 h, 48 h, 72 h in 5% CO2 at 37 °C. After a specified time of incubation, the medium
cytotoxicity evaluation. Briefly, the conditioned media were introduced to osteoblast cell cultures and
was removed and 100 μL of NR solution (40 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) was
incubated for 24 h, 48 h, 72 h in 5% CO2 at 37 ◦ C. After a specified time of incubation, the medium was
introduced to the wells of the plate. The cells were incubated with NR for 2 h at 37 °C. After
removed and 100 µL of NR solution (40 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) was introduced
incubation, the dye was removed, the wells were rinsed with PBS (Sigma-Aldrich) and left to dry at
to the wells of the plate. The cells were incubated with NR for 2 h at 37 ◦ C. After incubation, the dye
room temperature. Subsequently, 150 μL of de-stain solution (50% ethanol 96%, 49% deionized water,
was removed, the wells were rinsed with PBS (Sigma-Aldrich) and left to dry at room temperature.
1% glacial acetic acid; POCH, Gliwice, Poland) was introduced to each well. The plate was vigorously
Subsequently, 150 µL of de-stain
shaken in a microtiter solution
plate shaker (50%
for 30 minethanol 96%,was
until NR 49% deionized
extracted water,
from the 1% glacial
cells acetic acid;
and formed a
POCH, Gliwice,solution.
homogenous Poland) Next,
was introduced
the value ofto each well. The plate
NR absorbance was was vigorously
measured shaken in a microtiter
spectrometrically using a
plate shaker for
microplate 30 min
reader until NR was
(Multi-scan GO,extracted
Thermofrom the Scientific,
Fisher cells and formed
Waltham,a homogenous
MA, USA)solution. Next,
at 540 nm
Materials 2018, 11, 971 6 of 16

the value of NR absorbance was measured spectrometrically using a microplate reader (Multi-scan
GO, Thermo Fisher Scientific, Waltham, MA, USA) at 540 nm wavelength. The absorbance value of
cells not treated with extracts was considered 100% of potential cellular growth (positive control).

2.4.2. Osteoblast’s Growth and Multiplication on Implants

1mL containing 1.5 × 105 cfu of osteoblasts was introduced to a well of a 24-well plate containing
series I and II BFS structures and incubated for 7, 14, and 30 days at 37 ◦ C/5CO2 . The medium was
changed every 48 h or earlier if it changed color from red to green (internal medium indicator showing
pH change). All specimens were cultured in a static culture. The number of adhered cells was counted
as presented in Section 2.4.1, and selected samples were dedicated for SEM analysis.

2.4.3. Scanning Electron Microscopy

To assess cell growth on implants, a SEM (Carl Zeiss GmbH, Oberkohen, Germany) analysis was
performed. The implants incubated in the presence of osteoblasts for the periods given in Section 2.4.2.
Then, they were fixed using 3% glutaraldehyde (POCH) for 15 min at room temperature. Then,
the samples were rinsed twice with phosphate buffer (PBS; Sigma-Aldrich, Germany) to remove the
fixative. The next step was dehydration in increasing concentrations of ethanol (25%, 60%, 95%, and
100%; POCH) for 5 min in each solution. After rinsing off the ethanol, the samples were dried. Then,
the samples were covered with gold and palladium (60:40; sputter current, 40 mA; sputter time, 50 s)
using a Q150T Quorum machine (Quorum Technologies Ltd., Laughton, UK) and examined under a
Zeiss EVO MA25 scanning electron microscope (Carl Zeiss GmbH, Oberkohen, Germany).

2.5. Ability of S. epidermidis to form Biofilm

The impact of surface modifications of Ti6Al7Nb scaffolds on ability of S. epidermidis strain was
investigated. The S. epidermidis strains were cultured on McConkey agar plates and then incubated in
the TSB liquid medium (Biocorp, Warsaw, Poland) overnight. Subsequently, the strain liquid culture
was diluted to obtain suspension of 2 × 108 cells/mL. The samples were incubated in the bacterial
presence for 24 h/37 ◦ C in the aerobic conditions and then vigorously rinsed using 0.9% NaCl and
subjected to vortex mixing for 1 min. Bacterial colonies were counted and number of bacterial cells
forming biofilm on the implants was assessed. All measures were repeated three times.

2.6. Statistical Analysis

Statistical calculations were performed using Statistica software (10, Statsoft, Krakow, Poland)
and Mann–Whitney test. Statistical significance was defined as p ≤ 0.05.

