materials

Article
3D Powder Printed Bioglass and β-Tricalcium
Phosphate Bone Scaffolds
Michael Seidenstuecker 1, * ID , Laura Kerr 1,2 , Anke Bernstein 1 , Hermann O. Mayr 1 ,
Norbert P. Suedkamp 1 , Rainer Gadow 3 ID , Peter Krieg 3 , Sergio Hernandez Latorre 1 ,
Ralf Thomann 4 , Frank Syrowatka 5 and Steffen Esslinger 3, * ID
1 Department of Orthopedics and Trauma Surgery, Medical Center—Albert-Ludwigs-University of Freiburg,
Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany;
laurak1@hotmail.co.uk (L.K.); anke.bernstein@uniklinik-freiburg.de (A.B.);
hermann.mayr@uniklinik-freiburg.de (H.O.M.); norbert.suedkamp@uniklinik-freiburg.de (N.P.S.);
sergio.latorre@uniklinik-freiburg.de (S.H.L.)
2 School of Engineering, James Watt South Building, University of Glasgow, Glasgow G12 8QQ, UK
3 Institute for Manufacturing Technologies of Ceramic Components and Composites, University of Stuttgart,
Allmandring 7b, D-70569 Stuttgart, Germany; Rainer.Gadow@ifkb.uni-stuttgart.de (R.G.);
peter.krieg@ifkb.uni-stuttgart.de (P.K.)
4 FMF—Freiburg Materials Research Center, University of Freiburg, Stefan-Meier-Str. 21, D-79104 Freiburg,
Germany; ralf.thomann@fmf.uni-freiburg.de
5 Interdisciplinary Center of Materials Science (CMAT), Martin Luther University Halle, Heinrich Damerow
Str. 4, D-06120 Halle, Germany; syrowatka@cmat.uni-halle.de
* Correspondence: Michael.seidenstuecker@uniklinik-freiburg.de (M.S.);
Steffen.Esslinger@gsame.uni-stuttgart.de (S.E.); Tel.: +49-761-270-261-04 (M.S.); +49-711-685-683-17 (S.E.)

Received: 23 October 2017; Accepted: 18 December 2017; Published: 22 December 2017

Abstract: The use of both bioglass (BG) and β tricalcium phosphate (β-TCP) for bone replacement
applications has been studied extensively due to the materials’ high biocompatibility and ability to
resorb when implanted in the body. 3D printing has been explored as a fast and versatile technique
for the fabrication of porous bone scaffolds. This project investigates the effects of using different
combinations of a composite BG and β-TCP powder for 3D printing of porous bone scaffolds.
Porous 3D powder printed bone scaffolds of BG, β-TCP, 50/50 BG/β-TCP and 70/30 BG/β-TCP
compositions were subject to a variety of characterization and biocompatibility tests. The porosity
characteristics, surface roughness, mechanical strength, viability for cell proliferation, material
cytotoxicity and in vitro bioactivity were assessed. The results show that the scaffolds can support
osteoblast-like MG-63 cells growth both on the surface of and within the scaffold material and do not
show alarming cytotoxicity; the porosity and surface characteristics of the scaffolds are appropriate.
Of the two tested composite materials, the 70/30 BG/β-TCP scaffold proved to be superior in terms
of biocompatibility and mechanical strength. The mechanical strength of the scaffolds makes them
unsuitable for load bearing applications. However, they can be useful for other applications such as
bone fillers.

Keywords: 3D printing; bone scaffolds; biocompatibility in vitro; bioglass; β-TCP

1. Introduction
To tackle the complications associated with autograft and allograft methods, bone graft substitutes,
known as bone scaffolds, were developed. Various artificial materials have been used to bridge bone
defects and these are discussed below. Synthetic bone scaffolds only possess, at most, two of the
four ideal graft material characteristics; osteointegration and osteoconduction [1]. However, there are

Materials 2018, 11, 13; doi:10.3390/ma11010013 www.mdpi.com/journal/materials

Materials 2018, 11, 13 2 of 21

a wide variety of challenges relating to the development of a successful graft material. According
to Bose et al. [2], there are four key requirements for an “ideal” bone scaffold. Biocompatibility:
The scaffold should be able to support normal cellular activity without inducing any local and systemic
toxicity in the host tissue. Mechanical Properties: The mechanical properties of a bone scaffold should
be similar to that of the host tissue it is replacing. It is also important that the modulus of elasticity is
similar. Pore size: Scaffolds must have interconnected porosity for the supply of essential nutrients
and oxygen, as well as bone tissue ingrowth. Bioresorbability: Scaffolds should be able to degrade
in vivo, ideally at a rate similar to that of new tissue growth.
Most other literature agrees on these requirements [3,4], while Polo-Corrales et al. [5] also advocate
the importance of incorporating growth factors into the scaffolds by methods such as controlled release
of injection to enhance bone growth.
Calcium phosphates are both osteointegrative and osteoconductive and form a hydroxyapatite
(HA) layer when implanted into the body. The main advantage of using calcium phosphates is that
they are highly biocompatible and there are no reports of systemic toxicity or foreign body reactions
following their implantation [1,6]. The degradability of ceramics is determined, inter alia, on water’s
solubility. The calcium to phosphate ratio is crucial in this regard. HA has a Ca/P ratio of 1.67.
Other calcium phosphate ceramics such as β-tricalcium phosphate (β-TCP) have a more favorable
1.5 ratio and thus faster degradability [7,8]. Bioglass (BG) contains minerals which occur in the body
and the quantity of calcium and phosphorous oxides present are similar to that of natural bone [9].
When BG comes into contact with body fluid, it forms a HA-like layer on its surface. The layer has
a similar mineral composition to that of natural bone and this allows the BG to form a strong bond
with surrounding biological tissue [10,11]. It has been used to form coatings on metal implants which
improve osteointegration and to fill voids in bone grafts [1]. BG [9] and β TCP [12] were both used as
bone replacement materials. These are the materials explored in this project.
3D powder printing was developed by Michal J. Cima and co-workers [13] in 1993, based on the
conventional inkjet printing mechanism. It is an additive manufacturing method used to print the data
from a CAD model. The model is printed in layers, composed from a starch-based powder bed and
glued together with a liquid binder [14]. The technique allows for close control in the fabrication of
complex 3D geometries [15]. A 3D CAD file is converted into cross sections between 0.01 and 0.2 cm
thick and these cross sections are printed as slices layered on top of each other from the bottom of the
model to the top.
One particularly well developed use for 3D printing in medicine is bone replacement. Bone
scaffolds can be fabricated by a variety of methods, including chemical or gas foaming, solvent casting,
freeze drying and thermally induced phase separation [16,17]. Due to the ability to precisely control
geometry and porosity, it is possible to create an implantable material with bone-like characteristics [14].
Much of the literature on the subject agrees that 3D printing is superior to these other methods,
primarily due to the control over pore size, shape and interconnectivity which it provides [18]. Because
of the high variability between individual bone defects, the customization of models allowed by 3D
printing is also particularly advantageous in bone scaffold applications for creating implants specific to
a particular defect. Another advantage of using 3D printing to create bone scaffolds is the wide range
of materials which can be used with this fabrication method; it allows for the use of both polymers and
ceramics, as well as composite combinations of these materials [19]. According to Butscher et al. [20],
the only limitation for the use of a material is its availability in powder form. This is obviously
in reference to the ability of the printer to create the scaffold geometry, and does not consider the
scaffold’s ability to act as an efficient bone replacement, but it demonstrates the diversity of 3D printing
regardless. Butscher et al. [20] states that the geometries which can be created are only restricted by
the resolution of the 3D printing. The resolution depends mostly on the layer thickness, which is
ultimately controlled by the properties of the powder itself. Each different material in powder form
has a different particle size, and this impacts the printing process parameters and the binder required.
Furthermore, the powder particle size impacts upon the mechanical strength of the scaffold, as particle
size has a direct influence on porosity, and mechanical strength is directly influenced by porosity [21].
Materials 2018, 11, 13 3 of 21

