materials

Article
Rheological and Mechanical Properties of
Thermoresponsive Methylcellulose/Calcium
Phosphate-Based Injectable Bone Substitutes
Öznur Demir Oğuz ID
and Duygu Ege * ID

Institute of Biomedical Engineering, Boğaziçi University, Rasathane St., Kandilli, 34684 İstanbul, Turkey;
oznur.demir@boun.edu.tr
* Correspondence: duygu.ege@boun.edu.tr; Tel.: +90-216-516-3438




Received: 26 February 2018; Accepted: 27 March 2018; Published: 14 April 2018


Abstract: In this study, a novel injectable bone substitute (IBS) was prepared by incorporating a
bioceramic powder in a polymeric solution comprising of methylcellulose (MC), gelatin and citric
acid. Methylcellulose was utilized as the polymeric matrix due to its thermoresponsive properties
and biocompatibility. 2.5 wt % gelatin and 3 wt % citric acid were added to the MC to adjust the
rheological properties of the prepared IBS. Then, 0, 20, 30 and 50 wt % of the bioceramic component
comprising tetracalcium phosphate/hydroxyapatite (TTCP/HA), dicalcium phosphate dehydrate
(DCPD) and calcium sulfate dehydrate (CSD) were added into the prepared polymeric component.
The prepared IBS samples had a chewing gum-like consistency. IBS samples were investigated
in terms of their chemical structure, rheological characteristics, and mechanical properties. After
that, in vitro degradation studies were carried out by measurement of pH and % remaining weight.
Viscoelastic characteristics of the samples indicated that all of the prepared IBS were injectable
and they hardened at approximately 37 ◦ C. Moreover, with increasing wt % of the bioceramic
component, the degradation rate of the samples significantly reduced and the mechanical properties
were improved. Therefore, the experimental results indicated that the P50 mix may be a promising
candidates to fill bone defects and assist bone recovery for non-load bearing applications.

Keywords: methylcellulose; injectable bone substitutes; calcium phosphate cement; calcium sulfate;
citric acid; gelatin; bone; rheological studies; injectability; mechanical properties

1. Introduction
Since the late 1980s, there is ongoing research on the development of injectable bone substitutes
(IBS) [1–3]. The most commonly studied IBS’s in dentistry and orthopedic applications are calcium
phosphate cements (CPC) due to their excellent physical, mechanical and biological properties [3–5].
Since CPCs are biocompatible, bioactive, biodegradable and osteoconductive, there is tremendous
research on their further development. CPCs are injectable, and they harden in vivo, after taking
the shape of the defect site. Two of the most commonly used bioceramics for the production of
CPCs are dicalcium phosphate dihydrate (DCPD, CaHPO4 .2H2 O) and tetra-calcium phosphate (TTCP,
Ca4 (PO4 )2 O) [4,6,7]. After a mixture of TTCP and DCPD is injected into the defect site, it transforms
into hydroxyapatite (HA) [6–9]. Pure CPC bone substitutes have many drawbacks; such as the
possibility of collapse under physiological conditions, poor degradability, as well as weak torsional
and bending strength [10–12]. The addition of calcium sulfates (CSD) to CPCs may overcome some of
these drawbacks, including the improvement of their injectability, mechanical strength, degradability,
and osteointegration. CSD form after mixing α-calcium sulfate hemihydrate with distilled water. The
addition of CSD into CPC leads to improvement of plasticity and the compression strength of the
CPC [11,13,14].

Materials 2018, 11, 604; doi:10.3390/ma11040604 www.mdpi.com/journal/materials

Materials 2018, 11, 604 2 of 17

To adjust the rheological properties, including injectability, setting temperature, mechanical

