Acta Biomaterialia 156 (2023) 146–157

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Full length article

Composites consisting of calcium phosphate cements and mesoporous

bioactive glasses as a 3D plottable drug delivery system✩
Richard Frank Richter, Tilman Ahlfeld, Michael Gelinsky∗, Anja Lode∗
Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital Carl Gustav Carus and Faculty of Medicine, Technische Universität Dresden,
Dresden, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Calcium phosphate cements (CPC) and mesoporous bioactive glasses (MBG) are two well studied bioma-
Received 15 October 2021 terial groups widely under investigation on their applicability to treat bone defects in orthopaedics and
Revised 18 December 2021
maxillofacial surgery. Recently the extrusion properties of CPC-MBG composites using a pasty CPC based
Accepted 14 January 2022
on a hydrophobic carrier-liquid were studied in our group demonstrating that such composites are suit-
Available online 19 January 2022
able for low temperature 3D plotting. Based on this work, we show in this study that by variation of
Keywords: the MBG content in the composite the degradation of the final scaffolds can be influenced. Furthermore,
Calcium phosphate cement by modifying the cement phase and/or the MBG with therapeutically active ions like strontium, the re-
Mesoporous bioactive glass leased ion concentration can be varied over a wide range. In a second step the MBG was functionalized
Drug delivery exploiting the high specific surface area of the glass as a carrier system for proteins like lysozyme or
Additive manufacturing grow factors. We developed a protocol that allows the incorporation of protein-laden MBG in CPC pastes
3D plotting
without impairing the extrudability of the CPC-MBG composites. Additionally, we found that released
proteins from pure MBG or 3D plotted composite-scaffolds maintained their biological activity. Therefore,
the combination of CPC and MBG allows the creation of a highly flexible composite system making it a
promising candidate for bone tissue engineering.

Statement of Significance

Calcium phosphate cements and mesoporous bioactive glasses are two promising degradable biomateri-
als for the regenerative treatment of bone defects. The combination of both materials to a 3D printable
composite enables the creation of implants with patient specific geometry. By varying the composition of
the composite, the degradation behaviour can be influenced and especially the release of therapeutically
active ions is tailorable over a wide range. We demonstrated this for strontium, as it has been shown
to stimulate bone formation. Moreover, the bioactive glass can be used as a carrier system for drugs
or growth factors and we show the successful combination of such functionalised glass particles and a
cement paste without affecting the printability.
© 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction and they possess excellent biocompatibility [1–5]. A specific class

Calcium phosphates (CaP) are bioactive, ceramic biomaterials
that have been used in many applications for the treatment of
bone defects. The positive effect of soluble and insoluble CaP
phases for osteogenesis has been evidenced in vitro and in vivo
✩
Part of the Special Issue on Biofabrication for Orthopedic, Maxillofacial, and
Dental Applications, guest-edited by Professors Hala Zreiqat, Khoon Lim, and Debby
Gawlitta.
∗
Corresponding authors. Postal address: Technische Universität Dresden, Medi-
zinische Fakultät TFO, PF 133, Fetscherstraße 74, 01307 Dresden.
E-mail addresses: michael.gelinsky@tu-dresden.de (M. Gelinsky), anja.lode@tu-
dresden.de (A. Lode).
of CaP are self-setting calcium phosphate cements (CPC) that are
a formulation consisting of a liquid phase and at least one CaP
precursor [6] with hydraulic reactivity, for example α -tricalcium
phosphate (α -TCP). Immediately after contact with aqueous me-
dia, these CPC start self-induced dissolution-precipitation reac-
tions and form non-stochiometric calcium-deficient hydroxyapatite
(CDHA) at neutral pH. Typically, self-setting powder/liquid-cements
get prepared by mixing the precursor powder with an aqueous liq-
uid obtaining an injectable material that can be applied through a
nozzle into a bone defect. Because of the ongoing setting reaction,
the processing time of these CPC is limited.