Materials 2018, 11, 13 3 of 21

The porosity can be decreased, and therefore the mechanical strength increased, by using an optimized
combination of different particle sizes [22]. In addition to the powder used, the printing process
decreased, and therefore the mechanical strength increased, by using an optimized combination of
itself also affects
different particlethesizes
mechanical properties
[22]. In addition of powder
to the the scaffolds
used, produced.
the printingThe mechanical
process itself alsoproperties
affects theof
scaffolds created by 3D printing can depend largely on the structural and process
mechanical properties of the scaffolds produced. The mechanical properties of scaffolds created parameters, rather
by
than purely on the properties of the materials used [23]. The material’s mechanical
3D printing can depend largely on the structural and process parameters, rather than purely on the properties can
be significantly
properties of altered following
the materials usedprinting
[23]. The process due mechanical
material’s to complexproperties
interactioncan events. Significant
be significantly
factors in the mechanical strength of printed parts include the concentration
altered following printing process due to complex interaction events. Significant factors in of binder used andthethe
layer orientation
mechanical duringofprinting
strength printed [24,25]. According
parts include to Chumnanklang
the concentration et al.used
of binder [24];and
the the
higher
layerthe
binder concentration
orientation the higher
during printing the mechanical
[24,25]. According tostrength of the printed
Chumnanklang partthe
et al. [24]; will be. In
higher theaddition,
binder
concentration
Vlasea the higherthe
et al. [25] explores theeffect
mechanical
of layer strength of theduring
orientation printedprinting
part willon
be.the
In addition,
mechanical Vlasea et al.
properties
[25] explores the effect of layer orientation during printing on the mechanical properties
of the part. The Aim of this study was to explore to what extent the fabrication technique for a biphasic of the part.
The Aimout
composite of of
this
BGstudy was to isexplore
and β-TCP to what
appropriate forextent the fabrication
development technique
of scaffolds for usefor in
a biological
biphasic
composite like
applications, out bone
of BGregeneration.
and β-TCP is appropriate for development of scaffolds for use in biological
applications, like bone regeneration.
2. Results
2. Results
2.1. Sample Characterization
2.1. Sample Characterization
The diameter and height of the scaffolds were measured using Vernier calipers. The mean
diameters The diameter
were and to
calculated height of the
be 10.19 scaffolds
± 0.06 mm forwereBG,measured
10.41 ± 0.25using
mmVernier
for β-TCP,calipers.
11.09The mean
± 0.02 mm
diameters were calculated to be 10.19 ± 0.06 mm for BG, 10.41 ± 0.25 mm for β-TCP,
for the 50/50 and 10.60 ± 0.02 mm for 70/30 scaffolds. This difference was deemed to be statistically 11.09 ± 0.02 mm
for the 50/50
significant and 10.60
(p < 0.05). The ±mean0.02 mm for 70/30
heights were scaffolds.
calculatedThis difference
to be 2.88 ± 0.1wasmmdeemed
for BG, to 2.45
be statistically
± 0.16 mm
significant (p < 0.05). The mean heights were calculated to be 2.88 ± 0.1 mm
for β-TCP, 2.84 ± 0.12 mm for the 50/50 scaffolds and 2.94 ± 0.26 mm for the 70/30 scaffolds; for BG, 2.45 ± 0.16 mm for
a small
β-TCP, 2.84 ± 0.12 mm for the 50/50 scaffolds and 2.94 ± 0.26 mm for the 70/30 scaffolds; a small
difference which was considered not to be statistically significant (p > 0.05). Figure 1 shows different
difference which was considered not to be statistically significant (p > 0.05). Figure 1 shows different
views of the scaffolds captured under a stereo microscope. A statistically significant difference in the
views of the scaffolds captured under a stereo microscope. A statistically significant difference in the
weight of the materials was detected (p < 0.05). The mean weight was calculated to be 251.92 ± 12.9 mg
weight of the materials was detected (p < 0.05). The mean weight was calculated to be 251.92 ± 12.9
for BG, 190.3 ± 7.13 mg for β-TCP, 223.85 ± 15.56 mg for the 50/50 scaffolds and 238.67 ± 18.91 mg
mg for BG, 190.3 ± 7.13 mg for β-TCP, 223.85 ± 15.56 mg for the 50/50 scaffolds and 238.67 ± 18.91 mg
for the 70/30 scaffolds. The dimensions of the 3D printed Samples are summarized in Table 1.
for the 70/30 scaffolds. The dimensions of the 3D printed Samples are summarized in Table 1.

(a) (b)
Figure 1. Side view (a); and top view (b) of 3D printed scaffold. The scale bar is 1 mm. Images were
Figure 1. Side view (a); and top view (b) of 3D printed scaffold. The scale bar is 1 mm. Images were
taken with Olympus (Tokyo, Japan) SZ-61.
taken with Olympus (Tokyo, Japan) SZ-61.

Table 1. Dimensions of 3D printed Samples (n = 10).

Table 1. Dimensions of 3D printed Samples (n = 10).
BG β-TCP 50/50 70/30
Diameter (mm) 10.19
BG ± 0.06 10.41
β-TCP± 0.25 11.09 ±
50/500.02 10.60 ± 0.02
70/30
Height (mm) 2.88 ± 0.10 2.45 ± 0.16 2.84 ± 0.12 2.94 ± 0.26
Diameter (mm) 10.19 ± 0.06 10.41 ± 0.25 11.09 ± 0.02 10.60 ± 0.02
Weight
Height (mm) (mg) 2.88
251.92 ± 12.90 2.45
± 0.10 190.30 ± 7.13 223.85
± 0.16 2.84 ±± 15.56
0.12 238.67
2.94± ±
18.91
0.26
Weight (mg) 251.92 ± 12.90 190.30 ± 7.13 223.85 ± 15.56 238.67 ± 18.91
Materials 2018, 11, 13 4 of 21

Materials 2018, 11, 13 4 of 21

2.1.1. EDX Analysis
2.1.1. EDX Analysis
The spectrum produced from EDX analysis of the BG showed the presence of oxygen (O), silicon
The spectrum produced from EDX analysis of the BG showed the presence of oxygen (O),
(Si), calcium (Ca), phosphorous (P) and small traces of sodium (Na), magnesium (Mg), aluminum (Al)
silicon (Si), calcium (Ca), phosphorous (P) and small traces of sodium (Na), magnesium (Mg),
and fluorine (F). The composites showed the same elements but in different compositions. The spectra
aluminum (Al) and fluorine (F). The composites showed the same elements but in different
are shown in Figure 2. The mean mass percentage of each element present in the tested materials
compositions. The spectra are shown in Figure 2. The mean mass percentage of each element present
is shown in Table 2. In addition, the mean Ca/P ratio for each material was calculated. The mean
in the tested materials is shown in Table 2. In addition, the mean Ca/P ratio for each material was
Ca/P ratio for 50/50 was calculated as 1.9. For 70/30, the mean Ca/P ratio was calculated to be
calculated. The mean Ca/P ratio for 50/50 was calculated as 1.9. For 70/30, the mean Ca/P ratio was
2.0.calculated
Compared with
to be 2.0.those results,with
Compared the those spectrum
β-TCPresults, showed
the β-TCP only Ca,showed
spectrum O, P with
onlyCa/P ratio
Ca, O, of 1.5.
P with
TheCa/P
pureratio
BG of
showed a Ca/P ratio of 5.3, as expected for a 45S5 BG. The elemental compositions
1.5. The pure BG showed a Ca/P ratio of 5.3, as expected for a 45S5 BG. The elemental of all
samples are summarized
compositions in Table
of all samples 2.
are summarized in Table 2.

Figure
Figure 2. 2.
EDXEDX Spectra
Spectra ofof3D
3Dprinted
printedScaffolds:
Scaffolds:(a)
(a)BG;
BG;(b)
(b) β-TCP;
β-TCP; (c)
(c) 50/50;
50/50;and
and(d)
(d)70/30;
70/30;performed
performed
as area scan (area 0.81 mm
as area scan (area 0.81 mm ). 2 2).

Table 2. Mean elemental composition for the samples (ZAF corrected).

Table 2. Mean elemental composition for the samples (ZAF corrected).
Material BG β-TCP 50/50 70/30
Material ElementBG Atom % β-TCP Atom % Atom % 50/50Atom % 70/30
Element F2O **
Atom % 2.8 Atom %0 2.0
Atom % 2.5 Atom %
Na2O 3.8 0 1.7 2.6
F2 O ** 2.8 0 2.0 2.5
MgO 4.8 0 3.8 5.3
Na2 O 3.8 0 1.7 2.6
MgO Al2O3 4.8 0.8 00 0.8 3.8 1.0 5.3
Al2 O3 SiO2 0.8 41.5 00 18.8 0.8 27.1 1.0
SiO2 PO 4 41.5 7.4 39.1
0 25.5 18.8 20.3 27.1
PO4 CaO 7.4 38.9 60.9
39.1 47.4 25.5 41.2 20.3
CaO 38.9 5.3
Ca/P ratio 60.9
1.5 1.9 47.4 2.0 41.2
Ca/P
Philips ESEM XLratio 5.3 was used for1.5
30 with EDX unit 1.9
the integral measurement, 2.0(live time), area
10 min
Philips
0.81 ESEM
mm2; XL 30 with
** only forEDX used for the integral measurement, 10 min (live time), area 0.81 mm2 ; ** only
unit waspurposes.
Calibration
for Calibration purposes.
Materials 2018, 11, 13 5 of 21

Materials 2018, 11, 13 5 of 21

2.1.2. 3D Laser Scanning

2.1.2. 3D Laser Scanning
The mean maximum surface profile height (Rz) and mean roughness (Ra) for each material type
The mean
are shown maximum
in Table 3, alongsurface profile
with the height
standard (Rz) and
deviation mean
and roughness
percentage (Ra) for
relative each material
standard type
deviation for
are shown
each in Table Figure
measurement. 3, along3 shows
with the standard
the surface deviation
roughnessand percentage
of the relative
four different standard deviation
samples.
for each measurement. Figure 3 shows the surface roughness of the four different samples.
Table 3. Surface Roughness of the 3D printed Scaffolds (selected parameters) (n = 10).
Table 3. Surface Roughness of the 3D printed Scaffolds (selected parameters) (n = 10).
Material BG β-TCP 50/50 70/30
Material BG β-TCP 50/50 70/30
Parameter Rz (µm) Ra (µm) Rz (µm) Ra (µm) Rz (µm) Ra (µm) Rz (µm) Ra (µm)
Parameter Rz (µm) Ra (µm) Rz (µm) Ra (µm) Rz (µm) Ra (µm) Rz (µm) Ra (µm)
Mean
Mean 78.33
78.33 11.82
11.82 207.29
207.29 26.08
26.08 183.81
183.81 28.92
28.92 194.50
194.50 27.61
27.61
SD 9.17 1.58 37.69 7.01 8.64 4.71 38.62 8.86
SD
% RSD
9.17
3.24
1.58
0.56
37.69
13.33
7.01
2.48
8.64
3.06
4.71
1.66
38.62
13.66
8.86
3.13
% RSD 3.24 0.56 13.33 2.48 3.06 1.66 13.66 3.13

The
The3D 3Dscaffolds
scaffoldsmade
madeofofthethepure
puresubstance
substanceBGBG showed
showed thethe
smallest roughness,
smallest Ra 11.82
roughness, ± 1.58
Ra 11.82 µm
± 1.58
for
µmBG forvs.BG vs. ±
28.92 4.71±µm
28.92 4.71 27.61or±27.61
or µm 8.86 µm for µm
± 8.86 the composites. Compared
for the composites. to BG, β-TCP
Compared to BG,showed
β-TCPa
higher
showed a higher roughness (Ra) of 26.08. Figure 4 shows the variation in surface roughness onof
roughness (Ra) of 26.08. Figure 4 shows the variation in surface roughness on different areas
the scaffolds
different viaof
areas 3Dthe
reconstructions
scaffolds via 3D of the scaffold surfaces.
reconstructions of theThe highest
scaffold peaks, shown
surfaces. in red,peaks,
The highest are in
the 200–300
shown µmare
in red, range. There
in the is a significant
200–300 µ m range. difference between the
There is a significant mean values
difference of Rathe
between and (p = values
mean 0.05) as
well as a significant difference between the mean values of Rz (p = 0.05).
of Ra and (p = 0.05) as well as a significant difference between the mean values of Rz (p = 0.05).