properties of IBS, CPC and CSD mixture may be incorporated into a polymeric matrix. The presence
of a polymeric matrix may also assist the permeation of body fluids into bone substitutes; therefore
promoting three-dimensional cell migration, cell growth and ultimately ossification [4,15]. Cellulose
is a promising polymer for this purpose; however, it is not water-soluble due to the presence
of intra-molecular hydrogen bonding; which restricts its range of biomedical applications [16–18].
Therefore, hydrophilic and water-soluble derivatives of cellulose have emerged [19]. Methylcellulose
(MC), which is one of the cellulose ether derivatives, has been extensively studied for biomedical
applications. It also shows the potential for production of IBS due to its thermoresponsive properties [16].
Research shows that MC enhances injectability, mechanical properties, and osteoconductivity of
CPC-based IBS [20–23]. In their study, Liu et al. used 8, 10 and 12 wt % of MC to prepare injectable,
biocompatible and biodegradable hydrogels. Overall, 10 wt % MC led to the most suitable rheological
properties for biomedical applications including viscosity, setting time and storage modulus [24].
In addition to that, like MC, gelatin has been added to CPC pastes; which does not only
mimics the organic matrix of bone, but also contributes to the hardening of CPC and improves
the workability of cement pastes [25,26]. Moreover, the addition of gelatin into MC enables gelation
at the physiological temperature and also provides improved cell-surface interactions due to the
presence of its arginine-glycine-aspartate or RGD group [26–28]. Nishinari et al. [29] observed that the
interaction between the non-substituted hydroxyl group in methylcellulose and the carboxyl group in
gelatin reduces the gelling temperature of MC. [9,30]. In many studies, 2–3 wt % gelatin was added in
hydrogels to improve their biocompatibility. Additionally, gelatin addition improved the mechanical
properties of hydrogels [3,31,32].
Experimental studies show that the addition of different salts to MC solution also reduces
the gelling temperature to the physiological temperature [16,19,33]. Citric acid is a cost-effective,
non-toxic additive which optimizes the physical properties of IBS; such as degree of swelling, setting
time, viscosity and mechanical properties [9,30]. The carboxylate group in citric acid reacts with the
hydroxyl groups of MC and the amino groups of gelatin via esterification. Citric acid acts as a liquefier
and therefore also enhances the injectability and workability of CPC pastes [34,35]. It also improves
the strength of CPC pastes due to its high salting-out effect according to the Hofmeister series [9].
In previous studies, citric acid was incorporated in calcium phosphate cements up to 3 wt%. With
an increase of wt % of citric acid, setting time and injectability decreased which was described as
acceptable for clinical applications [36].
In the current study, IBS was prepared by mixing MC, gelatin, citric acid, and different wt % of
bioceramic comprising of CPC and CSD. As previous studies indicated improved physical properties
for use of 10 wt % MC, 3 wt % citric acid and 2.5 wt % gelatin in prepared scaffolds; in this study,
these values were kept constant. With this novel combination of polymers and CPC, an ideal IBS
for orthopedic applications can be developed which stimulates new bone formation. 20, 30, and
50 wt % of CPC/CSD–based bioceramic powder was added in the polymeric matrix to produce IBS.
The chemical structure of the samples was investigated using X-ray Diffraction (XRD). The physical
handling and mechanical properties of the IBS samples were analyzed with rheological studies,
compressive strength measurements and injectability tests. After that, the in vitro degradation studies
were carried out. Overall, the experimental studies suggest that MC/bioceramic powder-based IBS
were promising in terms of their rheological, mechanical and degradation properties for future bone
treatment applications.

2. Materials and Methods

2.1. Materials
Methylcellulose (MC, viscosity 15 cps, Mw 14 kDa, DS 1.5–1.9), gelatin (from bovine skin, gel
strength ~225 g Bloom, Type B), sodium citrate tribasic dihydrate (SC, C6 H5 Na3 O7 Mw 294.10 g/mol,
Materials 2018, 11, 604 3 of 17

mp: >300 ◦ C (lit.), pH 7.0–9.0 at 25 ◦ C), calcium hydrogen phosphate dihydrate (DCPD, CaHPO4 .2H2 O,
Mw 172.09 g/mol, d:2.31 g/mL), monetite (CaHPO4 , Mw 136.06 g/mol), calcium carbonate (CaCO3 ,
Mw 100.09 g/mol, d: 2.93 g, ≤30 µm particle size) and phosphate buffer saline (PBS, tablets, pH
7.4 at 25 ◦ C) were purchased from Sigma Aldrich (Sigma Aldrich, Taufkirchen, Germany). Calcium
sulfate dihydrate (CSD, CaSO4 ·2H2 O, Mw 172.17 g/mol) was purchased from Merck (Merck KGaA,
Darmstadt, Germany).