3. Results

3.1. Mechanical Properties of BFS

Our studies were conducted in relation to the types of structure (Figure 3) and build orientation as
well as loading direction in regard to manufacturing direction (Figure 4). For that purpose 15 specimens
from each orientation were analyzed (five for each loading direction). Maximum compression stress
of 158 MPa was obtained for type A1 specimens. In this group we also observed the highest
anisotropy of values depending on the direction of applied loading force (Table 2), which was
respectively: 85 ± 31 MPa (1), 158 ± 14 MPa (2), 82 ± 30 MPa (3).
The most even results were obtained for type A3 specimens for which the determined mean
values were 105 ± 4 MPa. Young’s modulus of porous Ti6Al7Nb structure A3 was at the level of
3 GPa, which is between that of trabecular and cortical bone and similar for those achieved in the
literature [32]. Such a manner of sample orientation on the build platform enables the achievement of
uniform geometric deviations and the highest relative values of equivalent compressive strength. The
results have shown that the mechanical properties of the manufactured structures are not accidental
Materials 2018, 11, 971 7 of 16

and depend on load direction and manufacturing method. Porous BFSs feature sufficient mechanical
strength and low stiffness. Changing the dimensions of elementary cells (pores), strut thickness, and
their orientation enables to scale the structures in relation to the designed equivalent strength of the
Materials
structure and 2018,
to11,optimize
x FOR PEER
it REVIEW
in terms of clinical requirements. 7 of 16

thickness, and their orientation enables to scale the structures in relation to the designed equivalent
Table 2. Process parameters used to manufacture test specimens
strength of the structure and to optimize it in terms of clinical requirements.
BFS Types
Force Direction Table 2. Process parameters used to manufacture test specimens
Parameters
A1 A2 A3 B C
BFS Types
Force Direction σ [MPa]
Parameters 85 ± 31 ±6
50A2 100 ±3 32 ± 5 29 ± 5
1 A1 A3 B C
E [MPa] 2430 ± 727 1752 ± 671 2860 ± 174 855 ± 130 817 ± 108
σ [MPa] 85 ± 31 50 ± 6 100 ± 3 32 ± 5 29 ± 5
1
σ [MPa]
E [MPa] 158 ± ±14727 1752
2430 51 ± 6
± 671 107
2860 ± 4 855 ± 29
± 174 130± 2 817 ± 10829 ± 9
2
E [MPa]
σ [MPa] 743158 ± 270
± 14 213951 ± 107 ± ±
± 6173 3009 ± 2 ± 195 29 ± 9648 ± 78
4 202 29817
2
E [MPa]
σ [MPa] 743
82 ± ±30270 2139 ± 173
28 ± 7 3009 ± 202
108 ± 5 817 ± 81
195± 2 648 ± 7828 ± 8
3 σ [MPa] 219382±±867 30 28±± 7112 108 ± ±
5 145 812356
± 2 ± 12928 ± 790
8 ± 102
E [MPa] 485 3303
3
E [MPa] 2193 ± 867 485 ± 112 3303 ± 145 2356 ± 129 790 ± 102

3.2. CT Reconstruction
3.2. CT Reconstruction
Thanks to technical
Thanks computed
to technical computedtomography
tomography (CT)(CT)reconstruction,
reconstruction, thethe internal
internal geometry
geometry of theof the
fabricated BFSs
fabricated waswas
BFSs evaluated.
evaluated.A-type
A-typesamples weresubjected
samples were subjected to to testing
testing as they
as they displayed
displayed the best
the best
mechanical
mechanicalperformance
performance andandshow
showgeometry-dependent anisotropy
geometry-dependent anisotropy of of properties.
properties. During
During the first
the first
stagestage
of theof CT
the CT analysis,
analysis, thethe geometryof
geometry ofthe
the designed
designed BFS
BFSstructures
structures was compared
was comparedwithwith
the actual
the actual
geometry
geometry of fabricated
of the the fabricated structures.
structures. TheThe actualmodels
actual models(reconstructed,
(reconstructed, STLSTL format)
format)were
werefitted
fittedtoto the
the nominal models (designed, CAD format) using the ‘best-fit’ method. The
nominal models (designed, CAD format) using the ‘best-fit’ method. The above enabled to determineabove enabled to
determine the deviations between design geometry and models obtained on the basis of CT
the deviations between design geometry and models obtained on the basis of CT reconstruction for
reconstruction for the manufactured elements in differing positions relative to the build platform
the manufactured elements in differing positions relative to the build platform (Figure 5).
(Figure 5).

Figure
Figure 5. 5. Geometry deviations
Geometry deviations ofofactual models
actual fromfrom
models nominal model: (a)
nominal ‘A1 type’
model: (a) specimen,
‘A1 type’(b)specimen,
‘A2
type’
(b) ‘A2 specimen,
type’ (c) ‘A3
specimen, (c)type’ specimen,
‘A3 type’ (d) ‘B type’
specimen, (d) ‘Bspecimen, (e) ‘C type’
type’ specimen, (e) specimen (the most (the
‘C type’ specimen
mostappropriate
appropriatescale waswas
scale used for each
used sample
for each so thesogeometric
sample deviations
the geometric were easy
deviations to analyze
were easy toand
analyze
compare).
and compare).