(a) (b)

(c) (d)
Figure 3. 3D Laserscanning Image of Samples Surface; Differences in the particle size of the used
Figure 3. 3D Laserscanning Image of Samples Surface; Differences in the particle size of the used
granules can be seen, as well as the presence of only a few sinter bridges: (a) BG; (b) β-TCP; (c) 50/50;
granules can be seen, as well as the presence of only a few sinter bridges: (a) BG; (b) β-TCP; (c) 50/50;
and (d) 70/30. Scale bar = 100 µ m. Images were taken with Keyence VK-X210 (Keyence, Osaka,
and (d) 70/30. Scale bar = 100 µm. Images were taken with Keyence VK-X210 (Keyence, Osaka, Japan).
Japan). Magnification, 400×.
Magnification, 400×.
 Materials
Materials 2018,11,
2018, 11,1313 6 of621
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Materials 2018, 11, 13 6 of 21

Figure 4. 3D reconstruction of Surface of composites: depending on the different composition and

Figure
Figure 4. 4.3D3Dreconstruction
reconstructionofofSurface
Surface ofof composites: dependingon
composites: depending onthe
thedifferent
different composition
composition andand
particle size of used granules, a different surface roughness can be observed: (a) BG; (b) β-TCP;
particle
particlesizesizeofofused
usedgranules,
granules, aa different
different surface roughnesscan
surface roughness canbebeobserved:
observed:(a)(a) BG;
BG; (b)(b) β-TCP;
β-TCP;
(c) 50/50; and (d) 70/30. Images were taken with Keyence VK-X210. Magnification, 400×.
(c)(c)
50/50;
50/50;andand(d)(d)70/30. Imageswere
70/30. Images weretaken
takenwith
withKeyence
Keyence VK-X210.
VK-X210. Magnification,
Magnification, 400×.
400×.
2.1.3. Mercury Porosimetry
2.1.3.
2.1.2. MercuryPorosimetry
Mercury Porosimetry
The pore size distribution for β-TCP, BG, 50/50 and 70/30 materials are shown in Figure 5.
The
The
The poresize
sizedistribution
measurements
pore distribution
were similar forfor
for β-TCP, BG, 50/50
all materials
β-TCP, BG, 50/50
and and
and70/30
show materials
that the
70/30 areof
majority
materials areshown
pores
shown in in
Figure
present are 5. 5.
Figure
The measurements
macropores with were similar
diameter 1–15forµ all
m. materials
The resultsand
alsoshow that
demonstratethe majority
the of
presence
The measurements were similar for all materials and show that the majority of pores present pores
of present
some areare
larger
macropores
macroporeswith
withdiameter
diameters 1–15 µ m. 15
between Theand
results
60 µ also
m, demonstrate
along with some the presence(2–100
mesopores of some
nm) larger
and
macropores with diameter 1–15 µm. The results also demonstrate the presence of some larger
macropores
microporeswith diameters
(<2 nm) between end
at the opposite 15 and 60scale
of the µ m, for
along withMeasurements
β-TCP. some mesopores (2–100
of other nm) and
parameters
macropores with diameters between 15 and 60 µm, along with some mesopores (2–100 nm) and
micropores
obtained (<2
fromnm)the at the opposite
mercury end ofanalysis
porosimetry the scalearefor β-TCP.inMeasurements
outlined of other
Table 4. The average parameters
pore radius
micropores (<2 nm) at the opposite end of the scale for β-TCP. Measurements of other parameters
was between 5.80 and 12 µ m. Total porosity was in the region of 46–75%.
obtained from the mercury porosimetry analysis are outlined in Table 4. The average pore radius
obtained from the mercury porosimetry analysis are outlined in Table 4. The average pore radius was
was between 5.80 and 12 µ m. Total porosity was in the region of 46–75%.
between 5.80 and 12 µm. Total porosity was in the region of 46–75%.
300 50 500 50

45 45
300 250 50 500
400
50
40 40
Specific Volume [mm³/g]

45
[mm³/g][mm³/g]

45
35 35
Relative Volume [%]

Relative Volume [%]

250 200
40 400
300 40
30 30
Specific Volume [mm³/g]

VolumeVolume

200 150 35 35
Relative Volume [%]

Relative Volume [%]

25 25
300
200
3020 20 30
100
150
Specific Specific

2515 15 25
100
200
50
100 2010 10 20

155 0 5 15
0 100
50
100 0 10
-3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3
10 10 10 10 10 10 10 10 10 10 10 10 10 10
5 0 5
0 Pore radius ranges [µm] Pore radius ranges [µm]
0 0
10-3 10-2 10-1 100 101 102 103 10-3 10-2 10-1 0
101 102 103
(a) (b)10
Pore radius ranges [µm] Pore radius ranges [µm]

(a) (b)

Figure 5. Cont.
 Materials 2018, 11, 13 7 of 21
Materials 2018,
Materials 11,
2018, 11,1313 7 of721
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50 50
50 50
800 45 800 45
800 45 800 45
40 40
40 40
/g]

Specific Volume [mm³/g]

/g]

Specific Volume [mm³/g]

600 35 600 35
[mm³

Relative Volume [%]

Relative Volume [%]

600 35 600 35
Volume[mm³

Relative Volume [%]

Relative Volume [%]

30 30 30 30
Specific Volume

400
400 25 25 400 400 25 25

20 20 20 20
Specific

200
200 15 15 200 200 15 15

10 10 10 10

00 5 5 0 0 5 5

0 0 0 0
-3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3
10
10-3 10
10-2 1010-1 10100 10 101 10 102 10 103 10 10-3 10 10-2 10 10-1 10 10010 10110 1010
2
103
Pore
Poreradius ranges
radius [µm]
ranges [µm] Pore radius rangesranges
Pore radius [µm] [µm]

(c)
(c) (d) (d)
Figure
Figure 5.
5.Pore
Pore size
sizedistribution:
distribution:(a) pure BG; (b)(b)
pure β-TCP; (c) 50/50; and and
(d) 70/30. Because of theof the
Figure 5. Pore size distribution: (a) (a) pure
pure BG;BG;
(b) purepure β-TCP;
β-TCP; (c) 50/50;
(c) 50/50; and (d)(d) 70/30.
70/30. Because
Because of the
different particle sizes and milling grades of the used granules, different pore size distributions can
different particle sizes and milling grades of the used granules, different pore size distributions cancan
different particle sizes and milling grades of the used granules, different pore size distributions
be observed.
be be observed.
observed.
Table 4. Mercury Porosimetry parameters for the scaffolds (n = 3).
Table
Table 4. Mercury
4. Mercury Porosimetry
Porosimetry parameters
parameters forfor
thethe scaffolds
scaffolds (n =(n3).
= 3).
Material BG β-TCP 50/50 70/30
Material BG β-TCP 50/50 70/30
Total cumulative
Materialvolume (mm³/g) 232.50 BG 319.62β-TCP788.18 50/50713.91 70/30
Total cumulative volume (mm³
Total specific surface area (m²/g) /g) 232.50
0.09 319.62
0.06 788.18
1.08 1.19713.91
3 232.50 319.62 788.18
TotalTotal
cumulative
specific
Average volume
pore radius(mm
surface area /g)/g) 5.84
(µ m)(m² 0.09 11.99 0.06 10.571.08 9.58713.91
1.19
Total specific surface area 2 0.09 0.06 1.08 71.299.58
1.19
Average
Total porosity (%)(m(µ/g)
pore radius m) 5.84 63.73
46.99 11.99 74.1210.57
Average
Bulk
pore
Total radius
porosity
density
(µm)
(%)
(g/cm³ )
5.84
2.02
11.99 10.57 9.58
46.99 1.9963.73 0.9474.12 0.9971.29
Total
Bulkporosity
density (%) 46.99 63.73 74.12
0.94 3.4871.29
Apparent density(g/cm³
(g/cm³ ) 3.812.02 5.49 1.99 3.63 0.99
Bulk density (g/cm3 ) 2.02 1.99 0.94 0.99
Apparent
Sample volumedensity (g/cm³
correction ) 0.983.81 none5.49 0.953.63 0.95 3.48
Apparent density (g/cm3 ) 3.81 5.49 3.63 3.48
Sample volume correction 0.98 none 0.95 0.95
Sample volume correction 0.98 none 0.95 0.95
2.1.4. Compression Test
2.1.4. Compression Test
The results ofTest
2.1.3. Compression the compression tests for the BG, β-TCP, 50/50 and 70/30 scaffolds are shown in
TableThe
5. The original
results of theplot, produced by
compression teststhefor
Zwick “Test
the BG, Expert50/50
β-TCP, II” software
and 70/30 (Zwick Roellare
scaffolds Group,
shown in
TheGermany),
Ulm, results of reported
the compression
a maximum testsforce
for the BG, than
higher β-TCP, the 50/50
true and 70/30force
maximum scaffolds
that arematerial
the shown in
Table 5. The original plot, produced by the Zwick “Test Expert II” software (Zwick Roell Group,
Table 5. The
could originalThis
withstand. plot,
was produced
due by presence
to the the Zwick of “Test Expert II”channels
the large software in(Zwick Roell Group,the Ulm,
Ulm, Germany), reported a maximum force higher thanporethe true maximum the scaffolds:
force thatwhenthe material
Germany),
powder reported
structure wasa maximum force higher than the true maximum force that the material could
could withstand. Thiscompressed,
was due to the the large pores
presence ofclosed,
the large thus forming
pore a more
channels solidscaffolds:
in the structurewhen
that the
withstand.
provided This wasresistance
greater due to the to presence
the appliedof the large
force. For pore
this channels
reason, in first
the the scaffolds:
peak in thewhen the powder
resultant plot
powder structure was compressed, the large pores closed, thus forming a more solid structure that
structure
was takenwasto compressed,
be the the large
maximum pores
force closed,
which thethus forming
scaffold a more
could solid structure
withstand. Figure 6 that
showsprovided
the plot
provided greater resistance to the applied force. For this reason, the first peak in the resultant
greater resistance to the
Force-Deformation applied
curves force.
for the For this
different reason, the first peak in the resultant plot was taken to
samples.
was taken to be the maximum force which the scaffold could withstand. Figure 6 shows the
be the maximum force which the scaffold could withstand. Figure 6 shows the Force-Deformation
Force-Deformation curves for the different samples.
curves for80
the different samples. 80