https://doi.org/10.1016/j.actbio.2022.01.034
1742-7061/© 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
R.F. Richter, T. Ahlfeld, M. Gelinsky et al.
In the last decade, it was investigated how CPC can be utilized
for extrusion-based additive manufacturing, commonly termed as
direct ink writing, robocasting or 3D plotting. Suitable formula-
tions were developed that allow extrusion for a prolonged time
enabling the fabrication of anatomically relevant three-dimensional
constructs with defined porosity. For example α -TCP-based precur-
sors were mixed with gelatine or Pluronic 127 and CPC constructs
of high shape fidelity were achieved [7–9]. These CPC retard the
formation of CaP precipitates within the needle without problems
such as filter pressing and needle clogging [10]. The cementous
formulation applied in this experimental study takes usage of a
water-immiscible, biocompatible oil mixture, which acts as a tem-
porary carrier liquid (cl) and is exchanged with water during the
setting reaction [11]. The setting reaction is retarded until exposure
to an aqueous environment (e.g. water-saturated atmosphere) and
thus this CPC allows limitless extrusion through nozzles and can
be plotted with high accuracy [12,13]. The possibility to fabricate
patient specific implants with this CPC formulation was for exam-
ple demonstrated by Korn et al. for a rat alveolar defect model and
showed how the scaffold geometry can influence the bone forma-
tion [14]. However, the scaffolds showed no distinct sign of degra-
dation, pointing out the disadvantage of a slow resorption of this
CDHA-forming cement and the need for a further material modi-
fication. Additionally, this plottable CPC also cannot per se act as
carrier material for biomolecules such as growth factors or antibi-
otics rather than dip coating preventing spatiotemporal release of
multiple biologically active factors. Any mixing with an aqueous
protein solution would start the setting reaction and thus prevent
plotting.
Another group of bioactive materials with high potential for
bone tissue engineering is the class of bioactive glasses (BG). In
particular, the Bioglass 45S5 originally developed by Larry L. Hench
[15] has attracted much attention in the last decades. Similar to
CaP they have an excellent biocompatibility and were shown to
form stable bonds to bone and soft tissue [16]. A specific group of
BG are mesoporous bioactive glasses (MBG). These MBG are syn-
thesised via sol-gel methods and possess a highly ordered intrinsic
pore structure with channels of diameters in the low nanometre
range (3–5 nm) resulting in a superior high specific surface area
(20 0–50 0 m²/g) with high binding affinity and capacity for pro-
teins [17,18]. This distinguishes MBG from CPC as excellent carrier
materials for biomolecules. However, MBG are usually obtained as
a powdery material and need a suitable matrix to be delivered into
the respective bone defect.
The aim of a composite is to combine the advantages of the
materials used or to compensate for the disadvantages of indi-
vidual components. Herein, we investigated whether CPC can act
as a suitable matrix to carry protein-functionalized MBG. CPC-
MBG composite systems have been studied only rarely in litera-
ture. Li et al. have used polyvinyl alcohol (PVA) as binder for CPC
and MBG powders to create an extrudable CPC-MBG composite;
protein-laden constructs facilitated bone formation and vascular-
ization in vivo [19,20]. Our group recently developed an injectable
CPC-MBG composite that could be functionalized with growth fac-
tors and revealed promising stimulation of bone growth in a pilot
study in vivo [21,22]. Additionally, cl-based CPC-MBG composites
can be used for 3D plotting [23]. Remarkably, both the CPC and the
MBG are highly modifiable regarding their chemical composition:
For example, therapeutically active strontium ions were incorpo-
rated without disturbing their intrinsic physical properties [23,24].
In particular, XRD measurements showed that the incorporation of
strontium did not change the final composition or the mechan-
ical properties of the hardened cement compared to strontium-
free CPC [24]. For CPC-MBG composites also no other phases apart
from hydroxyapatite and small residues of the precursor compo-
nents could be detected [21]. This was visually confirmed by SEM
147
Acta Biomaterialia 156 (2023) 146–157
imaging, as no distinct changes in microstructure were observed
[23,25].
Despite these promising achievements, fundamental interac-
tions were not investigated thoroughly so far, e.g. the influence of
the MBG composition or the MBG concentration within the com-
posite on material properties such as degradation behaviour, pore
development or release of bone-stimulating ions (e.g. Ca2+ , Sr2+ ).
Further, the potential of protein release from 3D plotted CPC-MBG
structures with varying MBG compositions and concentrations has
only been poorly characterized. Taking into account also the Sr-
modified variants of CPC and MBG, even more interactions and im-
pact on degradation behaviour and protein release profiles must be
expected.
Herein, we investigated the basic relationships between these
parameters by thorough studies using 3D plotted structures of dif-
ferent CPC and Sr-modified CPC matrices in combination with dif-
ferent concentrations of MBG and Sr-modified MBG. The high ge-
ometric variety obtained by 3D plotting, as well as the possibil-
ity to combine all components in different concentrations, result
in full control about release profiles of therapeutically active ions
and proteins. This indicates that the combination of (Sr-)CPC and
(Sr-)MBG are a highly flexible material toolbox that can be tailored
directly to the patient’s needs.
2. Materials and methods
2.1. Sample preparation
2.1.1. Synthesis of bioactive glass
MBG powders were synthesized using a template-induced, self-
assembling sol-gel method based on the protocol developed by
Yan et al. [26]. In a typical synthesis for the calcium modified
MBG (gCa, molar ratio of Si/Ca/P = 80:15:5), the sol was prepared
by dissolving 4.0 g of the non-ionic block copolymer Pluronic
P123 (molecular weight Mw = 5800, Sigma-Aldrich, Steinheim,
Germany) in 60 g ethanol (96%) and stirring at room tempera-
ture for 1 h. Afterwards, 6.7 g of tetraethyl orthosilicate (99%,
Sigma-Aldrich, Steinheim, Germany), 0.73 g of triethyl phosphate
(99.8%, Sigma-Aldrich, St. Louis, MO, USA), 1.4 g of calcium nitrate-
tetrahydrate (Merck, Darmstadt, Germany) dissolved in 6 ml deion-
ized water and 1.0 g of 0.5 M HCl were added. The complete sol
was stirred at room temperature for 24 h. The solution was then
casted in petri dishes and left for drying at room temperature for
24 h to undergo an evaporation-induced self-assembly process. The
obtained gel was dried at 60 °C for 3 h, grinded and then calcined
at 700 °C for 7 h. The glass particles were then again grinded and
sieved using a 45 μm sieve. Strontium modified MBG (gSr) was
produced in the same way whereas the calcium nitrate was com-
pletely substituted with strontium nitrate. Due to the lower solu-
bility, strontium nitrate was dissolved beforehand in 20 ml deion-
ized water.
2.1.2. Preparation of protein-loaded MBG
Protein loaded MBG powder was prepared by mixing protein
solution and MBG (gCa) in a ratio of 1 ml per 10 mg. As model
proteins, lysozyme (70,0 0 0 U/mg, Sigma-Aldrich, Overijse, Bel-
gium) and rhVEGF165 (PeproTech, Rocky Hill, NJ, USA) were used.
Lysozyme (concentration of 100 mg/ml) was dissolved in deion-
ized water and VEGF (concentration of 200 or 1000 ng/ml) in
phosphate-buffered saline (PBS). The protein solution was mixed
with the MBG using an overhead shaker (neoLab Rotator, neoLab
Migge GmbH, Heidelberg, Germany) for 3 h at 25 rpm at room
temperature.
For all release experiments and the fabrication of CPC-MBG
composites, the protein-loaded MBG was collected by centrifuga-
tion, washed with PBS, frozen at −20 °C and subsequently freeze-
R.F. Richter, T. Ahlfeld, M. Gelinsky et al. Acta Biomaterialia 156 (2023) 146–157

Table 1
Single CPC (cX) components, CPC mixtures (cX-cX) and CPC-MBG composites (cX-gX) tested for mass degradation and/or porosity (X = Ca or Sr).

CPC component MBG component

Label
CPC modification Percentage [wt%] of Calcium/Strontium modified CPC MBG modification Added amount of MBG [wt%] Degradation medium

cCa Ca 100/0 – – PBS

cSr Sr 0/100 – – PBS, HEPES
cCa25-cSr75 Ca/Sr 25/75 – – PBS
cCa50-cSr50 Ca/Sr 50/50 – – PBS
cCa75-cSr25 Ca/Sr 75/25 – – PBS
cCa-gCa(8) Ca 100/0 Ca 8 PBS
cCa-gSr(8) Ca 100/0 Sr 8 PBS
cSr-gCa(4) Sr 0/100 Ca 4 PBS, HEPES
cSr-gCa(8) Sr 0/100 Ca 8 PBS, HEPES
cSr-gCa(16) Sr 0/100 Ca 16 PBS, HEPES
cSr-gSr(4) Sr 0/100 Sr 4 PBS
cSr-gSr(8) Sr 0/100 Sr 8 PBS
cSr-gSr(16) Sr 0/100 Sr 16 PBS

Table 2
Single CPC (cX) and MBG (gX) components, CPC mixtures (cX-cX) and CPC-MBG composites (cX-gX) tested for ion release in PBS (X = Ca or Sr).