The highest geometry deviations (in the range of −504 to 573 μm and −334 to 429 μm) were
observed for B and C type specimens respectively. For ‘A1 type’ specimens majority of the deviations
Materials 2018, 11, 971 8 of 16

The2018,
Materials highest
11, x FORgeometry
PEER REVIEW deviations (in the range of −504 to 573 µm and −334 to 429 µm) 8were of 16
observed for B and C type specimens respectively. For ‘A1 type’ specimens majority of the deviations
were
were revealed
revealed
Materials
within
within
2018, 11,
limits
limits
x FOR PEER
−−227
227 to 355 µm.
REVIEW μm. The lowest deviations
deviations range
rangewere
weredisplayed
displayedforforA2 A2 (−16 226
8 of(−226
to 312 µm) and for A3 ( − 124 to
μm) and for A3 (−124 to 226 μm) type 226 µm) type (Figure 5). Based
Based on the results obtained at this stage, it
was decided
decided
were revealed that
that for further
for further
within testing,
limits −227testing, A-type
to 355A-type samples—characterized
μm. Thesamples—characterized
lowest deviations range were by the highest
the displayed manufacturing
highest manufacturing
for A2 (−226
accuracy—would
to 312 μm) and for beA3used. Thickness
(−124Thickness analysis
to 226 μm)analysis of the
type (Figure
of the fabricated
5).fabricated
Based on theBFSresults showedat
strutsobtained that
thisthe largest
stage, it
diameters
was decided werethatnotedfor for strutstesting,
further oriented horizontally
A-type on the build
samples—characterized platform.
oriented horizontally on the build platform. by the It was
highest also revealed
manufacturing that
they were nearly twice
accuracy—would be used.as thick as theanalysis
Thickness design values. The wall
of the fabricated
The wallBFS
thickness of the
the struts
struts showed
thickness of struts in ‘A3
that the
in ‘A3
largesttype’
type’
diameters
specimen were
with the noted
lowest for geometry
struts oriented horizontally
deviations was inon thetherange
buildbetween
range platform.100
between It was
100 andalso
and 250revealed
250 μm (Figure
µm (Figure that6). 6).
they
The meanwere nearly
diameter twice
of the as thick
struts inas the
this design
case was values.
at the The
level
diameter of the struts in this case was at the level of 225 μm. wall
of thickness
225 µm. of the struts in ‘A3 type’
specimen with the lowest geometry deviations was in the range between 100 and 250 μm (Figure 6).
The mean diameter of the struts in this case was at the level of 225 μm.

Figure 6. (a) Example of wall thickness analysis (‘A3 type’ specimen); (b) cross-section showing the
Figure 6. (a) Example of wall thickness analysis (‘A3 type’ specimen); (b) cross-section showing the
thickness changes
Figure 6.changes of the struts
(a) Example in the A3 specimen.
thickness of theofstruts
wall thickness
in the A3 analysis
specimen.(‘A3 type’ specimen); (b) cross-section showing the
thickness changes of the struts in the A3 specimen.
Next, we determined total porosity and volume of individual pores of the specimen with the
Next,
lowest Next,
we determined
geometry deviations.total
we determined Theporosity
total geometry
porosity
and volume
of the
and
of individual
examined
volume
pores
BFS typepores
of individual
of the specimen
A3 structure with
and the with
of the specimen volume the
the of
lowest
the geometry
individual
lowest deviations.
pores
geometry The
was determined
deviations. geometry of
(Figure
The geometry the
the examined BFS type A3 structure and the volume of of
of 7). examined BFS type A3 structure and the volume
the individual pores was determined (Figure
the individual pores was determined (Figure 7).7).

Figure 7. Porosity analysis of BFS structure: (a) CT image thresholding for BFS structure geometry,
Figure 7. Porosity analysis of BFS structure: (a) CT image thresholding for BFS structure geometry,
Figure Porositywith
(b) BFS7.structure analysis of BFS
porosity structure:
values, (a) CT
(c) pores image
after thresholding
the separation for BFS
process structure
with geometry,
colors indicating
(b) BFS structure with porosity values, (c) pores after the separation process with colors indicating
(b) BFS
their structure
differing with porosity values, (c) pores after the separation process with colors indicating their
volumes.
their differing volumes.
differing volumes.
512
512pores
poreswere
wereregistered;
registered; the
the smallest
smallest ofof which
which had
had aa volume
volumeofof0.110.11mm³,
mm³,whilewhileforforthethe
largest—0.18
512 poresmm³—the nominal volume of 3
largest—0.18 were registered;
mm³—the nominalthevolume of a
smallest single
a single porehad
of which
pore wasa0.2
was 0.2 mm³.Our
volume
mm³. Our analysis
of analysis
0.11 mmhas has shown
, while
shown for that
thatthe
allallidentified
largest—0.18 3 —the
pores had a smaller actual volume. However, it should 3 . be noted that no blocked pores
identified pores had a smaller actual volume. However, it should be noted that no blocked poresall
mm nominal volume of a single pore was 0.2 mm Our analysis has shown that
were
wereidentified
identified identifiedand,
pores had a consequently
and, smaller actual itit
consequently has
has been
volume. confirmed
However,
been thatall
it should
confirmed that all channels
bechannels
noted that are
areno fully
blocked
fully unobstructed.
pores were
unobstructed.
Figure
Figure8 8shows
showsaahistogram
histogramof ofpore
pore volume
volume distribution.
distribution.
Materials 2018, 11, 971 9 of 16

identified and, consequently it has been confirmed that all channels are fully unobstructed. Figure 8
shows
Materialsa2018,
histogram
11, x FORofPEER
poreREVIEW
volume distribution. 9 of 16