70 70

80 60 80
60

70 50 70
50
Force [N]
Force [N] Force [N]

60 40 60
40
30 50
50
30
Force [N]

20 40
40
20
10 30 TCP 1
30
10 BG 1
BG 2 0
TCP 2
20 TCP 3
0
20 BG 3
-10
10 TCP 1
10 0 1 2 3 4 5BG1 0 1
10 2
20 3
30 4
40
BG 2 0 Deformation [%] TCP 2
Deformation [%]
0 BG 3 TCP 3
-10
0 1 (a)
2 3 4 5 0
(b)
1
10 2
20 3
30 4
40

Deformation [%] Deformation [%]

(a) (b)

Figure 6. Cont.
 Materials
Materials 2018,
2018, 11,11,
13 13 8 of821
of 21

18
16
16
14
14
12
12
Force [N]

10

Force [N]
10
8
8
6
6
4
4
50/50_1 70/30_1
2 50/50_2 2 70/30_2
50/50_3 70/30_3
0 0

0 1 2 3 4 0 1 2 3 4
Deformation [%] Deformation [%]

(c) (d)
Figure 6. Force-Deformation curves: (a) pure BG; (b) pure β-TCP; (c) 50/50; and (d) 70/30.
Figure 6. Force-Deformation curves: (a) pure BG; (b) pure β-TCP; (c) 50/50; and (d) 70/30. Experiments
Experiments performed at Zwick Roell Universal Testing machine Z005 using Test Xpert II Software
performed at Zwick Roell Universal Testing machine Z005 using Test Xpert II Software (n = 3).
(n = 3).
Table 5. Failure load and compressive strength of the 3D printed Scaffolds (n = 3).
Table 5. Failure load and compressive strength of the 3D printed Scaffolds (n = 3).
SampleSample BG BG β-TCPβ-TCP 50/50
50/50 70/30
70/30
FailureFailure load (N) 51.90 ±
load (N) 51.90 ± 17.10
17.10 54.3054.30 ± 14.50 14.30
± 14.50 14.30
±± 1.10
1.10 15.10
15.10 ±±1.10
1.10
Compressive
Compressive strength
strength 0.64 ±0.64
(MPa) (MPa) 0.21± 0.21 0.64 ±
0.64 ± 0.17 0.15
0.17 ±±
0.15 0.01
0.01 0.17 ±±0.01
0.17 0.01

The mean compression strength was calculated to be: 0.64 ± 0.21 MPa for BG; 0.64 ± 0.17 MPa for
The mean compression strength was calculated to be: 0.64 ± 0.21 MPa for BG; 0.64 ± 0.17 MPa
β-TCP; 0.15 ± 0.01 MPa for the 50/50 composition; and 0.17 ± 0.01 MPa for the 70/30 scaffolds. A
for β-TCP; 0.15 ± 0.01 MPa for the 50/50 composition; and 0.17 ± 0.01 MPa for the 70/30 scaffolds.
comparison of failure load and compressive strength of the different 3D printed scaffolds is shown
A comparison of failure load and compressive strength of the different 3D printed scaffolds is shown
in Table 5. There is a significant difference between the mean failure load for all different samples
in Table 5. There is a significant difference between the mean failure load for all different samples
(p = 0.05). Additionally, there is a significant difference between the mean compressive strength of
(p = 0.05). Additionally, there is a significant difference between the mean compressive strength of the
the four different samples (p = 0.05).
four different samples (p = 0.05).

2.2.2.2. Scaffold
Scaffold Biocompatibility
Biocompatibility

2.2.1.
2.2.1. Live/Dead
Live/Dead Assay
Assay
TheThe results
results fromfromthethe live/dead
live/dead assay,
assay, shown
shown in in Table
Table 6, illustrate
6, illustrate thethe quantity
quantity of of live
live andand dead
dead
cells
cells present
present at 73,and
at 3, 7 and
10 10
daysdays following
following seeding.
seeding. Live
Live cells
cells appear
appear luminous
luminous green
green andanddeaddead cells
cells
appear
appear red.red.
TheThe
areas areas
withwith the highest
the highest visiblevisible concentration
concentration of live
of live cells cells
from from
each each are
scaffold scaffold
shownare
in shown in theItimages.
the images. is clearItfrom
is clear
the from
images thethat
images
live that
cellslive
are cells are present
present on the
on the pure pure substances,
substances, as well as
as well
on theas composite
on the composite
materials materials at 3,10
at 3, 7 and 7 and
days.10There
days.isThere is a notable
a notable increase increase in the quantity
in the quantity of live of
livepresent
cells cells present
as the as the period
period increases,
increases, as moreas more luminous
luminous green green
spots spots
can can be identified,
be identified, which
which is an
is an
indication
indication that
that cellscells
areare
ableable to grow
to grow andand multiply
multiply on the
on the materials.
materials. Comparing
Comparing the the
50/50 50/50
andand 70/30
70/30
compositions
compositions and andthethe samples
samples of of pure
pure BGBG and and β-TCP,
β-TCP, moremore
livelive cells
cells areare present
present on on
thetheBGBG or 70/30
or 70/30
composition
composition than
than thethe TCP
TCP oror 50/50composition.
50/50 composition.However,
However,after after1010 days,
days, nearly
nearly the same quantity of
of living cells could
could be be found
foundon oneach
eachdifferent
differentsample.
sample.InInaddition,
addition,the theconcentration
concentration ofof live
live cells
cells is is
higher
higher around
around thethe openings
openings to to
thethe large
large porous
porous channels
channels in in
thethe scaffolds,
scaffolds, which
which appear
appear black
black in in
thethe
images.
images. Very
Very fewfew deaddead cells
cells areare identifiable
identifiable in these
in these images
images (see(see Table
Table 6 or6 Figure
or Figure
7). 7).

BG β-TCP 50/50 70/30

3 days
Materials 2018, 11, 13 9 of 22
Materials 2018, 11, 13 9 of 21

BG β-TCP 50/50 70/30

3 days

7 days

10 days

Figure 7. Live/dead assay images of the inner surface of the scaffolds, captured with Olympus BX 51
Figure 7. Live/dead assay images of the inner surface of the scaffolds, captured with Olympus BX 51
fluorescence microscope, broadband blue excitation with interference band-eliminator filter
fluorescence microscope, broadband blue excitation with interference band-eliminator filter U-MWIB3:
U-MWIB3: excitation filter Olympus BP460-495, beam splitter Olympus DM505, band-eliminator
excitation filter Olympus BP460-495, beam splitter Olympus DM505, band-eliminator filter BA510F;
filter BA510F; living cells, green; dead cells, red. The scale bar is 400 µm.
living cells, green; dead cells, red. The scale bar is 400 µm.

Table 6. Comparison of living and dead cells per mm²2 on the different materials.
Table 6. Comparison of living and dead cells per mm on the different materials.
Time BG TCP 5050 7030
Time BG Cells/mm² TCPCells/mm² 5050
Cells/mm² 7030
Cells/mm²
Alive
Cells/mm 2 Dead Alive
Cells/mmDead
2 Alive Dead
Cells/mm 2 AliveCells/mm
Dead2
3 days Alive Dead Alive Dead Alive Dead Alive Dead
Outer surface 80 ± 9 7±2 68 ± 7 5±1 79 ± 8 6±1 70 ± 5 5±2
3 days
Inner surface 208 ± 44 6±4 321 ± 42 6 ± 3 119 ± 42 3±1 72 ± 13 8±4
Outer surface 80 ± 9 7±2 68 ± 7 5±1 79 ± 8 6±1 70 ± 5 5±2
7 days
Inner surface 208 ± 44 6±4 321 ± 42 6±3 119 ± 42 3±1 72 ± 13 8±4
Outer surface 634 ± 48 62 ± 14 126 ± 3 18 ± 3 480 ± 17 52 ± 22 576 ± 119 74 ± 4
7 days
Inner surface634 ±
Outer surface
19748± 15 62 ±
3214
± 23 126
191±±314 184 ±± 13 164 ± 17 16 ± 11
480 ± 17 52 ± 22
178 ±2
576 ± 119
24 ± 74
3 ±4
10 days
Inner surface 197 ± 15 32 ± 23 191 ± 14 4±1 164 ± 17 16 ± 11 178 ± 2 24 ± 3
Outer surface 670 ± 167 0 636 ± 63 8 516 ± 28 10 ± 3 534 ± 31 10 ± 3
10 days
OuterInner surface670 ±
surface 277
167± 32 0 0 278
636 ± ±6311 80 289
516±±7228 0 ± 3 251534
10 ± 20
± 31 0 10 ± 3
Inner surface 277 ± 32 0 278 ± 11 0 289 ± 72 0 251 ± 20 0
2.2.2. Cell Proliferation Assay
2.2.2.The
Cell WST-1
Proliferation
assayAssay
was divided into scaffold and remaining cells: Figure 8a shows the
proliferation of the cells on
The WST-1 assay was divided the 3D printed scaffolds
into scaffold after removing
and remaining cells: them
Figurefrom the well
8a shows theplate used in
proliferation
the assay.
of the cellsFigure
on the8b3D shows the scaffolds
printed proliferation
afterofremoving
the cells that
themremained
from theinwellthe plate
well plate,
used inwhich were
the assay.
moving through the pores of the 3D printed scaffolds and settled at the walls of
Figure 8b shows the proliferation of the cells that remained in the well plate, which were moving the cell culture plate.
As shown
through theinpores
Figure 8a, 3D
of the there was no
printed significant
scaffolds difference
and settled at theinwalls
cell viability
of the cellamong
culturecomposites after
plate. As shown
three and8a,
in Figure seven
theredays
was and of cultivation.
no significant Afterinseven
difference days ofamong
cell viability incubation, MG 63
composites cells
after hadand
three a higher
seven
metabolic activity than at three days, which can be ascribed to the increased proliferation
days and of cultivation. After seven days of incubation, MG 63 cells had a higher metabolic activity of the cells.
Each experiment showed higher absorbance for the 70/30 scaffolds than for the 50/50
than at three days, which can be ascribed to the increased proliferation of the cells. Each experiment scaffolds at all
time
showedpoints. These
higher results correspond
absorbance for the 70/30very well with
scaffolds thanthe
formeasuring of total specific
the 50/50 scaffolds area.
at all time points. These
results correspond very well with the measuring of total specific area.
Materials 2018, 11, 13 10 of 21