Label Cement component MBG component

CPC modification Percentage [wt%] of Calcium/Strontium modified CPC MBG modification Added amount of MBG [wt%]

cCa Ca 100/0 – –
cSr Sr 0/100 – –
cCa25-cSr75 Ca/Sr 25/75 – –
cCa50-cSr50 Ca/Sr 50/50 – –
cCa75-cSr25 Ca/Sr 75/25 – –
cCa-gCa(8) Ca 100/0 Ca 8
cCa-gSr(8) Ca 100/0 Sr 8
cSr-gCa(4) Sr 0/100 Ca 4
cSr-gCa(8) Sr 0/100 Ca 8
cSr-gCa(16) Sr 0/100 Ca 16
cSr-gSr(4) Sr 0/100 Sr 4
cSr-gSr(8) Sr 0/100 Sr 8
cSr-gSr(16) Sr 0/100 Sr 16
gCa – – Ca –
gSr – – Sr –

dried for 24 h (Christ alpha 1–2, Martin Christ, Osterode am Harz,

Germany). For the composite fabrication, the protein-loaded MBG
was repeatedly sieved through a 45 μm sieve to remove any ag-
glomerates that may have formed after freeze-drying. For the VEGF
experiments all steps were performed under sterile conditions and
the MBG was gamma-sterilized (25 kGy) before loading.

Fig. 1. Representative images of 3D plotted scaffolds consisting of 4 layers with a

2.1.3. Preparation of plottable pastes
Ready-to-use paste CPC (cCa) and strontium modified paste CPC
(cSr) as well as carrier liquid (cl) were obtained from INNOTERE
GmbH (Radebeul, Germany). The CPC paste had a solid content of
86 wt% and a corresponding content of 14 wt% carrier liquid. The
cement precursors consisted of 60 wt% α -tricalcium phosphate, 26
wt% calcium hydrogen phosphate, 10 wt% calcium carbonate and 4
wt% hydroxyapatite for cCa and 57.3 wt% α -tricalcium phosphate,
24.8 wt% calcium hydrogen phosphate, 14.1 wt% strontium carbon-
ate and 3.8 wt% hydroxyapatite for cSr. For the strontium con-
taining CPC the calcium carbonate phase was completely substi-
tuted with strontium carbonate [24]. The carrier liquid consisted
of 80.4 wt% Miglyol 812, 14.7 wt% Cremophor ELP and 4.9 wt%
Amphisol A. For cement pastes consisting of both CPC variants, re-
spective parts of cCa and cSr (25/75 wt%, 50/50 wt%, 75/25 wt%)
were mixed manually. For CPC-MBG composites, parts of (protein-
laden) MBG (4, 8 and 16 wt%) were added, based on the amount
of CPC paste. To maintain the extrudability of the paste, parts of
cl were added respectively as described earlier [23]. All materials
and composites analysed are shown in Table 1-4 for the respective
experiments.
90° layer-to-layer orientation (A) and a hexagonal geometry (B) exemplarily from a
CPC-MBG composite consisting of strontium modified CPC with the addition of 16
wt% calcium modified MBG (scale bars 2 mm). Fluorescence image of a 3D plot-
ted scaffold (C), overlay of blue (auto fluorescence of CPC) and red (MBG particles
stained with 5-TAMRA) channel, exemplarily from a CPC-MBG composite consist-
ing of strontium modified CPC with the addition of 16 wt% calcium modified MBG
(scale bar 500 μm). (For interpretation of the references to colour in this figure leg-
end, the reader is referred to the web version of this article.)
2.1.4. Scaffold fabrication
Scaffolds were produced by 3D plotting using a multichannel
3D plotter (BioScaffolder 2.1, GeSiM mbH, Radeberg, Germany).
Scaffolds (12 mm x 12 mm) consisting of 4 layers with a layer-to-
layer orientation of 90°, a layer thickness of 0.3 mm and a strand
distance of 1.2 mm were (Fig. 1A) plotted with a conical 410 μm
needle (Globaco, Rödermark, Germany). Scaffolds for VEGF release
had a hexagonal geometry (outer radius of 8 mm) and a strand
distance of 1.0 mm (Fig. 1B). All other geometry parameters re-
mained the same. Afterwards, plotted scaffolds were incubated in
water-saturated atmosphere at 37 °C (humidity > 95%) for 3 days.
To exemplarily visualise the distribution of the glass particles in
the cement matrix, the MBG was stained with a fluorescent dye
(5-TAMRA, 1:10 in 0.1 M sodium bicarbonate buffer, Sigma Aldrich,

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R.F. Richter, T. Ahlfeld, M. Gelinsky et al. Acta Biomaterialia 156 (2023) 146–157

Table 3
Protein-containing CPC-MBG composites used for ion release in PBS.

Label CPC modification MBG modification Added amount of MBG [wt%] Protein loaded Protein loading concentration

cSr-gCa(4)[Lyz_100] Sr Ca 4 Lysozyme 100 mg/ml

cSr-gCa(8)[Lyz_100] Sr Ca 8 Lysozyme 100 mg/ml
cSr-gCa(16)[Lyz_100] Sr Ca 16 Lysozyme 100 mg/ml