Figure 8.
Figure 8. Histogram of pore
pore volume
volume distribution
distribution obtained
obtained using
using Avizo
Avizo8.0.
8.0.

On the basis of the information obtained about the volume of individual pores, porosity of the
On the basis of the information obtained about the volume of individual pores, porosity of the
fabricated structure was determined, on the basis of Equation (1).
fabricated structure was determined, on the basis of Equation (1).

=1−
VBFS (1)
P = 1− + (1)
VBFS + VP
where is the volume of the BFS structure and is the pore volume. Table 3 includes a
where V is the volume of the BFS structure and
summary of the analysis of BFS structure geometry.
BFS VP is the pore volume. Table 3 includes a summary
of the analysis of BFS structure geometry.
Table 3. Results of BFS structure geometry analysis using CT
Table 3. Results of BFS structure geometry analysis using CT
BFS Strut Diameter Total Porosity P Volume of Single Pore Total Surface
Type
BFS Type A3 A3Strut Diameter
µm µm % P%
Total Porosity Volume ofmm³
Single Pore mm3 mm²
Total Surface mm2
CAD
CAD model
model 150
150 85%
85%
0.202
0.202
431.34431.34
CT data
CT data 225 225 56%
56% 0.150
0.150 849.49849.49

Total porosity of the fabricated structure was 56% and it was 29% lower than the porosity
Total porosity of the fabricated structure was 56% and it was 29% lower than the porosity planned
planned in the design. This discrepancy was related to the geometry of the fabricated struts whose
in the design. This discrepancy was related to the geometry of the fabricated struts whose diameters,
diameters, due to the nature of the SLM process, were higher than those designed. The achieved
due to the nature of the SLM process, were higher than those designed. The achieved porosity suggests
porosity suggests that bone cells may be more prone to grow into the pores, which will be
that bone cells may be more prone to grow into the pores, which will be investigated further below.
investigated further below.
3.3. In-Vitro Cell Response
3.3. In-Vitro Cell Response
BFS cytotoxicity was evaluated using NRU cytotoxicity assay procedure as per PN-EN ISO
BFSfor
10993-5, cytotoxicity was evaluated
specimens incubated underusing NRUconditions
controlled cytotoxicity(37 assay
◦ C/5%procedure
CO2 ) at 100% as per PN-EN
humidity ISO
under
10993-5,
static for specimens
conditions. The lackincubated
of toxicityunder
of the controlled
investigated conditions (37 materials
implantation °C/5% CO 2) at 100% humidity
was confirmed also in
under static conditions. The lack of toxicity of the investigated implantation
macroscopic observation of the behavior of cells cultured in the presence of TiAl7Nb alloy materials wason confirmed
which a
also in macroscopic observation of the behavior of cells cultured in the presence of TiAl7Nb
clear increase in culture density after 48 h and progressing colonization of the area of the investigated alloy on
which ainclear
sample increasewith
comparison in culture density
the image afterafter
obtained 48 h24and
h ofprogressing colonization
incubation. Biological of the
studies area ofthat
revealed the
investigated sample in comparison with the image obtained after 24 h of
BFSs manufactured using SLM technology have low toxicity and high survival of osteoblasts equal toincubation. Biological
studiesand
75.4% revealed
90.5%,that BFSs
before manufactured
(non-CP samples) using
and SLM
after technology have low(CP
chemical polishing toxicity and high
samples), survival
respectively.
of evaluate
To osteoblasts theequal
growth toand
75.4% and 90.5%,ofbefore
development cells on(non-CP
the testedsamples) anda SEM
implants, after analysis
chemicalwas polishing (CP
conducted.
Osteoblasts were visualized on all tested surfaces (Figures 9–11). The number of cells changed witha
samples), respectively. To evaluate the growth and development of cells on the tested implants,
SEM The
time. analysis was
longer theconducted.
cell cultureOsteoblasts were cells
lasted, the more visualized
were seenon all
ontested surfacesfields
the analyzed (Figures 9–11). The
of observation.
number of cells changed with time. The longer the cell culture lasted, the more cells were seen on the
analyzed fields of observation.
Materials 2018, 11, 971 10 of 16
Materials 2018, 11, x FOR PEER REVIEW 10 of 16
Materials 2018, 11, x FOR PEER REVIEW 10 of 16
Materials 2018, 11, x FOR PEER REVIEW 10 of 16

a) b)
a)
a) b)
b)