2.2.3. LDH Assay

The results of the Lactate dehydrogenase (LDH) absorbance, measured at 24, 48 and 72 h following
seeding, are shown in Figure 9. All measured values were normalized to the cell control. Compared
to all other samples the 70/30 showed the lowest LDH activity over time. The composites showed
more stable LDH activity than the scaffolds made of pure substances. The LDH activity was higher for
the 50/50 scaffolds than the 70/30 scaffolds in all experiments. The β-TCP showed the lowest LDH
activity after 24 h, but this value increased to the value of the 50/50 after three days. BG showed a
Materials
similar 2018, 11, 13 10 of 21
Materials result,
2018, 11,but
13 with a shifted curve; with the highest LDH activity after 72 h. 10 of 21

Figure 8. Differences in WST-1 Assay of MG-63 cells after cultivation on the different
differentsamples after
after3,3,
Figure 8. Differences
Figure 8. Differences inin WST-1
WST-1 Assay
Assay of
of MG-63
MG-63 cells
cells after
after cultivation
cultivation on
on the
the different samples
samples after 3,
77and
and 10
10 days.
days. Cell
Cellculture
culture grade
grade plastic
plastic was
was used
used as
as aacontrol.
control.Significant
Significant difference
difference ***
***(p(p==0.001).
0.001).
7 and 10 days. Cell culture grade plastic was used as a control. Significant difference *** (p = 0.001).
(a)
(a)Cells on the Scaffolds, after removing it out of the cell culture plates; and (b) cells remaining in the
(a) Cells
Cells on
on the
the Scaffolds,
Scaffolds, after
after removing
removing itit out
out of
of the
the cell
cell culture
culture plates;
plates; and
and (b)
(b) cells
cells remaining
remaining in in the
the
same
same cell culture
culture plates,
plates, which
which were
were moving
moving through
through the the pores
pores of of
the the
3D 3D printed
printed scaffolds
scaffolds and and
settled
same cell culture plates, which were moving through the pores of the 3D printed scaffolds and
settled
at at theof
the walls walls of the
the cell cell culture
culture plate (nplate
= 30).(n = 30).
settled at the walls of the cell culture plate (n = 30).
[%]

300
control)[%]

300
Cell control
Cell control
cellcontrol)

BG ***
250 BG ***
250 TCP
TCP
5050
5050
(Normalizedtotocell

200 7030
200 7030
*** ***
*** ***
LDH-Activity(Normalized

150
150

100
100
LDH-Activity

50
50

0
0 24 48 72
24 48 72
Time [h]
Time [h]
Figure 9. LDH Activity of MG-63 cells on the samples measured after 24, 48 and 72 h using LDH
Figure 9. LDH Activity of MG-63 cells on the samples measured after 24, 48 and 72 h using LDH
Figure 9. LDH
(normalized Activity
to cell of MG-63
control). cellsdifference
Significant on the samples
*** (p = measured
0.001), (n =after
30). 24, 48 and 72 h using LDH
(normalized to cell control). Significant difference *** (p = 0.001), (n = 30).
(normalized to cell control). Significant difference *** (p = 0.001), (n = 30).
2.2.4. Immersion in SBF
2.2.4. Immersion in SBF
2.2.4. Immersion in SBF
ESEM images of the scaffold surfaces after immersion in SBF for 28 days resulted in formation
ESEM images of the scaffold surfaces after immersion in SBF for 28 days resulted in formation
ESEMFigure
of crystals. images10a
of the
showsscaffold surfaces
the crystal after immersion
formation in SBFoffor
on the surface 28 days
pure resulted
BG (red in formation
arrows) of
and Figure
of crystals. Figure 10a shows the crystal formation on the surface of pure BG (red arrows) and Figure
crystals.
10b FigureThe
on β-TCP. 10a crystal
shows the crystal formation
formation on the surface
on BG is especially of pure BG
interesting due(red arrows)
to the and Figure
formation 10b
of small
10b on β-TCP. The crystal formation on BG is especially interesting due to the formation of small
on β-TCP.
nano sized The crystal
crystals andformation on BG
larger crystals is especially
after immersioninteresting
in SBF fordue to theβ-TCP
28 days. formation of show
did not small such
nano
nano sized crystals and larger crystals after immersion in SBF for 28 days. β-TCP did not show such
sized
big crystals
crystals, and larger
because of itscrystals after
different immersion
solubility. Onlyinsmall
SBF for 28 days. β-TCP
(nano-sized) did
crystals not show
could such On
be found. big
big crystals, because of its different solubility. Only small (nano-sized) crystals could be found. On
the 50/50 scaffold (Figure 10c), long, jagged crystals, as on the BG, are visible on the material;
the 50/50 scaffold (Figure 10c), long, jagged crystals, as on the BG, are visible on the material;
however, apatite nanocrystals could not be observed at higher magnification. An elementary
however, apatite nanocrystals could not be observed at higher magnification. An elementary
analysis via EDX (Figure A1 in Appendix) showed that those crystals in Figure 10c could be HA.
analysis via EDX (Figure A1 in Appendix) showed that those crystals in Figure 10c could be HA.
Ciobanu et al. [26] reported similar HA crystals on titanium surfaces. Figure 10d shows crystal
Ciobanu et al. [26] reported similar HA crystals on titanium surfaces. Figure 10d shows crystal
formation on the surface of a 70/30 scaffold with increasing magnification. Apatite nanocrystals
formation on the surface of a 70/30 scaffold with increasing magnification. Apatite nanocrystals
Materials 2018, 11, 13 11 of 21

crystals, because of its different solubility. Only small (nano-sized) crystals could be found. On the
50/50 scaffold (Figure 10c), long, jagged crystals, as on the BG, are visible on the material; however,
apatite nanocrystals could not be observed at higher magnification. An elementary analysis via EDX
(Figure A1 in Appendix A) showed that those crystals in Figure 10c could be HA. Ciobanu et al. [26]
reported similar HA crystals on titanium surfaces. Figure 10d shows crystal formation on the surface
of a 70/30 scaffold with increasing magnification. Apatite nanocrystals could be observed in this
image, as pointed
Materials by the red arrows.
2018, 11, 13 11 of 21

(a) (b)

(c) (d)
Figure 10. Low-vac SEM images showing crystal formation on scaffold surfaces: (a) BG; (b) β-TCP;
Figure 10. Low-vac SEM images showing crystal formation on scaffold surfaces: (a) BG; (b) β-TCP;
(c) 50/50; and (d) 70/30. Red arrows indicate the HA crystals after immersion in SBF for 28 days.
(c) 50/50; and (d) 70/30. Red arrows indicate the HA crystals after immersion in SBF for 28 days.
Images were taken with FEI Quanta 250 FEG, Large Field Detector, 5 kV, 100 Pa.
Images were taken with FEI Quanta 250 FEG, Large Field Detector, 5 kV, 100 Pa.
After immersion in SBF for 28 days, changes in diameter, height and weight of the samples
Afterbe
could immersion
observed.inThe
SBFhighest
for 28 changings
days, changes in diameter,
in weight could beheight and weight
observed for the of the samples
composites: could
12.03 ±
be observed.
3.63% for The highest
50/50 changings
and 13.67 ± 4.91%infor
weight
70/30could be observed
(see Table for the
7). For the composites:
composites, 12.03in±diameter
changes 3.63% for
(1.98
50/50 and 13.67 ±
± 0.25% for4.91%
50/50for
and 2.36 ±(see
70/30 0.56% for7).
Table 70/30) were
For the also found.changes
composites, Smaller in
changes (1.98 ±(0.12
in height
diameter ±
0.25%
0.02% for 50/50 and 0.24 ± 0.12% for 70/30) were measured.
for 50/50 and 2.36 ± 0.56% for 70/30) were also found. Smaller changes in height (0.12 ± 0.02% for
50/50 and 0.24 ± 0.12% for 70/30) were measured.
Table 7. Degradation of 3D printed Samples after immersion in SBF for 28 days (n = 3).
Table 7. Degradation of 3D printed Samples after immersion in SBF for 28 days (n = 3).
BG β-TCP 50/50 70/30
∆ Diameter (%) 0 1.96 ± 0.03 1.98 ± 0.25 2.36 ± 0.56
∆ Height (%) BG0.54 ± 0.05 β-TCP 0 0.12 ±50/50
0.02 0.24 ± 0.1270/30
∆ Diameter∆(%)
Weight (%) 0 8.08 ± 0.97 1.96
0.61
± ±0.03
0.03 12.03
1.98±±3.63
0.25 13.67 ±2.36
4.91± 0.56
∆ Height (%) 0.54 ± 0.05 0 0.12 ± 0.02 0.24 ± 0.12
∆ Weight (%)
3. Discussion 8.08 ± 0.97 0.61 ± 0.03 12.03 ± 3.63 13.67 ± 4.91