2.2. Preparation of Injectable Bone Substitutes (IBS) Samples

2.2.1. Preparation of the Polymeric Solution

Methylcellulose (MC) solution was prepared by dissolving 6 g of MC powder in distilled water at
90 ◦C until MC’s complete dissolution. The prepared solution was stored at 4 ◦ C overnight to obtain a
clear MC solution [33]. 1.875 g of gelatin was dissolved in 12.5 mL of distilled water at 50 ◦ C and was
allowed to cool down before use [24]. 2.25 g of sodium citrate dihydrates (SC) solution was prepared
by dissolving SC salts in 12.5 mL distilled water at room temperature [19]. Then all solutions were
blended to a final concentration of 8.0, 2.5, and 3.0 wt % of MC, gelatin, and SC, respectively.

2.2.2. Preparation of the Bioceramic Powder Mixture

The bioceramic component of IBS samples consist of TTCP/HA-based powder, DCPD, and CSD.
Analytical grade DCPD and CSD were used without any further purification. TTCP/HA-based powder
was synthesized by heating an equi-molar mixture of monetite (CaHPO4 ) and calcium carbonate
(CaCO3 ) at 1500 ◦ C for 6 h, using a 5 ◦ C/min heating rate and 10 ◦ C/min cooling rate [37]. CPC mixture
was prepared by mixing an equi-molar mixture of DCPD and TTCP/HA-based powder and then this
CPC mixture was added to the CSD in a 4 to 1 mass ratio [38].
After this, different wt % of the bioceramic powder phase was added to the polymeric component.
Table 1 shows the compositions of prepared IBS samples.

Table 1. Composition of the injectable bone substitutes (IBS) samples.

Abbreviation Gelatin (wt %) SC (wt %) MC (wt %) Bioceramic Powder Component (wt %)

P0 2.5 3 8 0
P20 2.5 3 8 20
P30 2.5 3 8 30
P50 2.5 3 8 50

2.3. Characterization of IBS Samples

2.3.1. XRD Analysis

XRD (Rigaku D/MAX-Ultima+/PC, Austin, TX, USA) analysis was performed to examine the
synthesized powder and lyophilized IBS samples at 40 kV and 30 mA with a step size of 0.01◦ between
20◦ and 40◦ in a fixed time mode at Bogaziçi University, Istanbul, Turkey [33].

2.3.2. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

Functional groups of the synthesized powder were detected by using a Perkin Elmer FTIR
spectrometer (Perkin Elmer, Waltham, MA, USA) from 4000 to 400 cm−1 using the KBr pellet technique
at Yildiz Technical University, Istanbul, Turkey [39].

2.3.3. Injectability Measurements

Injectability of the samples was qualitatively evaluated by extruding the IBS samples through
a disposable syringe, using an 18-gauge needle, in PBS at 37 ◦ C. Each syringe was filled with
Materials 2018, 11, 604 4 of 17

approximately 2 g of IBS, which was then extruded from the syringe manually at a constant
speed [24,39].

2.3.4. SEM Analysis

The morphology and the internal porous structure of the lyophilized IBS samples were observed
by using Scanning Electron Microscopy (SEM) (Zeiss, Evo LS10, Oberkochen, Germany) with 10 kV
accelerating voltage at Yildiz Technical University, Istanbul, Turkey [30]. Samples were coated with
gold-palladium before the experiment.