Figure Osteoblasts
9. 9.
Figure Osteoblastsafter
afterseven
sevendays
daysofofincubation
incubation on on the
the tested
tested implants. Samplesnot
implants. Samples notsubjected
subjected
Figure 9. Osteoblasts after seven days of incubation on the tested implants. Samples not subjected
Figure samples)
(non-CP
(non-CP 9.samples)
Osteoblasts
(a) after
(a)and
and seven days
subjected
subjected to of incubation
tochemical
chemical on the
polishing
polishing (CP tested
(CP implants.
samples) Samples
(b).(b).
samples) Blue arrow
Blue not subjected
indicates
arrow BFS
indicates
(non-CP samples) (a) and subjected to chemical polishing (CP samples) (b). Blue arrow indicates BFS
(non-CP
material; samples)
green (a)
arrow and subjected
indicate to chemical
adhered cells, polishing
while red(CP samples)
ones (b). Blue
extracellular arrow
matrix
BFS material; green arrow indicate adhered cells, while red ones extracellular matrix produced indicates
produced BFS
byby
material; green arrow indicate adhered cells, while red ones extracellular matrix produced by
material; green
osteoblasts,
osteoblasts, SEM.
SEM. arrow indicate adhered cells, while red ones extracellular matrix produced by
osteoblasts, SEM.
osteoblasts, SEM.

a) b)
a)
a) b)
b)

Figure 10. Osteoblasts after 14 days of incubation on tested implants. Samples non-subjected (non-CP
Figure
Figure Osteoblasts
10.10. Osteoblastsafter
after14
14days
daysof
ofincubation
incubation on tested implants.
implants.Samples
Samplesnon-subjected
non-subjected(non-CP
(non-CP
Figure 10.(a)
samples) Osteoblasts after 14
and subjected to days of incubation
chemical polishingon tested
(CP implants.
samples) (b). Samples non-subjected
Red arrows (non-CP
indicate increasing
samples)
samples) (a)(a)
and
andsubjected
subjectedtotochemical
chemicalpolishing
polishing (CP
(CP samples) (b). Red
samples) (b). Red arrows
arrowsindicate
indicateincreasing
increasing
samples)
(please (a) anditsubjected
compare to Figureto9) chemical
amount ofpolishing (CP matrix
extracellular samples) (b). Redby
produced arrows indicate
osteoblasts, increasing
SEM.
(please compare
(please compare it it
totoFigure
Figure9)9)amount
amountofofextracellular
extracellularmatrix
matrix produced osteoblasts,SEM.
produced by osteoblasts, SEM.
(please compare it to Figure 9) amount of extracellular matrix produced by osteoblasts, SEM.

a) b)
a)
a) b)
b)

Figure 11. Osteoblasts after 30 days of incubation on tested implants. Samples not subjected (non-CP
Figure 11. Osteoblasts after 30 days of incubation on tested implants. Samples not subjected (non-CP
Figure
samples) Osteoblasts
11.11.
Figure Osteoblasts
(a) after
after3030
and subjected todays
daysofofincubation
chemical polishingon
incubation on tested
tested
(CP implants.
samples) Samples
(b). Samples
Red notindicate
not
arrow subjected
subjected (non-CP
(non-CP
increasing
samples) (a) and subjected to chemical polishing (CP samples) (b). Red arrow indicate increasing
samples)
samples)
amount (a) and
of(a) subjected
and subjected
extracellular totochemical
matrix chemical
embedding polishing
polishing (CP
(CP samples)
osteoblasts, samples) (b). Red
SEM. Red arrow
arrow indicate
indicateincreasing
increasing
amount of extracellular matrix embedding osteoblasts, SEM.
amount
amountof of
extracellular matrix
extracellular matrixembedding
embeddingosteoblasts,
osteoblasts,SEM.
SEM.
 Materials 2018, 11, x FOR PEER REVIEW 11 of 16
Materials 2018, 11, 971 11 of 16

A clear difference between the analyzed series may be observed. Samples subjected to chemical
polishing (b) were settled
A clear difference between by the
osteoblasts
analyzedmore eagerly
series may be than those not
observed. subjected
Samples to chemical
subjected polishing.
to chemical
This trend
polishing wassettled
(b) were observed for every analyzed
by osteoblasts time point
more eagerly of incubation
than those (7, 14,
not subjected toand 30 days).
chemical Since the
polishing.
BFS samples subjected to chemical polishing displayed a higher value of design
This trend was observed for every analyzed time point of incubation (7, 14, and 30 days). Since the BFS porosity (total
samples subjected to chemical polishing displayed a higher value of design porosity (total porosity in
porosity of the structure was 61%, so the chemical etching process applied caused a 5% increase
structure
of the porosity
structure relative
was 61%, so thetochemical
the initial value),process
etching the osteoblasts had more
applied caused a 5%surface
increase toinadhere to and
structure
propagate within. On the other hand, there was a high amount of loosely adhered
porosity relative to the initial value), the osteoblasts had more surface to adhere to and propagate powder in the
samples
within. notother
On the subjected
hand,tothere
polishing, which
was a high probably
amount prevented
of loosely the adherence
adhered powder inof osteoblasts.
the samples not CFU-
osteoblasts, calculated by the NR test, increased systematically with the time
subjected to polishing, which probably prevented the adherence of osteoblasts. CFU-osteoblasts, of incubation (Figure
12).
calculated by the NR test, increased systematically with the time of incubation (Figure 12).