A difference in weight was expected due to the difference in composition and the results reflect
this; although the density of bioglass is more than 10% lower than the density of β-TCP, the scaffolds
with a higher bioglass content were ~15% heavier than those with equal BG/β-TCP content. This is a
result of the mass ratio of the mixed powder, where the fine fraction is as high as the coarse one. This
leads to worse flowability, but also higher green density of the printed specimen. Although the
Materials 2018, 11, 13 12 of 21

3. Discussion
A difference in weight was expected due to the difference in composition and the results reflect
this; although the density of bioglass is more than 10% lower than the density of β-TCP, the scaffolds
with a higher bioglass content were ~15% heavier than those with equal BG/β-TCP content. This is
a result of the mass ratio of the mixed powder, where the fine fraction is as high as the coarse one.
This leads to worse flowability, but also higher green density of the printed specimen. Although the
scaffolds were printed with identical dimensions, the mean diameter of the 50/50 scaffolds was ~5%
larger than the mean diameter of the 70/30 scaffolds. This difference is likely a result of the different
powder compositions used in the printing process. Vorndran et al. [27] demonstrated that grain size
can affect particle interconnectivity and sintering behavior. Based on this study, it is possible that larger
grains were present in the 50/50 powder composition, which may have resulted in scaffolds with poor
particle interconnectivity. Smaller grain size in the 70/30 material could have resulted in scaffolds
with greater interconnectivity, thus showing larger shrinkage during sintering and therefore smaller
diameters. Furthermore, a higher temperature for sintering β-TCP scaffolds is required compared to
bioglass, which is already melting at about 1200 ◦ C. Although the sintering temperature of the 50/50
scaffolds was 100 K higher than the sintering temperature of the 70/30 scaffolds, the higher mass
content of β-TCP requires even higher temperatures in the oven. This leads to a lower densification
of the scaffolds compared to those with a higher bioglass content. Despite the statistically significant
difference in scaffold diameters, the difference remains small at ~5%, and no significant difference in
height was detected.
EDX analysis detected all elements which were expected to be present in the β-TCP and BG
composites; calcium, phosphorous, silicon, sodium and oxygen. Hence, silicon is the main component
of BG thus considerably increasing the silicon content of the composite material. Differences also exist
in the calcium content of the materials. The mean Ca/P ratio for the 50/50 material was 1.86, whereas
for the 70/30 material it was 2.03. The increase in Ca/P ratio for the 70/30 material was expected,
as BG has a higher Ca/P ratio than β-TCP. As expected, the BG (45S5) showed a Ca/P ratio of 5.27 [28]
and the β-TCP a ratio of 1.55 [7]. This information provides an interesting prelude to the immersion in
SBF experiment. Based on studies into the effect of Ca/P content on apatite formation, it is expected
that by increasing the Ca/P ratio of pure β-TCP with the addition of BG, apatite formation on the
material surface should occur when the material is immersed in SBF (see Figure A1 in Appendix A).
After immersion in SBF for 28 days, a degradation of the samples from 0.61% to 13.67% could be
observed. The samples with the highest specific surface area showed the highest degradation.
3D laser scanning results indicate that the average roughness of the 50/50 scaffolds was higher
(Ra 40.11 ± 3.18 µm) than for the 70/30 scaffolds (Ra 38.23 ± 10.25 µm); however, the difference was
not hugely significant with only a ~5% increase in the 70/30 Ra. Zareidoost et al. [29] and Li et al. [30]
also discovered significantly higher cell growth on samples with higher roughness. High macro- and
micro-roughness can increase osteoblast differentiation and hence bone formation on the surface. Based
on these results it is expected that cells should successfully proliferate on the surfaces of both samples.
A few sinter bridges, where particles have joined together, can be observed; however, many more
individual particles are apparent. This poor particle interconnectivity should be noted as it may
affect the mechanical strength of the scaffolds. Hence, the study by Vorndran et al. [27] indicated that
scaffolds with small, well interconnected particles showed higher mechanical strength than those with
larger, individual particles.
Although the mercury porosimetry results reported the total porosity, average pore radius and
total cumulative volume to be slightly higher for the 50/50 scaffold than the 70/30 scaffold, for the
purpose of this project the differences can be considered negligible. It can therefore be concluded that
the scaffolds have been successfully printed with similar porosities and that difference in porosity is not
a factor which could influence other experiments carried out using the scaffolds. Lawrence et al. [28]
postulated that a porosity is very important for cell colonization in 3D scaffolds. The total porosity of
the scaffolds (between 70% and 75%) is in the region of that for many similar publications, although
Materials 2018, 11, 13 13 of 21

Polo-Corrales et al. [5] believes that a higher porosity is ideal. Moreover, the scaffolds contain a range
of macropores, mesopores and micropores which has been proven to encourage osteoblast activity in
addition to aiding various types of essential bone growth. In addition to the wide range of macro and
microporosity which enhances cell behavior, the large pore channels running throughout the scaffolds
should aid supply of essential nutrients and formation of blood vessels. Comparing the mercury
porosimetry results to the information on porosity found in literature, the porosity characteristics of the
scaffolds are encouraging [5]. Based on this, it is reasonable to expect the cell culture experiments to
show cell growth into both types of scaffold material. With reference to the study by Zhang et al. [31] it
is possible that incorporating controlled nanoporosity into the scaffolds via nanofabrication techniques
could further improve the porosity characteristics of the scaffolds by allowing closer control over
the surface porosity with the aim of improving cell adhesion. For a faster osseointegration, the 3D
scaffolds could additionally be coated with nanoparticles of growth factors, as already described by
Gong et al. [32] and Yi et al. [33]. This would promote bone growth on a nanoscale. The high porosity
of the constructs could even serve for a guided bone growth.
The compression tests found the 70/30 scaffolds to be ~13% stronger in compression than the
50/50 scaffolds. This is in-keeping with the fact that BG has superior mechanical properties when
compared to β-TCP and therefore the scaffolds with a higher BG content should be stronger in
compression. However, both scaffolds had considerably weaker compression strength than the other
3D printed or plotted scaffolds, which showed compression strengths ranging from 7.4 to 11 MPa
and of natural bone, which has a compression strength ranging 2–20 MPa (cancellous bone) and
100–200 MPa (cortical bone) [34]. Roohani-Esfahani et al. also reported a 3D plotted scaffold with
50–200 MPa [35]. In contrast to this, the strongest of the scaffold had a compression strength of just
0.171 MPa, which does not compare to even the weakest forms of cancellous bone. Again, referring to
Vorndran et al. [27], poor interconnectivity may be related to low mechanical strength.
A higher concentration of live cells was present on the 70/30 materials and the 50/50 materials
than on the scaffolds out of 100% BG or 100% β-TCP. This is particularly obvious in the images captured
after seven days. In addition, the concentration of live cells increased over 3, 7 and 10 days. These
results indicate that the osteoblast-like MG-63 cells can survive and proliferate on both materials.
The assay results also revealed that cells appeared to survive and proliferate with greatest success
around the large pore channels in the scaffolds. Karageorgiou et al. [36] and Zhang et al. [31]
demonstrated that cell infiltration and distribution within the whole scaffold will greatly affect the
overall performance of the resulting construct. The images in Figure 7 show the areas on the scaffolds
with the highest concentration of living cells. Overall, the live/dead assay results are an encouraging
indication of good biocompatibility in both materials and agree with other extensive studies proving
the biocompatibility of both BG and β-TCP [37]
In summary, there was a higher MG-63 cell proliferation on the 70/30 scaffolds, suggesting that
greater suitability of this scaffolds in vivo. Moreover, an increase in absorbance on both the 50/50 and
70/30 scaffolds was observed between three and seven days in each of the experiments. This was
expected to increase further between 7 and 10 days, as in the MG63 cell control, but, conversely,
the results show an increase in absorbance following 10 days of incubation. It is likely that this a result
of scaffold dissolution; as the scaffold degrades with time the surface area for cells to attach to and
differentiate on is decreased. With this lack of space, overcrowding occurs and it is impossible for
cell proliferation to continue. Due to the faster degradation rate of BG, it is likely that the decrease in
proliferation will be greater in the 50/50 scaffolds, as they have a higher β-TCP content and should
therefore show greater degradation; however further investigation into the solubility of the scaffolds
would be necessary to confirm this.
With reference to scaffold behaviour, the results from the LDH assay mainly demonstrate that
the 50/50 scaffolds showed a higher LDH activity, than the 70/30 scaffolds. This is demonstrated in
Figure 9. Compared to the cell control the LDH activity was lightly increased for 70/30. A higher
value could be found for the 50/50.
Materials 2018, 11, 13 14 of 21

Figure 8 also demonstrates the increase in the detected absorption, and therefore cytotoxicity,
of the scaffolds with time. Similar to the scaffold degradation discussed in the previous section, as the
scaffold dissolves over time more cells will become detached from the scaffold surface and thus will be
unable to survive. This increase in cell death with time is detected by the LDH assay as an increase
in cytotoxicity, when in fact it could potentially be a result of scaffold degradation. For this reason,
it can be concluded that the cytotoxicity reported in the LDH assay may not be reliable and therefore is
unsuitable for comparison to similar experiments. Overall, the LDH assay is effective in indicating
that the 50/50 material has higher levels of cytotoxicity than the 70/30 scaffold. Given the higher
viability of the 70/30 scaffolds indicated in the WST assay, this is the result which was expected.
The LDH measurements were made after 1, 2 and 3 days. The WST measurements were made after 3,
7, and 10 days. The higher LDH activity is equivalent to a higher cell death rate at the beginning of
the experiment. This results in a low cell count on the scaffolds. Thereafter, the cells show a normal,
comparable to the control, albeit less growth. Compared to the cell control, the absorbance of the
scaffolds in WST shows smaller values. This is because there are fewer cells on and in the scaffolds
than in the cell culture plate, that was used as control. For correct values the absorbance of the scaffolds
shown in Figure 8a, should be combined with the absorbance of cells that remained in the cell culture
plates in Figure 8b.