2.3.5. Rheological Measurements

Rheological measurements of IBS samples were performed by using a stress-controlled rheometer
with a parallel plate geometry (diameter: 15 mm) (Anton Paar, MCR302, Graz, Austria) at Bogaziçi
University, Istanbul, Turkey. The mechanical properties of the samples were measured at 0.1% strain
and 10 rad/s frequency within the linear viscoelastic region of IBS. The oscillation amplitude and
frequency sweep were carried out at 37 ◦ C. A temperature sweep was performed from 15 to 45 ◦ C at a
heating rate of 2 ◦ C/min in order to determine the gelation and setting temperatures. A time sweep
was carried out at 37 ◦ C to investigate the gelation kinetics. Finally, the shear thinning properties of
IBS samples were analyzed both at 25 and 37 ◦ C [24,40].

2.3.6. Compressive Strength Measurements

IBS samples were molded into columns of 7.0 mm of diameter and 10.0 mm length. The
compressive strength of the samples was measured using a Geratech SH-500 (Geratech, Taiwan)
and SH-20 testing device (Geratech, Taiwan) at Bogaziçi University, Istanbul, Turkey. The compressive
strength of IBS samples was taken at 15% strain. Measurements were taken on days 1,3, 5 and 7 and 14
in an atmosphere of 100% humidity at 37 ◦ C (n = 5) [41].

2.3.7. pH Changes
IBS samples were molded into discs, and after setting of the cement phase, they were immersed
in PBS solution at 37 ◦ C. The pH values of the PBS solution were measured at different time intervals
including 5,10, 20, 30, 60, and 120 min, 1,3, 5, 7, 14, 21 and 28 days (n = 3) [41].

2.3.8. In Vitro Degradation

The degradation behavior of the IBS samples was measured in PBS at 37 ◦ C. IBS samples were
immersed in 12 well plates. At pre-determined times, the IBS samples were lyophilized and weighed.
The remnant dry weight was calculated using Equation (1) where W0 is the initial weight of the dry
IBS samples and Wt is the dry weight of the IBS samples after t days of incubation [24].

Wt
% = × 100 [%] (1)
W0

3. Results and Discussion

3.1. Analysis of the Synthesized Powder

The TTCP/HA-based powder was synthesized as described in Section 2.2.2. Figure 1 shows the
XRD analysis of the synthesized powder. The relevant Miller Indices of TTCP, HA and monetite are
also presented.
Figure 1 shows that TTCP and hydroxyapatite peaks were detected since the powder was
furnace-cooled [9,37,42,43]. A trace amount of monetite and calcium oxide (CaO) were also found
from the XRD analysis. The matched peaks with JCPDS file No. 25-1137, No. 09-0432 and No. 09-0080
is given in Figure S1.
Materials 2018, 11, 604 5 of 17

Figure 1. XRD (X-ray Diffraction) pattern of synthesized (tetracalcium phosphate/hydroxyapatite)

TTCP/HA-based powder.

Figure 2 shows the FTIR spectrum of the synthesized powder.

Figure 2. Fourier transform infrared (FTIR) spectrum of TTCP/HA-based powder by using KBr
pellet method.

In FTIR spectrum, absorption peaks were found at 452, 471, 502, 567, 600, 628, 958, 987, 1044,
1091, 1625, 1922, 2001, 2077, 3435, 3570 and 3643 cm−1 . As labelled in Figure 2, PO4 3− bands of
TTCP were detected in the spectrum which were found similar with that of literature [43–45]. OH−
Materials 2018, 11, 604 6 of 17

stretching bands located at 628 and 3570 cm−1 indicated the presence of HA [42,46]. Absorption
bands between 3000–3600 cm−1 indicated H2 O adsorbed. The peaks located at 1625, 1922, 2000 and
2077 cm−1 indicated the presence of carbonate content. These peaks were possibly found due to carbon
dioxide absorption from the atmosphere [47]. Therefore, FTIR results supported the results observed
from XRD spectrum which indicated synthesis of TTCP/HA-based powder [37,44].

3.2. Injectability of IBS Samples

Figure 3 shows that the mixture of bioceramic and polymeric components have a chewing
gum-like consistency after mixing.

Figure 3. Chewing gum-like consistency of P50 samples after mixing of bioceramic and
polymeric components.