Figure
Figure 12. Number
12. Number of cells
of cells withwith regard
regard to analyzed
to analyzed series
series of specimens.
of specimens.

Regardless
Regardless of the
of the timetime point
point analyzed,
analyzed, thethe number
number of of cells
cells waswas higherfor
higher forthe
thesamples
samplestested
tested in
in comparison to the control sample. After 14 days, the number of cells on BFS structures exceeded the
comparison to the control sample. After 14 days, the number of cells on BFS structures exceeded
the number
number of ofcells
cellsininthe
thecontrol
controlsample
samplebyby
70–90%,
70–90%,while after
while 30 days
after of incubation
30 days of incubationthis value was 75–
this value
was 75–100% higher in comparison to the control sample. The obtained result suggests that the BFSBFS
100% higher in comparison to the control sample. The obtained result suggests that the
accelerates
accelerates the the osteoblastic
osteoblastic differentiation
differentiation andand
cancan promote
promote the the formation
formation of extracellular
of extracellular matrix
matrix at at
laterlater stage.
stage.

3.4. 3.4. Microbiological

Microbiological TestsTests
TheThe analyzed
analyzed strainstrain wasto able
was able form to formbiofilm
strong strongstructures
biofilm on
structures onofboth
both types typesregardless
scaffold, of scaffold,
regardless of the condition of the surface. However, quantitative cultures revealed
of the condition of the surface. However, quantitative cultures revealed a significantly decreased a significantly
decreased
number number
of microbial of microbial
cells on surfacecells on surface
of BFS of to
subjected BFS subjected
chemical to chemical
polishing polishing(Figure
(CP samples) (CP samples)
13).
(Figure 13). (KW
(KW test, p < 0.05). test, p < 0.05).
Materials 2018, 11, 971 12 of 16
Materials 2018, 11, x FOR PEER REVIEW 12 of 16

Materials 2018, 11, x FOR PEER REVIEW 12 of 16

Figure
Figure 13.13. S. epidermidis
S. epidermidis biofilm
biofilm formation
formation on on non-CP
non-CP andand
CPCP samples.
samples.
Figure 13. S. epidermidis biofilm formation on non-CP and CP samples.
3.5. Example of Application
3.5. Example of Application
3.5. Example of Application
The capabilities, based on processing biocompatible metal alloys, to produce objects with
The capabilities, based on processing biocompatible metal alloys, to produce objects with
diversified structure,based
The capabilities, designed and manufactured
on processing to support
biocompatible metal the growth
alloys, of functional
to produce objectsbone
withtissue
diversified structure, designed and manufactured to support the growth of functional bone tissue with
diversified structure,
with geometries designed
defined and manufactured
by computer 3D models,tohave
support theagrowth
created potentialof for
functional
solving bone
manytissue
problems
geometries defined by computer 3D models, have created a potential for solving many problems in
with geometries defined
in implantology. Basedby oncomputer
CT data,3D themodels,
degreehave created
of bone a potential
tissue damagefor andsolving many
the size andproblems
shape of the
implantology. Based on CT data, the degree of bone tissue damage and the size and shape of the bone
inbone
implantology.
resection was determined. Then, obtained external geometry of the bone defect was of
Based on CT data, the degree of bone tissue damage and the size and shape the with
filled
resection was determined. Then, obtained external geometry of the bone defect was filled with the
bone resection was determined. Then, obtained external geometry of the bone defect
the replicated BSF structure. According to obtained results, we have chosen the ‘A3 type’ sample aswas filled with
replicated BSF structure. According to obtained results, we have chosen the ‘A3 type’ sample as they
the replicated
they display BSFthe structure. According
best geometrical to obtained
accuracy and results,
do notwe havegeometry-dependent
show chosen the ‘A3 type’ sample
anisotropyas of
display the best geometrical accuracy and do not show geometry-dependent anisotropy of properties.
they display the best geometrical accuracy and do not show geometry-dependent
properties. Examples of customized jaw and facial implants for oncological treatment designed anisotropy of on
Examples
properties. of customized
Examples ofjaw and facialjaw
customized implants
and for oncological
facial implants fortreatment designed
oncological on the
treatment basis ofonthe
designed
the basis of the results of this study are shown in Figure 14.
results of this
the basis study
of the are of
results shown in Figure
this study 14. in Figure 14.
are shown