Table 4 and the supernatant was collected. The remaining protein concen-
MBG modification and protein loading concentration used for protein loading tests.
tration (cend ) was measured using Bradford assay. Therefore, 50 μl
Protein loading of the sample was mixed with 200 μl Bradford reagent (Carl Roth,
Label MBG modification Protein loaded concentration Karlsruhe, Germany) and incubated for 5 min in the dark. The
gCa[Lyz_1] Ca Lysozyme 1 mg/ml lysozyme concentration was determined by adsorption measure-
gCa[Lyz_10] Ca Lysozyme 10 mg/ml ment at 595 nm (Infinite M200 Pro, Tecan, Männedorf, Schweiz)
gCa[Lyz_50] Ca Lysozyme 50 mg/ml using a respective calibration line. The adsorbed protein amount
gCa[Lyz_100] Ca Lysozyme 100 mg/ml
(mprotein ) and the loading efficiency (Ep ) were calculated by the fol-
gSr[Lyz_1] Sr Lysozyme 1 mg/ml
gSr[Lyz_10] Sr Lysozyme 10 mg/ml lowing equations:
gSr[Lyz_50] Sr Lysozyme 50 mg/ml
gSr[Lyz_100] Sr Lysozyme 100 mg/ml
mprotein = (cstart − cend ) × Vps
gCa[VEGF_200] Ca VEGF 200 ng/ml
gCa[VEGF_1000] Ca VEGF 1000 ng/ml cstart − cend
Ep = × 100%
cstart
The protein loading capacity of MBG (gCa) was additionally
St. Louis, MO, USA) before mixing with the cement paste and plot-
studied for VEGF using the same loading protocol as described. Ac-
ting (Fig. 1C).
cordingly, two VEGF concentrations, 200 and 1000 ng/ml in PBS
were used. MBG and protein solution were mixed for 3 h and the
2.2. Sample characterization
remaining VEGF concentration in the supernatant was quantified
by a sandwich ELISA (PeproTech, Rocky Hill, NJ, USA) using a cali-
2.2.1. Porosity and mass degradation
bration line obtained from a dilution series of VEGF standard solu-
The initial porosity and mass of the plotted scaffolds were mea-
tion. All analysed MBG-protein combinations are shown in Table 4.
sured followed by incubation in 2 ml PBS at 37 °C. Additionally,
scaffolds of cSr and cSr-gCa-composites were incubated in 2 ml
2.2.4. Protein release and activity assay
HEPES buffer and treated similarly. Every 3 to 4 days, supernatants
The release of lysozyme from pure MBG (20 mg of gCa) and
were exchanged completely with fresh buffer solution. After 28
plotted scaffolds was analysed by incubating the samples in 2 ml
days, the scaffolds were air-dried and the final porosity and mass
PBS under cell culture conditions. At each time point, the su-
was determined. Additionally, mass change for pure gCa and gSr
pernatant was collected completely and replaced with fresh PBS.
was obtained as well. All analysed materials are shown in Table 1.
Lysozyme concentrations (cLyz ) were measured using Bradford as-
Porosity of plotted scaffolds was determined by helium-
say. The activity of lysozyme was determined using an assay based
pycnometry (Ultrapyc 1200e, Quantachrome Instruments, Boynton
on the lysis of the cell wall of Micrococcus lysodeikticus (Sigma
Beach, FL, USA). The Porosity (P) was calculated from the quotient
Aldrich, St. Louis, MO, USA). Therefore, 50 μl of the sample and
of apparent volume (Vapp ) and the obtained pycnometric volume
200 μl of bacteria suspension (bacteria in PBS with an initial ab-
(Vpyc ) according to the following equation:
sorption of 0.6 to 0.8) were mixed and the decrease of absorption
Vapp − Vpyc was measured at 450 nm over 10 min at 29 °C. The activity (Avol )
P= × 100%
Vapp of released lysozyme was calculated using a calibration line. Con-
sidering the released concentration of lysozyme, the specific activ-
2.2.2. Ion release ity (Aspec ) was calculated as follows:
Calcium, strontium and silicon (silicate) ion release from pure
Avol [U/ml]
MBG samples (m = 20 mg) and plotted scaffolds was studied over Aspec [U/mg] =
cLyz [mg/ml]
28 days. The analysed protein-free groups are listed in Table 2 and
protein-containing groups are listed in Table 3. All samples were The release of lysozyme from pure MBG was measured after 2,
incubated in 2 ml PBS and stored at 37 °C. After 1, 4, 7, 10, 18, 6, 12, 24 and 168 h respectively, and activity was determined for
21, 24 and 28 days, supernatants were exchanged completely with the time points 2 h, 24 h and 168 h. For plotted scaffolds, the re-
fresh PBS and the collected supernatants were frozen at −20 °C lease of lysozyme was measured after 2 and 6 h and after 1, 4, 7,
until ion content measurement. The thawed supernatants were 10, 14, 17, 21, 24, 28, 35, 42 and 49 days whereas the activity was
analysed using inductively coupled plasma-optical emission spec- determined for the time points 2 h and 1 day followed by addi-
troscopy (ICP-OES, Plasma Quant PQ 90 0 0 Elite, Analytik Jena, Jena, tional weekly investigations.
Germany). Therefore, 1 ml of the supernatant was diluted 1:10 and The release of VEGF from pure MBG (20 mg of gCa) and plotted
acidified with 2% nitric acid. scaffolds (cSr-gCa(16)[VEGF_10 0 0]) was studied by immersing the
samples in 2 ml (MBG) or 1 ml (plotted scaffolds) of Endothelial
2.2.3. Protein uptake Cell Basal Medium MV (PromoCell, Heidelberg, Germany) with 15%
The protein loading capacity and loading kinetics for two dif- heat inactivated foetal calf serum and 1% Penicillin-Streptomycin
ferent MBG types (gCa and gSr) was studied using lysozyme. The and quantified by a sandwich ELISA. For the pure MBG the super-
same loading protocol was used as described earlier (see 2.1.2) natant was collected after 2 h, exchanged completely and collected
mixing 20 mg of MBG and 2 ml of protein solution (Vps ) with repeatedly after additional 22 h (time point 24 h). For plotted scaf-
varying protein concentration (cstart = 1, 10, 50, 100 mg/ml). Every folds the supernatant was exchanged completely after 1, 4, 7, 10,
30 min during the mixing, the respective samples were centrifuged 14, 17 and 21 days. The biological activity of the released VEGF