4. Materials and Methods

The bioglass (BG) was obtained from Colorobbia, Italy, the β-tricalcium phosphate (β-TCP) from
Budenheim, Germany and the dextrin from Carl Roth, Germany. The chemical composition of the
bioglass was 47.3 wt % SiO2 , 28.6 wt % CaO, 15.2 wt % P2 O5 , 4.9 wt % Na2 O, 2.5 wt % MgO and
1.5 wt % F. The scaffolds used in the project were composed of specific combinations of BG and
β-TCP scaffolds: 50/50 and 70/30 BG/β-TCP ratios, pure BG and pure β-TCP. As the particle size
of the powders was initially too large, it was necessary to mill the powder using grinding balls until
the medium particle size was smaller than 2 µm and 90% of the grains were smaller than 5 µm
diameter. Moreover, this was suggested to improve the mechanical strength of the scaffolds [21].
The raw materials were milled into a water based suspension containing 65 wt % ceramics. For the BG
suspensions, DOLAPIX A88 (Zschimmer & Schwarz, Lahnstein, Germany) was used as dispersant.
The suspensions which contained β-TCP were stabilized with DOLAPIX CE64 (Zschimmer & Schwarz,
Germany). The suspensions were granulated in a spray dryer (GEA Niro, Gladsaxe, Denmark) at
220 ◦ C using PVA 22000 (Fluka, Buchs, Switzerland) as a binder. The samples were produced with
an Inkjet 3D Printer (ZPrinter 310 Plus, Z Corporation, Rock Hill, SC, USA). To increase the green
density of the printed scaffolds the powder contained a mixture of a fine (medium diameter at about 5
to 10 µm) and a coarse (medium diameter of the granules at about 35 to 40 µm) fraction. The mass
ratio of the coarse to fine powder was 75:25 for all powders except of the 50/50 BG/β-TCP scaffolds,
where the mass ratio is 50:50. The powder was also mixed with 10–15 wt % dextrin, a starch based
substance which acts as a binder when combined with the water based output from the inkjet (zb60,
Z Corporation, Rock Hill, SC, USA), as dextrin is water soluble and its dissolution allows the particles
to bind together [38]. The layer thickness was 100 µm and the binder/volume-ratio for the shell and
the core was 0.4 and 0.2, respectively. After the binder was dried, the samples were de-powdered
and sintered to improve the mechanical properties. The sintering protocol differed for each different
material composition used. The sintering temperatures for the pure β-TCP was 1250 ◦ C [39]. The BG
and the 50/50 scaffolds were heated to 350 ◦ C and kept at this temperature for 60 min. The temperature
was then increased to 950 ◦ C and the scaffolds were then exposed to this temperature for 120 min
before cooling down gradually in the oven. The process was similar for the 70/30 scaffolds except
that in the second stage of the process they were heated to 1050 ◦ C rather than 950 ◦ C and kept at this
temperature for 140 min. A sinter shrinkage of around 10–15% could be observed for the scaffolds.
The scaffolds consisting of pure β-TCP were also debindered for 60 min at 350 ◦ C and then heated to
Materials 2018, 11, 13 15 of 21

1250 ◦ C, keeping this temperature for 240 min. To avoid cracks during the cooling, the temperature
was decreased to 200 ◦ C with a cooling rate of 100 K/h.

4.1. Sample Characterization

The ceramics (3 samples of each) were tempered at 105 ◦ C for 24 h to ensure that no water
was present in the samples. The test was then carried out in accordance with DIN 66133. For this
purpose, the samples were transferred into a glass vessel and this was installed in the Pascal 140/440
porosimeter. The pressure control and evaluation were carried out by PASCAL software. The results
from both measurements were then combined into a diagram by the software [40]. The SEM/ESEM
images were taken with FEI Quanta 250 FEG (FEI, Hillsborough, OR, USA) and Philips ESEM XL 30
FEG (Amsterdam, The Netherlands). The EDX measurements were carried out on a Philips ESEM XL
30 FEG with EDX unit and the EDX measurements in Appendix A were carried out on a FEI Quanta
250 FEG with Oxford EDX unit. The measurement conditions were 10 kV accelerating voltage and
10 min measurement time (live time). A Keyence 3D Laserscanning Microscope (Keyence VK-X210)
was used to measure the surface roughness. All experiments were carried out at room temperature.
The uniaxial compression tests were carried out at a Zwick/Roell Z005 (Zwick/Roell, Ulm, Germany)
materials testing machine with Test Expert II Software. A standard compression test was carried
out with 1 N pre force and testing speed of 10 mm/min. Stop criterion was break or 20% length
deformation. Origin 2016 Professional (OriginLab, Northampton, MA, USA) was used for all following
statistical calculations.

4.2. Cell Culture Experiments

The cell culture experiments revealed the effect of the composite on MG-63 cells (ATCC CRL-1427)
via live–dead assays and Giemsa staining. The live–dead cell staining kit II (Art. No. PKCA707-30002,
Promokine, Heidelberg, Germany) was obtained from Promokine and the azur eosin methylene
blue (Art. No. 109204, Merck, Lucerne, Switzerland) for Giemsa staining was obtained from Merck.
To sterilize the 3D printed scaffolds, they were heated up to 200 ◦ C for 4 h in a Drying oven (Memmert,
Schwabach, Germany, UN 100).

4.2.1. Cell Proliferation Assay

The scaffolds for use in the WST experiments were seeded in a 24 well plate. Each experiment
required 10 scaffolds of each material composition, with cells seeded in an empty well to act as a
control and a blank to account for background absorbance in the ELISA reader. The experiments
were repeated three times. The scaffolds and control well were seeded with cell solution containing
50,000 cells in 400 µL. Once seeded, the well plate was incubated at 37 ◦ C, 5% CO2 for 2 h and then
1.5 mL DMEM-F12 (Art. No. BE12-719F, Lonza, Basel, Switzerland) was added to each well. The WST
experiments were carried out 3, 7 and 10 days following seeding. The same procedure was repeated
at each interval: The DMEM-F12 was removed from each well and the scaffolds were washed with
DPBS (Art. No. 14190-094, Gibco, Grand Island, NE, USA) three times to remove all medium residues
from the pores. DMEM-F12 phenol red free (1 mL) (Art. No. 11039-021, Gibco, Grand Island, NE,
USA) was added to each cell-containing well, plus one empty well to act as a blank. WST reagent
(100 µL) (Art. No. 05015944001, Roche, Basel, Switzerland) was then added to all of the wells in use.
These volumes were sufficient to fully immerse the scaffolds. The well plate was then placed in an
incubator at 37 ◦ C, 5% CO2 for 2 h. After incubation, three 100 µL samples were taken from each
well and placed in a 96 well plate. The plate was placed in a Spectrostar Nano microplate reader
(BMG Labtech, Ortenberg, Germany) and subject to 60 s orbital shaking before the absorbance was
measured at wavelength λ = 450 nm with a reference λ = 600 nm. For the additional Giemsa stainings
(see Appendix A) Giemsa’s azur methylene blue solution (Art. No. 1.09204.0500, Merck, Switzerland
was used) according to the following protocol: washing the samples with DPBS (3x), fixing with
Materials 2018, 11, 13 16 of 21

methanol (Art. No. CP43.3 Carl Roth, Karlsruhe, Germany) for 10 min, washing again with DPBS (3x),
using the Giemsa solution 1:10 for 10 min followed by washing with water (3x) and air drying.

4.2.2. Lactate Dehydrogenase (LDH) Assay

The scaffolds for use in the LDH experiment were seeded in a 24 well plate. Each experiment
assessed 10 scaffolds from each material composition, a positive and negative control and a blank to
account for background absorbance in the ELISA reader. The experiments were repeated three times.
A 400 µL cell solution containing 50,000 cells was seeded onto each scaffold and additionally into two
empty wells to act as the positive and negative controls, respectively. One well was left empty for use
as a blank. The well plate was placed in an incubator at 37 ◦ C, 5% CO2 for 2 h. Following incubation,
1.5 mL DMEM-F12 phenol red free was added to each cell-containing well, plus one additional empty
well to act as the blank. Triton X-100 (10 µL) (Art. No. X100, Sigma Aldrich, Saint Louis, MO, USA,
was added to the positive control wells. The plates were then returned to the incubator. The LDH
experiments were carried out at 24, 48 and 72 h following seeding and the same procedure was repeated
at each interval: Three 100 µL samples were taken from each well into a 96 well plate. LDH reagent
(100 µL) was added to each well in use and the plate was incubated in darkness at room temperature
for 30 min. Following incubation, the plate was placed in a Spectrostar Nano microplate reader (BMG
Labtech, Ortenberg, Germany) and absorbance was measured at a λ of 490 nm with a reference λ of
600 nm. The measured values were normalised to the cell control (without any 3D printed sample) [26].