Figure14.14.
Figure Examples
Examples of of custom-made
custom-made implants
implants made
made of Ti6Al7Nb
of Ti6Al7Nb alloyalloy
filledfilled
with with
BFS. BFS.
Figure 14. Examples of custom-made implants made of Ti6Al7Nb alloy filled with BFS.
4.4.Discussion
Discussion
4. Discussion
Ensuringananappropriate
Ensuring appropriate interaction
interaction between
between thethe implant
implant andand its biological
its biological environment
environment posesposes
Ensuring
aa huge an
hugechallenge. appropriate
challenge.That interaction
Thatis iswhywhythethe between
properties
properties the
of ofimplant and its
biomaterials—such
biomaterials—such biological environment
as non-toxicity,
as non-toxicity, poses a
corrosion
corrosion
huge challenge.
resistance That
resistanceororcontrolled is
controlled why the properties
degradability,
degradability, and andof biomaterials—such
mechanical
mechanical andandbiologicalas non-toxicity,
biological corrosion
characteristics—have
characteristics—have resistance
for a for
longa long
ortime
controlled
been degradability,
regarded as and
crucial inmechanical
terms of and
selectionbiological
of an characteristics—have
appropriate
time been regarded as crucial in terms of selection of an appropriate biomaterial to a specific biomaterial for
to aa long
specifictime
been regarded
biomedical
biomedical as crucial Permanent
application.
application. inPermanent
terms ofand selection
and of andamage
irreversible
irreversible appropriate
damage tobiomaterial
to bone tissue
bone has to
tissue hasa motivated
specific
motivated biomedical
research on on
research
application.
filling
fillingthe Permanent
thecavities
cavitieswith
with and irreversible
biomaterials.
biomaterials. The damage
Theabove
above tofillings
bone
fillings aretissue has to
intended
are intended motivated
replace research
the
to replace pulp on serve
and
the pulp filling
and asthe as
serve
cavities with
aasuitable biomaterials.
foothold for new The above
tissue fillings
growth. Theare intended
above to
structuresreplace
should the
suitable foothold for new tissue growth. The above structures should have a porous architecture, pulp
have aand serve
porous as a suitable
architecture,
enabling
foothold
enabling the
for new
the penetration ofof
tissue growth.
penetration thethe
implant
The above
implant byby bone-forming
structures osteoblasts,
should
bone-forming have a which
osteoblasts,porous inarchitecture,
which time should
in time enable
enabling
should thethethe
enable
provision
penetration
provisionof of blood
ofthe supply
implant
blood supply and
by and innervation
bone-forming
innervation of the filled
osteoblasts,
of the filledcavity.
which in time should enable the provision of
cavity.
The
blood supply AM (additive
and manufacturing)
innervation of the filledprocess
cavity. enables
The AM (additive manufacturing) process enables the manufacture the manufacture of homogenous
of homogenous functional
functional
structures
The AM (scaffolds)
(additive having certain chemical,
manufacturing) process biological,
enables theand mechanicalofproperties
manufacture homogenous suited to the
functional
structures (scaffolds) having certain chemical, biological, and mechanical properties suited to the
expected (scaffolds)
structures actual load,havingdeformation,
certain and displacement
chemical, resulting
biological, and from an individual’s
mechanical properties anatomy
suited andthe
to
expected actual load, deformation, and displacement resulting from an individual’s anatomy and
physiology. Artificial bone scaffolds can be used in tissue engineering and can be helpful in the
physiology. Artificial bone scaffolds can be used in tissue engineering and can be helpful in the
Materials 2018, 11, 971 13 of 16