149
R.F. Richter, T. Ahlfeld, M. Gelinsky et al.
Table 5
Single MBG component and CPC-MBG composites used for protein release experiments.
Label CPC modification MBG modification
gCa[Lyz_100] – Ca
cSr-gCa(4)[Lyz_100] Sr Ca
cSr-gCa(8)[Lyz_100] Sr Ca
cSr-gCa(16)[Lyz_100] Sr Ca
gCa – Ca
gCa[VEGF_200] – Ca
gCa[VEGF_1000] – Ca
cSr-gCa(16)[VEGF_1000] Sr Ca
Fig. 2. Porosity before and after degradation in 2 ml PBS (A); accumulated release
of Sr2+ over 28 days (B) of plotted scaffolds made of different cements (cCa, cSr)
and cement mixtures (n = 6, mean ± standard deviation, ∗ ∗ p ≤ 0.01, ∗ ∗ ∗ p ≤ 0.001).
from MBG was analysed using an endothelial cell proliferation as-
say. Therefore, human dermal microvascular endothelial cells (HD-
MEC, PromoCell, Heidelberg, Germany) were expanded in Endothe-
lial Cell Growth Medium MV (PromoCell, Heidelberg, Germany);
2 × 10³ HDMEC (5th passage) were seeded per well into a 96-well
plate and cultured for 24 h to allow cell attachment. Afterward
the cells were exposed to the release samples for 3 d As control
groups, release samples of MBG without VEGF (gCa) and blank re-
lease medium (RM) were used. After the incubation period, cells
were washed with PBS and frozen at −80 °C until DNA quantifica-
tion. The DNA content was measured using the QuantiFluor assay
(Promega, Madison, WI, USA) and the cell number was calculated
with a calibration line. All analysed materials are shown in Table 5.
2.3. Statistics
All tests were performed with n = 6, except for the experiments
utilising VEGF that where repeated three times (n = 3). Results are
displayed as average ± standard deviation. Significance was tested
by one-way analysis of variance (ANOVA), followed by a multiple
comparison test (Tukey or Sidak test). Significant differences were
assumed for a significance level p < 0.05.
3. Results
3.1. Influence of cement composition on degradation
The influence of the cement composition on the degradation
behaviour was investigated by determining the change in poros-
ity as well as the strontium ion release. Therefore, plotted scaf-
folds of the single cement components (cCa and cSr) and various
mixtures of both cement components were incubated in PBS for
28 days. Porosity was measured before and after the degradation
period and ion release was analysed for multiple time points. As
shown in Fig. 2A, the cSr and cCa25-cSr75 scaffolds showed a sig-
nificantly higher initial porosity than the cCa scaffolds and groups
with up to 50 wt% of cCa. After degradation, the porosity increased
Acta Biomaterialia 156 (2023) 146–157
Added amount of MBG [wt%] Protein loaded Protein loading concentration
– Lysozyme 100 mg/ml
4 Lysozyme 100 mg/ml
8 Lysozyme 100 mg/ml
16 Lysozyme 100 mg/ml
– – –
– VEGF 200 ng/ml
– VEGF 1000 ng/ml
16 VEGF 1000 ng/ml
Fig. 3. Decrease in mass after incubation in 2 ml PBS (A) or 2 ml HEPES buffer (C)
for 28 days; Porosity before and after degradation in 2 ml PBS (B) or 2 ml HEPES
buffer (D) of plotted scaffolds made of different cements and CPC-MBG composites
(n = 6, mean ± standard deviation, ∗ ∗ p ≤ 0.01, ∗ ∗ ∗ p ≤ 0.001, ∗ ∗ ∗ ∗ p ≤ 0.0 0 01).
significantly for each material, but between the groups there was
no clear trend visible anymore. In terms of ion release (Fig. 2B), it
was observed that a decreasing amount of cSr in the mixture lead
to a decreasing amount of released Sr2+ (see Supplementary Fig-
ure 1 for accumulated calcium release). Surprisingly, the decrease
could not be related proportionally to the fractions of cSr used in
the mixture. Looking at the accumulated release after 28 days, the
cCa25-cSr75 composition still released 92% of Sr2+ (compared to
cSr samples), while the cCa50-cSr50 composition released 64% and
the cCa75-cSr25 composition released only 20% of Sr2+ .
3.2. Influence of glass type and glass amount on degradation
The influence of the glass type (gCa and gSr) and the amount of
glass added to the composite (4, 8 or 16 wt%) on the degradation
behaviour of plotted scaffolds consisting of respective composites
was investigated by determining the mass change and the porosity
after 28 days of incubation in 2 ml PBS in comparison to plotted
scaffolds of the pure cements (cCa and cSr). To evaluate the influ-
ence of the phosphate in the PBS, the change in porosity after in-
cubation in 2 ml HEPES buffer was measured for plotted cSr based
composites with different amounts of added gCa and compared to
pure cSr. As shown in Fig. 3A, an addition of at least 8 wt% of MBG
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R.F. Richter, T. Ahlfeld, M. Gelinsky et al.
Fig. 4. Accumulated release of Sr2+ in 2 ml PBS from plotted scaffolds made of Sr-
modified cement (cSr) and pure Sr-modified MBG powder samples (A); from CPC-
MBG composites based on cCa or cSr with 8 wt% addition of different MBG (B);
from cSr-based CPC-MBG composites with different addition (4, 8, 16 wt%) of Ca-
modified MBG (C); from cSr-based CPC-MBG composites with different addition (4,
8, 16 wt%) of Sr-modified MBG (D) (n = 6, mean ± standard deviation).
lead to a significant increase in mass loss regardless of the used
MBG type compared to the respective pure cement group. This is
in line with the determined initial porosity (Fig. 3B) as an addition
of at least 8 wt% MBG lead to a significantly higher initial porosity
of the plotted scaffolds compared to the pure cement groups. Inter-
estingly, no clear trend between all groups was detected after 28
days. Especially the glass-containing groups showed only negligi-
ble changes in porosity over time while the pure cement scaffolds
reached a similar porosity of around 70% just like all other ma-
terial groups. In contrast, there was a larger decrease in mass for
all material groups in HEPES buffer (Fig. 3C) and a larger change
of porosity over time for the MBG-containing groups after degra-
dation (Fig. 3D). The decrease in mass was significantly higher for
cSr-gCa(8) and cSr-gCa(16) compared to cSr and the significant dif-
ference of the initial porosity of cSr-gCa(8) and cSr-gCa(16) com-
pared to pure cSr was maintained after degradation. No distinct
changes in surface morphology were detected after the degrada-
tion period (SEM images not shown).
As another parameter of degradation, the ion release from plot-
ted CPC and composite scaffolds and pure MBG powder samples
was measured over a time span of 28 days. The accumulated stron-
tium release is shown in Fig. 4 (see Supplementary Figure 2 for
accumulated calcium, Supplementary Figure 3 for accumulated sil-
icon release and Supplementary Figure 4–5 for release profiles of
strontium, calcium and silicon). For a better comparison of the dif-
ferent materials, the data for the single components (Fig. 4A) is
normalised to the initial weight of the samples, while the other
data sets (Fig. 4B-D) are not normalised to have a better compari-
son between plotted scaffolds of the same geometry. After the first
four days, the gSr released more than hundred-fold Sr2+ compared
to cSr. At later time points, the difference between gSr and cSr de-
creased until a factor of 16 was reached after 28 days (Fig. 4A). Al-
though the pure gSr released high amounts of Sr2+ , the composite
cCa-gSr(8) released significantly less Sr2+ than the pure cSr or any
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Acta Biomaterialia 156 (2023) 146–157
Fig. 5. Protein uptake kinetics over a time span of 3 h for different initial lysozyme
concentrations and two different MBG types – gCa and gSr (A-D); Adsorbed amount
of lysozyme per mg MBG (E); Loading efficiency as a relation between initial pro-
tein concentration and adsorbed amount of protein (F); (n = 6, mean ± standard
deviation, ∗ p ≤ 0.05, ∗ ∗ p ≤ 0.01, ∗ ∗ ∗ ∗ p ≤ 0.0 0 01).
other composite based on cSr (Fig. 4B). Meanwhile the addition of
MBG to the cSr lead to a higher release of Sr2+ . At a constant level
of added MBG at 8 wt%, the addition of gCa revealed a significantly
improved Sr2+ release compared to cSr beginning at day 17. How-
ever, the addition of gSr showed an even higher release that was
not only significantly increased already from day 7 compared to
cSr, but also compared to cSr-gCa(8) from day 17 on. The influence
of the added MBG amount was studied for composites based on
cSr with either gCa (Fig. 4C) or gSr (Fig. 4D). The addition of 4 wt%
MBG showed only for cSr-gSr(4) on day 28 a significant difference
in the Sr2+ release compared to the pure cSr scaffolds, while no
significant effect was seen for cSr-gCa(4). The addition of 8 wt%
MBG led to a significantly higher Sr2+ release for both, gCa and
gSr. For 16 wt% MBG, the addition of gCa showed a significantly
higher release of Sr2+ from day 7 on compared to pure cSr scaf-
folds, although there was no significant effect visible in compari-
son to cSr-gCa(8). The addition of 16 wt% gSr led again to a higher
release of Sr2+ compared to the cSr group, that was already signif-
icant at day 1. Interestingly, unlike the gCa-containing composites,
there was also a significantly higher release in the first 10 days in
comparison to cSr-gSr(8).
3.3. Protein uptake of MBG
The protein uptake was studied for different lysozyme concen-
trations with gCa and gSr. Fig. 5A-D shows that a higher initial pro-
R.F. Richter, T. Ahlfeld, M. Gelinsky et al. Acta Biomaterialia 156 (2023) 146–157