4.2.3. Live–Dead Assay

The scaffolds for use in the live/dead assay were seeded in a 24 well plate. For each experiment,
at least 10 different samples of each composite/control were used, and the experiments were repeated
three times. A 400 µL cell solution containing 50,000 cells was seeded onto each scaffold and the
plate was placed in an incubator at 37 ◦ C, 5% CO2 for 2 h. Following incubation, 1.5 mL DMEM-F12
was added to each well in use and the plates were then returned to the incubator. The live/dead
experiments were carried out at 3, 7 and 10 days following seeding and the same procedure was
carried out at each interval: The scaffolds were moved to a new 24 well plate and washed three
times with DDPBS to remove any excess medium within the pores. The scaffolds were then cut
longitudinally through their centers with a small sterile razor blade. One half of each scaffold was
cut again in the transverse direction. One longitudinal and one transverse section of each scaffold
was placed in separate new wells. The live/dead solution was prepared, combining 8 mL DDPBS,
16 µL EthD III and 4 µL calcein out of the live/dead cell staining kit II. live/dead solution (1 mL)
was added to each scaffold-containing well. The plate was incubated at room temperature for 10
min. Following incubation, an Olympus BX 51 fluorescence microscope with U-MWIB3 broadband
blue excitation with interference band-eliminator filter: excitation filter Olympus BP460-495, beam
splitter Olympus DM505, band-eliminator filter BA510F was used to view the live and dead cells
simultaneously. For automatically cell counting ImageJ (National Institutes of Health, Bethesda, MD,
USA)version 1.47v according to Grishagin [41] was used. The cells were counted at 5 different positions
of 1 mm2 at the same scaffold. This procedure was repeated for all different scaffolds and for at least
5 different samples of each point of interest.

4.2.4. Preparation of Simulated Body Fluid

Simulated body fluid (SBF) (500 mL) was prepared as described by Jalota et al. [42]. The chemicals
used to prepare the SBF and the quantities which were added are outlined in Table 7. A beaker
of deionised water was placed on a magnetic stirrer at 37 ◦ C. Each chemical was weighed using
electronic scales and added to the deionized water in the order presented in Table 8. An electronic
pH meter (Mettler Toledo, EL20, Columbus, OH, USA) was then used to measure the exact pH of the
solution, and hydrochloric acid was added slowly until the solution reached a pH of 7.4. The beaker
was then covered with aluminum foil and left on the magnetic stirrer overnight. The following day,
Materials 2018, 11, 13 17 of 21

the solution was filtered through a 0.2 µm (pore size) filter and sealed under sterile conditions. At least
3 samples of each were immersed in SBF for 28 days. Before and after the SBF experiment, the samples
were weighed with a Sartorius scale. The diameter and height of the scaffolds were measured using
Vernier calipers.

Table 8. Composition of Tris-buffered SBF 27.

Reagent Obtained by Art. No. Quantity (g)

Sodium chloride (NaCl) Roth 3957.3 3.274
Sodium hydrogen carbonate (NaHCO3 ) Roth 6885.2 1.134
Potassium chloride (KCl) Sigma P5405 0.187
di-sodium hydrogen phosphate dihydrate (Na2 HPO4 ·2H2 O) Merck 6580.0500 0.089
magnesium chloride hexahydrate (MgCl2 ·6H2 O) Sigma M8266 0.071
calcium chloride dihydrate (CaCl2 ·2H2 O) Sigma C7902 0.184
sodium sulphate (Na2 SO4 ) Sigma-Aldrich 746363 0.0355
Tris ((CH2 OH)3 CNH2 ) Serva 37180 3.0285
1M HCl Roth CN63.1 Until pH 7.4

4.3. Statistics
Data were expressed as mean values ± standard deviation of the mean and analyzed by one-way
analysis of variance (ANOVA). The level of statistical significance was set at p < 0.05. For statistical
calculations, Origin 2016 Professional SR1 (OriginLab) was used. Each Experiment, except the
mechanical testing with three samples each, was repeated at least three times with a minimum
of 10 samples.

5. Conclusions
The results predominantly explore to what extent the fabrication technique is appropriate for
development of scaffolds for use in biological applications. The biocompatibility experiments indicated
that MG-63 cells could survive and proliferate on both material compositions. However, as indicated
by the lower than ideal Ca/P ratio, apatite distribution on the material surface is sparse and it is
doubtful that this would be sufficient for the material to form a strong bond with bone. The most
striking finding from the characterization experiments is the especially low mechanical strength of
the scaffolds in compression. It is clear from the information in the literature that, combined with the
laser images showing the poor particle interconnectivity and the increased sinter shrinkage of the
70/30 material, the poor mechanical strength is largely a result of printing process parameters rather
than properties of the materials or scaffold geometry. It is unlikely that it will be possible to increase
the mechanical strength of scaffolds of this nature to an extent where they would be suitable for use in
load bearing applications. However, bioactive degradable scaffolds could prove to be useful in other
non-load bearing applications such as acting as fillers in cases of tumor removal or similar applications
that require filling a gap in an area which is not subject to high stresses.

Acknowledgments: The article processing charge was funded by the German Research Foundation (DFG) and
the University of Freiburg in the funding program Open Access Publishing.
Author Contributions: Anke Bernstein and Rainer Gadow conceived and designed the experiments;
Michael Seidenstuecker, Laura Kerr, Sergio Hernandez Latorre and Steffen Esslinger performed the experiments;
Ralf Thomann and Frank Syrowatka performed additional measurements; Michael Seidenstuecker, Peter Krieg
and Laura Kerr analyzed the data; Norbert P. Suedkamp and Hermann O. Mayr contributed reagents/materials/
analysis tools; and Michael Seidenstuecker, Steffen Esslinger and Laura Kerr wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
 Author Contributions: Anke Bernstein and Rainer Gadow conceived and designed the experiments; Michael
Seidenstuecker, Laura Kerr, Sergio Hernandez Latorre and Steffen Esslinger performed the experiments; Ralf
Thomann and Frank Syrowatka performed additional measurements; Michael Seidenstuecker, Peter Krieg and
Laura Kerr analyzed the data; Norbert P. Suedkamp and Hermann O. Mayr contributed
reagents/materials/analysis
Materials 2018, 11, 13 tools; and Michael Seidenstuecker, Steffen Esslinger and Laura Kerr wrote 18
the
of 21
paper

Conflicts of Interest: The authors declare no conflict of interest.

Appendix A
Appendix A

(BG)
Materials 2018, 11, 13 18 of 22

Materials 2018, 11, 13 18 of 21

(TCP)

(TCP)

(5050)
(5050)

(7030)

Figure A1. EDX Spectrum of the Nanocrystals (from top to bottom: BG, TCP, 50/50 and 70/30):(7030)
Figure EDX Spectrum
A1. taken
Spectrum with of the 250
FEI Quanta Nanocrystals (from EDX
FEG with Oxford top to bottom: BG,100
TCP,
Pa, 50/50
minand 70/30):
Figure A1. EDX Spectrum of the Nanocrystals (from topunit, at 10 kV,
to bottom: BG, TCP, 10
50/50 livetime;
and 70/30):
Spectrum
BG: taken
Ca/P ratio with
= 1.69FEI Quanta
seems 250
to be HA. FEG with Oxford EDX unit, at 10 kV, 100 Pa, 10 min livetime;
= 1.72BG:
Spectrum taken with FEI Quanta 250TCP:
FEGCa/P
withratio = 1.70
Oxford EDXseems
unit,toatbe10HA;
kV,50/50: Ca/P
100 Pa, ratiolivetime;
10 min
Ca/P ratio
seems = 1.69
to be ratio seems
HA; and to
70/30:be HA.
Ca/P TCP:
ratio Ca/P
= 1.65 ratio = 1.70 seems to be HA; 50/50: Ca/P ratio
in== 1.72
BG: Ca/P = 1.69 seems to be HA. TCP:seems
Ca/P to be HA.
ratio The
= 1.70 tiny to
seems structures of the Ca/P
be HA; 50/50: Crystals
ratio the
1.72
seems to be HA; and
background 70/30:which
Ca/Pisratio = 1.65 seems to be HA. The tiny instructures of the Crystals in the
seems to beareHA;shining,
and 70/30: Ca/Pwhy
ratioNa, Mg,
= 1.65 Al and
seems to Si
be were found
HA. The tiny the spectra.
structures of the Crystals in the
background are shining, which is why Na, Mg, Al and Si were found in the spectra.
background are shining, which is why Na, Mg, Al and Si were found in the spectra.

(a) (b)
(a) (b)

Figure A2. Cont.

Materials 2018, 11, 13 19 of 21
Materials 2018, 11, 13 19 of 22

(c) (d)
Figure
Figure A2.A2. Giemsa
Giemsa StainingofofMG63
Staining MG63 cells
cells on
on the
theScaffolds
Scaffoldsafter 2424
after h: h:
(a) (a)
BG;BG;
(b) TCP; (c) 50/50;
(b) TCP; and
(c) 50/50;
(d) 70/30: Images taken with Olympus BX 51 with reflected light in 10× magnification. The staining
and (d) 70/30: Images taken with Olympus BX 51 with reflected light in 10× magnification. The
was according to the protocol given in Romeis [43].
staining was according to the protocol given in Romeis [43].

Because of the 3D structure of the samples, the Giemsa staining was an alternative to show the
Because
cell of the on
attachement 3Dthe
structure of the
ceramics. samples,
As shown the Giemsa
in Figures 3 andstaining was an
4, the surface of alternative
the samplesto
is show the
not flat.
cell Thus,
attachement on the ceramics. As shown in Figures 3 and 4, the surface of the samples is not
the The cell core is stained in violet and the cytoplasm dark blue. The images were taken with flat.
Thus, the TheBX
Olympus cell
51core
Lightismicroscope.
stained in violet and the cytoplasm dark blue. The images were taken with
Olympus BX 51 Light microscope.
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