expected actual load, deformation, and displacement resulting from an individual’s anatomy and
physiology. Artificial bone scaffolds can be used in tissue engineering and can be helpful in the
treatment of large bone cavities. Our results indicate the suitability of SLM technology for the
manufacture of biomechanical functional structures from Ti6Al7Nb alloy that can precisely fill the
bone loss. The mechanical properties obtained in our study are similar to those obtained for specimens
of similar geometry and porosity reported in the literature [33,34]. Depending on structure orientation,
its dimensions, architecture, and SLM process parameters, our experimentally determined substitute
modulus of elasticity was within the range from 485 to 3038 MPa, at the value of substitute compressive
strength from 28 to 158 MPa. On the basis of the above, we can claim that it is possible to predict
and suitably design material characteristics—such as compressive strength, stiffness, or maximum
load a structure can carry—by changing its geometric features. In line with the results reported in the
literature, the highest strength of materials manufactured using SLM, not subjected to heat treatment,
is observed for the build direction [24]. The above can also be observed in the case of BFSs analyzed
in this paper for which the highest compressive stress was observed for type A1 specimens when
the force was applied in the build direction. It was revealed that shape deviations which cause a
disproportion of thickness of individual struts result in varying mechanical properties obtained for
different directions of load application. Geometrical verification of the fabricated structures was made
using the CT method which enabled us to analyze and describe the manufactured structures in a
non-invasive manner [35]. A digital model facilitates the determination of strut thickness, porosity of
the global structure and material, comparison of the actual and nominal geometry, determination of
unit cell geometry parameters (diameter, the size of contact points of unit cells), and determination
of the surface area-volume ratio. The results are very precise and provide information on the
quality of the manufactured biomechanical functional structures and confirm the high accuracy
of the manufacturing process.
The observed shape deviations on the reconstruction images occur mainly in the bottom layers,
which is directly associated with the specific features of additive manufacturing. Thus, due to the
largest surfaces which are parallel to the build platform, the highest deviations were measured for the
specimen built parallel to the build platform (type A1 specimen). In the case of type A3 specimens,
due to the structure being inclined to the built platform evenly at all sides and the struts parallel to the
built platform being eliminated, the deviations are similar independently of their direction, and the
manufactured struts have similar diameters.
Material surface plays an extremely important role in the response of the biological environment
to an artificial medical device. For BFSs fabricated using the AM process, surface modification through
chemical etching enables to reduce the number of powder particles attached to the surface and
consequently to improve their surface quality. Additionally, such untreated surfaces are not always
suitable for biomedical applications and often specific surface properties need to be ensured to achieve
biological integration and bone formation. Chemical polishing described in this work and used for
surface modification of BFS scaffolds caused an increased amount of nitrogen and fluorine on the
scaffolds surface [30,31]. Nitrided layers are characterized by high resistance to abrasive wear, which
significantly improves tribological properties of the fabricated structures [36]. Additionally, it creates a
barrier, reducing the penetration of hydrogen into the material structure. Fluorine on the other hand
(in limited doses) stimulates bone metabolism and restructuring [37]. This type of surface also displays
antibacterial properties and significantly prevents the growth of S. epidermidis strain on the implant
surface, which was also confirmed by previous results of the authors’ research on staphylococcal and
pseudomonas cells.
Biological studies revealed that BFSs manufactured using SLM technology have low toxicity,
which is consistent with the literature data [38]. CFU-osteoblast colonies, calculated using the NR test,
increased systematically with time of incubation and after 30 days of incubation it was 75–100% higher
than the control sample. This is also confirmed by literature research which shows that the discs made
of Ti6Al7Nb manufactured with the use of SLM technology are biologically well tolerated, without
Materials 2018, 11, 971 14 of 16

producing any adverse reactions [39]. The condition and size of the culture as well as the morphology
of human osteoblasts during contact with the surface of a BFS sample indicate that the conditions for
implant incorporation into the recipient tissues listed in the introduction can be met.
The results suggest that the designed and manufactured BFSs are ideal for replacing bone tissue
loss. By enabling cell growth due to their open porosity, they effectively integrate with the surrounding
bone tissue. Moreover, the analyzed BFSs were manufactured from Ti6Al7Nb titanium alloy which is
characterized by low cytotoxicity. The next step of this investigation should undoubtedly be analysis
of osteoblast gene expression and protein level to estimate true nature of interaction between cell
and implant of specific porosity/geometry. However, it should be noted that the base-line results on
cytotoxicity and colonization ratio presented in this manuscript are of a highly promising nature.

5. Conclusions
The application of additive technologies opens up new possibilities for the design of modern
implants in the form of metallic scaffolds (BFS). Both their geometry and programmed mechanical
characteristics rely directly on the data obtained from medical CT scans. Based on the analysis of
five types of BSF structures (A1, A2, A3, B, C) made of titanium alloy, we observed the correlation
between applied technological parameters and obtained functional features. The mechanical tests
as well as geometrical accuracy analysis showed that the square shaped (A3) BFS structures were
characterized by the lowest deviations range and smallest anisotropy of mechanical properties.
Additionally, CT examinations revealed that the (A3) structure is characterized by open porosity
with simple connections between particular pores however, in vitro tests indicate the best method of
post-processing treatment (chemical polishing) enabling better cell adhesion capacity.
The results of the present study confirm the potential of the selective laser melting method for
producing custom-made implants from Ti6Al7Nb titanium alloy featuring not only high accuracy and
high geometric complexity but also controllable mechanical properties such as strength or stiffness.
The achieved distribution of internal porosity facilitates the growth of osteoblasts and enables the
achievement of predesigned biomechanical characteristics matching those of the bone tissue.

Author Contributions: P.S. conceived the project and designed the experiments. P.S., G.Z., and A.J. performed the
experiments and analyzed the data. E.C. supervised the research. P.S and AJ wrote the manuscript. All authors
discussed the results and revised the manuscript.
Funding: This work was supported by: Statutory grant 0401/0022/17 from Wroclaw University of Science and
Technology and ST-904 internal grant from Wroclaw Medical University.
Conflicts of Interest: The authors declare no conflict of interest.

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