Fig. 6. Released amount of lysozyme of plotted scaffolds made of Sr-modified cement (cSr) and different amounts of protein-laden MBG (gCa(x)[Lyz] with x = 4, 8, 16 wt%)
over a period of 49 days (A); Accumulated release of lysozyme from plotted scaffolds (B); Measured activity of released lysozyme from plotted scaffolds (C); Specific activity
of released lysozyme as a relation between the measured activity in a given volume of a release sample and the determined released lysozyme concentration (D); (n = 6,
mean ± standard deviation). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

tein concentration leads to a higher adsorbed protein content on
the glass surface. Especially for the higher protein concentrations,
a saturation of the adsorbed protein amount starts to set in after
120–150 min. After normalization of adsorbed lysozyme amount
to MBG mass, no clear trend is visible when comparing the two
glass types (Fig. 5E). Only for the highest initial protein concen-
tration, gCa adsorbed with 899 μg lysozyme per mg MBG signifi-
cantly more protein than gSr. For the loading efficiency, shown in
Fig. 5F, a reverse trend can be seen: higher initial protein concen-
trations led to decreased loading efficiencies from around 45% to
well below 10%. While gSr showed a significantly higher loading
efficiency for an initial lysozyme concentration of 10 mg/ml, gCa
showed again a significantly better result for the highest protein
concentration.
The uptake of the proangiogenic protein VEGF was stud-
ied using gCa. The initial protein concentrations were 200 and
10 0 0 ng/ml. After 3 h, the adsorbed amount of VEGF was deter-
mined as 393 ± 3 and 1973 ± 5 ng, respectively, leading to loading
efficiencies of 98 and 99% of the initial VEGF amount. That corre-
sponds to 19.7 and 98.7 ng VEGF per mg gCa, respectively.
3.4. Protein release and activity
MBG of the gCa composition were loaded with lysozyme for 3 h
(cstart = 100 mg/ml) and the release from pure MBG and the ac-
tivity of the released lysozyme was determined. Within the first
6 h, a total amount of up to 2.8 mg lysozyme was released (data
not shown). Afterwards, only small amounts were released, par-
tially below the detection limit of the used Bradford assay. In total,
around 11% of the adsorbed lysozyme were released after 7 days.
The specific activity of the released lysozyme was calculated for
the time point 2 h. For the other chosen time points (24 h and
168 h) the measured released concentration was below the detec-
tion limit and therefore a specific activity could not be calculated.
Nevertheless, an activity Avol of 156,497 ± 103 U/ml and therefore
a specific activity Aspec of 118,426 ± 9 U/mg was determined for
the time point 2 h. The specific activity of the used lysozyme batch
was 129,103 U/mg according to the manufacturer, so nearly 92% of
the initial activity remained after the whole loading, freeze-drying
and release process.
The protein release from plotted scaffolds was studied for cSr-
gCa composites containing 4, 8 and 16% of protein-laden gCa
(loaded similarly as before). Fig. 6A-B show that a higher amount
of protein-laden MBG in the composite led to a significantly higher
release of lysozyme. Within the first 6 h, a burst release for all
composites was observed and for up to 7 days released lysozyme
was detectable. The release after this initial phase depended sig-
nificantly on the MBG amount: For cSr-gCa(4)[Lyz] no released
lysozyme was observed anymore, while for the composite cSr-
gCa(8)[Lyz] only on day 28 released lysozyme was measurable. The
composite cSr-gCa(16) showed an ongoing release between day 14
and day 28. In contrast to the pure MBG, the overall release of
lysozyme was visibly decreased; after 49 days even the scaffolds
with the highest amount of lysozyme-laden MBG just released 1.1%
of the theoretically incorporated protein. As shown in Fig. 6C, the
activity of the released lysozyme decreased for all composites over
time and reached a similar level regardless of the incorporated
amount of MBG. The specific activity (Fig. 6D) was again only cal-

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R.F. Richter, T. Ahlfeld, M. Gelinsky et al. Acta Biomaterialia 156 (2023) 146–157

Fig. 7. Released amount of VEGF from pure MBG (gCa) samples after 2 and 24 h for different VEGF loading concentrations (A); Activity of released VEGF from MBG samples
determined by an endothelial cell proliferation assay – proliferation factor compared to the initial cell number of 2 × 10³ (B); (n = 3, mean ± standard deviation, ∗ ∗ p ≤ 0.01,
∗∗∗∗
p ≤ 0.0 0 01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Released amount of VEGF from plotted composite scaffolds made of Sr-modified cement (cSr) and 16 wt% VEGF-laden MBG (gCa) with an initial VEGF loading
concentration of 10 0 0 ng/ml over a period of 21 days (A); Accumulated release of VEGF from the plotted composite scaffolds over a period of 21 days (B); (n = 3, mean ±
standard deviation).

culated for time points with a released concentration above the Table 6
Accumulated ion concentrations released from cSr composites with different
detection limit. No clear trend was visible for the specific activity,
additions (4, 8, 16 wt%) of protein free and protein-laden gCa after 28 days
neither over time, nor between the different composites. Compared incubation in PBS (n = 6, mean ± standard deviation).
to the pure MBG, the specific activity decreased, but up to 57% of
Accumulated ion concentration after 28 days [mmol/l]
the initial activity was maintained.
Label
As a second protein, the growth factor VEGF was chosen and Calcium Strontium Silicate
the release and activity was studied for pure MBG samples loaded cSr-gCa(4) 1.4 ± 0.1 2.4 ± 0.1 6.5 ± 0.2
with two different initial VEGF concentrations. As shown in Fig. 7A, cSr-gCa(4)[Lyz] 1.5 ± 0.2 3.0 ± 0.1 6.2 ± 0.2
samples with a higher loading concentration also released more cSr-gCa(8) 1.7 ± 0.1 2.9 ± 0.2 7.4 ± 0.4
VEGF after 2 and 24 h. Maximum 6% in total of the theoretically cSr-gCa(8)[Lyz] 1.6 ± 0.1 2.9 ± 0.1 7.1 ± 0.3
cSr-gCa(16) 1.8 ± 0.1 2.8 ± 0.1 9.2 ± 0.3
adsorbed protein was released after 24 h. The activity of the re- cSr-gCa(16)[Lyz] 2.0 ± 0.1 2.8 ± 0.1 8.3 ± 0.2
leased VEGF was determined by an endothelial cell proliferation
assay. As shown in Fig. 7B, a higher amount of released VEGF led to
a stronger increase of proliferation. For both time points, there was lease in the first days, an ongoing release over a period of 21 days
a significant difference between the two VEGF containing groups was detected (Fig. 8A), which resulted in a total amount of released
and the two control groups (plain gCa and release medium (RM)), VEGF of 875 pg or 0.13% of the theoretically incorporated protein
as well as between the two VEGF groups themselves. Although (Fig. 8B).
not significant, interestingly, there was also a visible difference
between the RM and the VEGF-free MBG group, while the latter 3.5. Influence of protein loading on ion release
showed no change of the initial cell number (proliferation factor
approximately 1), the release medium alone resulted in a decrease The ion release for protein-laden scaffolds was measured for
in cell number, indicating a positive effect of the gCa release prod- plotted cSr-gCa composites with different amounts (4, 8, 16 wt%)
ucts onto the survival of HDMEC in absence of VEGF. of lysozyme (cstart = 100 mg/ml) containing MBG added to the
The VEGF release from plotted composite scaffolds was studied strontium modified cement and compared to protein free scaf-
for cSr-gCa(16) containing MBG loaded with an initial VEGF con- folds of the same composition. In Table 6, the accumulated ion
centration of 10 0 0 ng/ml. While the scaffolds showed a higher re- concentrations for calcium, strontium and silicate after 28 days of

153
R.F. Richter, T. Ahlfeld, M. Gelinsky et al.
incubation in PBS are shown. For the calcium release, there was
no significant impact of the lysozyme observed. Interestingly, cSr-
gCa(4)[Lyz] showed a significantly higher (p ≤ 0.0 0 01) strontium
release than the protein free composite. For cSr-gCa(8) and cSr-
gCa(16) there was no such difference detected. For silicate, all pro-
tein containing composites released slightly less compared to the
protein free composites which was significant (p ≤ 0.0 0 01) for the
cSr-gCa(16) composite.
4. Discussion
CaP materials are widely used bone substitutes in orthopaedic
and maxillofacial surgery and for the treatment of large osseous
cavities, injectable CPC formulations were developed that can be
applied easily in clinical applications [27,28]. As shown before, a
non-aqueous cl-based CPC keeps this good injectability but does
also possess a long-term storability and maintains its ready-to-
use character until contact with aqueous media [11,12]. Therefore,
this CPC is a highly suitable material for 3D plotting. Recently, we
showed that this CPC can be combined with mesoporous bioactive
glass, another material widely used to treat bone defects, and com-
posites applicable for 3D plotting could be achieved [23]. The pre-
vious study showed a linear relation between the added amount
of MBG and the additionally needed amount of carrier-liquid (cl).
In this study, we increased the maximum amount of MBG added
from 10 wt% to 16 wt% but needed more cl than expected to ob-
tain plottable composites (700 μl instead of 566 μl). This indicates
that for higher amounts of MBG such a linear relation is probably
not valid anymore. Nevertheless, this did not impair the plottability
and we were still able to fabricate scaffolds with this high amount
of MBG, suggesting that composites with an even larger MBG con-
tent might still be processable. Analysis of rheological parameters
showed that all pastes regardless of the amount of MBG added still
had a shear thinning behaviour (see Supplementary Figure 7), sup-
porting this idea.
Due to their high specific surface area, MBG degrade much
faster in a physiological environment than hydroxyapatite-forming
CPC. The incorporation of the glass particles into the cement
should not only increase the initial surface area and therefore
the initial porosity, but also benefits the degradation of the ce-
ment matrix by increasing its surface area after the glass degra-
dation. The measured decrease in mass for the single components
and different composites support this idea and the initial poros-
ity was also significantly increased for composites with an addition
of at least 8 wt% MBG regardless of cement or glass modification.
Interestingly, the composites changed their porosity only slightly
over time compared to the pure cement scaffolds. For comparison,
Schumacher et al. showed a reverse effect, while the incorporation
of up to 10 wt% MBG did not change the initial porosity, a signifi-
cantly higher porosity was observed after a degradation of 21 days
[21]. In contrast to the present study, bulk scaffolds based on a
powder-liquid formulation of the CPC and water as a degradation
medium were used, indicating that scaffold geometry and chem-
ical environment have a significant influence on the degradation
behaviour. For selected composites based on cSr, a phosphate free,
HEPES-based buffer system was used to examine the influence of
phosphates in the degradation medium and indeed a higher de-
crease in mass and an increase of porosity over time was observed
for all materials in the absence of phosphates. Probably, the release
of Ca2+ /Sr2+ from the composites and the phosphates from the PBS
formed precipitation products that could impair a further increase
in porosity. No occurring precipitation and a considerable increase
in porosity over time would also explain the higher decrease in
mass for the samples stored in HEPES buffer.
During degradation, the cement and MBG release ions which
impact bone metabolism, like silicate, calcium and phosphates
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Acta Biomaterialia 156 (2023) 146–157
[25,29]. By modification of the cement precursors or the bioac-
tive glass composition, also other ions can be released, amongst
many others strontium, magnesium, copper or zinc [24,30–34]. For
the treatment of bone defects, strontium is of particular interest,
because strontium ions were demonstrated to promote osteogenic
differentiation but also inhibit osteoclastic resorption [35–40]. Pre-
vious studies show that a strontium modification of CPC is a suit-
able method to obtain a Sr2+ release over a long time period while
reaching a therapeutically effective level [24,30]. We demonstrated
that by mixing strontium free and strontium modified CPC the re-
lease of Sr2+ is tailorable, can be improved by the incorporation of
MBG particles and even further increased by the incorporation of
strontium modified MBG. In contrast, Wagner et al. observed a de-
crease in Sr2+ release for the same material system, a combination
of strontium modified CPC and 10 wt% calcium modified MBG [25],
while we already found a significant increase for an addition of 8
wt% MBG. The addition of Sr-free MBG reduces the amount of cSr
and therefore the total amount of Sr2+ that can be released from
the cement matrix. At the same time the MBG counteracts this, as
the faster degradation of the glass leads to an increased surface
area of the cement and consequently to an improved degradation
of the cement and higher Sr2+ release. As bulk scaffolds were used
in the other study, this shows again the critical influence of the
scaffold geometry and the macroporous design. We could show
in a previous study, that due to the strongly increased surface-
to-volume ratio 3D plotted macroporous scaffolds release signifi-
cantly more ions compared to a bulk scaffold of similar mass [23].
This shows the great advantage of combining CPC-MBG compos-
ites with additive manufacturing, because it not only allows you
to create macroporous scaffolds, but also adjust the porosity and
therefore tailor the release kinetics to the desired level.
The possibility to functionalise the MBG as a carrier system for
proteins, growth factors or drugs is another key benefit of this ma-
terial and mesoporous silica based materials have shown in sev-
eral studies a great variety of applications for drug delivery [41].
Different groups used lysozyme as a model protein similar to our
present study [42,43], but there are also successful approaches
for bovine serum albumin [44], antibiotics like Gentamicin [45] or
drugs specific for the treatment of bone diseases like zoledronate
against osteoporosis [46]. In comparison, we observed similar load-
ing properties for the MBG used in combination with lysozyme as
Luo et al. for their MBG microspheres [42] and Henry et al. for
their silica nanofibers [43].
However, an inherent disadvantage of such MBG systems is that
due to their manufacturing process they are often present as gran-
ules, powders or nanoparticles and therefore lack options for scaf-
fold production. There are approaches to use binder materials like
PVA to form plottable MBG pastes [47] or the usage polyurethane
sponges as templates [48], but in these cases, the binder will either
remain in the final scaffold or the advantages of additive manu-
facturing are lost. Another possibility is to incorporate MBG into
a second material to overcome this drawback. For tissue engineer-
ing of bone tissue calcium phosphates are of great interest, espe-
cially calcium phosphate cements due to their good applicability.
Schumacher et al. even showed the combination of protein-laden
MBG and a CPC, but as this composite was based on a powder-
liquid cement system, it is not suitable for 3D plotting as the set-
ting reaction of the cement is too fast. There is again the possi-
bility to combine CPC and MBG powder with a binder like a PVA
solution to get a printable paste [20], but the processing time of
these pastes is limited as the PVA binder is an aqueous solution
and will nevertheless start the cement setting reaction [19]. The
possibility to fabricate large constructs in clinically relevant dimen-
sions might be challenging in this case. In addition, Li et al. added
the protein solution to the already prepared scaffolds. Vater et al.
[49] showed a similar dip-coating approach for cement-only sam-
R.F. Richter, T. Ahlfeld, M. Gelinsky et al.
Fig. 9. Overview of the hierarchically structured materials, investigated in the present study, and the influence parameters for degradation and release properties on each
feature level. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ples and the growth factors VEGF and BMP-2 (bone morphogenetic
protein 2), but both approaches lack the possibility of a controlled
spatial distribution of the protein. We herein showed the develop-
ment of a protocol that allows the incorporation of protein-laden
MBG into a 3D plottable CPC paste without impairing the plotta-
bility and mostly maintaining the biological activity of the protein
after the release from pure MBG and CPC-MBG scaffolds. Further-
more, the incorporation of protein did not additionally change the
ion release characteristics of the CPC-MBG composites. Such hi-
erarchically structured and highly flexible composites allow sub-
stantial control of degradation and release properties (Fig. 9). In
combination with multichannel plotting, it will be possible to pro-
duce CPC-MBG scaffolds releasing multiple biological factors in a
defined spatiotemporal manner, similarly as shown before for CPC-
hydrogel composite scaffolds [13].
Besides lysozyme, the developed protocol was tested with the
vascular endothelial growth factor (VEGF). VEGF chemotactically
attracts endothelial cells inducing migration and proliferation and
155
Acta Biomaterialia 156 (2023) 146–157
is therefore a key regulator in angiogenesis as it induces the for-
mation of pre-vascular, tubular structures [50,51]. The usage of
a growth factor like VEGF in a biomaterial system would enable
the possibility of in situ tissue engineering as the release into a
potential bone defect area could help neovascularisation and fur-
ther improve defect healing [52]. Similar to lysozyme, a success-
ful release from MBG and CPC-MBG samples was observed and
the VEGF maintained its biological activity. Interestingly, VEGF-free
MBG samples showed better results than the MBG-free control
group. Although not significant, it is possible that the ionic degra-
dation products of the MBG itself had a beneficial effect on HDMEC
proliferation. It is therefore also possible that the MBG additionally
improves the effect of the released VEGF. A similar effect was ob-
served for an alginate-gellan gum based hydrogel [13] and for an
alginate-based hydrogel, where a fourfold increase in activity was
observed [53]. The interaction between growth factors and scaffold
material and release products should therefore be subject of fur-
ther investigations.
R.F. Richter, T. Ahlfeld, M. Gelinsky et al.
5. Conclusion
Herein, we investigated 3D plottable composites of self-setting
calcium phosphate cements and mesoporous bioactive glasses for
the treatment of bone defects. The incorporation of MBG showed
beneficial effects regarding scaffold porosity and ion release. Ad-
ditionally, we successfully developed a protocol that allows the
functionalisation of the MBG particles as a carrier system for bi-
ological factors and the anhydrous combination with 3D plottable
CPC pastes without impairing the plottability. Released proteins,
lysozyme or VEGF, maintained their biological activity. Therefore,
the developed CPC-MBG system shows its high flexibility in many
ways, basically acting like a toolbox. The scaffolds can be additively
manufactured in a defect and thus patient specific way and the
release of therapeutic agents can be tailored to patient individual
doses.
Funding
This work was funded by German Research Foundation (DFG) as
part of the Collaborative Research Centre Transregio 79 (SFB/TR79,
subproject M2) and the project "New generation of 3D scaf-
folds for patient-specific therapies in orthopaedic applications" (GE
1133/24–1).
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgments
The authors thank Ortrud Zieschang for excellent technical as-
sistance.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.actbio.2022.01.034.
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