Since the IBS samples had a chewing gum-like consistency, IBS samples can be molded into the
desired shape of the complex bone defects. This consistency was achieved as a result of the presence
of the liquid phase [12]. Figure S2 shows that all of the IBS samples possess cohesive stability and
moldability. According to the extrusion videos, P30 and P50 samples had a higher stability than P20,
as P20 pastes had a tendency to disintegrate during the extrusion process. Figure S3 shows that all IBS
samples had high degree of injectability. Higher wt % of bioceramic components were also introduced
into the polymeric component; however, these samples could not be extruded through 18-gauge.
Therefore, the maximum wt % of bioceramic component was set at 50%.

3.3. Morphology of the IBS Samples

Figure 4 shows the morphologies of lyophilized hydrogels studied by SEM.
Figure 4 shows that all of the IBS samples had highly porous microstructure. When the wt %
of the bioceramic component increased, the pore size decreased. P0 revealed that scaffolds had an
interconnected, porous structure. SEM shows that bioceramics were well-adhered on the polymeric
phase. Moreover, the bioceramic component was found to be homogenously distributed in the
polymeric component [42,48] .

3.4. Rheological Measurements

Rheological measurements of IBS samples containing different wt % of the bioceramic component
were evaluated. Figure 5 shows the amplitude sweep measurement of IBS samples.
Materials 2018, 11, 604 7 of 17

Figure 4. SEM images of (a) P0, (b) P20, (c) P30, (d) P50 IBS samples at a magnification of 500×.

Figure 5. Amplitude-dependent variation of G’ and G” changes of P0, P20, P30 and P50 IBS samples at
a frequency of 10 rad/s at 37 ◦ C (results expressed as mean ± standard error, n = 3).

Compared with P0 samples, other IBS samples present a broader linear viscoelastic region
at 37 ◦ C; as the strain required to break the network structure of IBS samples slightly increased.
With the increase of wt % of the bioceramic component, the % strain required to break the network
structure of IBS decreased. Therefore, in order to maintain the structural integrity of IBS samples, 0.1%
strain was applied for frequency, temperature, and time sweep measurements. Figure 6 shows the
frequency-dependent rheological results performed in the linear viscoelastic region under 0.1% strain.
Materials 2018, 11, 604 8 of 17

Figure 6. Frequency-dependent variation of G0 and G” changes of P0, P20, P30 and P50 IBS samples at
a 0.1% strain at 37 ◦ C (results expressed as mean ± standard error, n = 3).

In the measured frequency range, all of the IBS samples had a higher storage modulus than the
loss modulus, confirming the gelation and stabilization of their structure after setting at 37 ◦ C [24,40].
The setting takes place in two stages. In the first stage, hardening occurs either by the hydration of the
salts in the powder component or by a chelate reaction between MC and citric acid. At this time, the
polymeric component and bioceramic components also have hydrogen bonds and ionic interactions.
In the second stage of cement setting, the hardening occurs via the transformation of the bioceramic
component to hydroxyapatite [9].
The rheological properties of the IBS samples were evaluated by the oscillatory rheometer as a
function of temperature and time. Temperature and time sweep measurements were taken to examine
the impact of bioceramic powder phase on the gelation and hardening mechanism. Figure 7 shows the
temperature-dependent changes of G0 and G” of IBS samples.

Figure 7. Temperature-dependent variation of G0 and G” of IBS samples at 0.1% strain and 10 rad/s
angular frequency (results expressed as mean ± standard error, n = 3).
Materials 2018, 11, 604 9 of 17

The exponential increase of storage modulus with temperature implies the phase transition of
the samples. The sol-gel transition temperature of the IBS samples shifted to a higher value with the
addition of 30 and 50% of bioceramic mixture. The shifting of gelation temperature might be due
to the change of the intra-molecular and inter-molecular interactions of MC chains [7]. The strong
hydrogen bond between MC chains and CPC causes a change in the temperature sensitivity of the MC
chains [49]. Figure 8 shows the gelation time at 37 ◦ C.

Figure 8. Time-dependent G0 and G” of IBS samples measured at 37 ◦ C, 0.1% strain, and 10 rad/s
frequency (results expressed as mean ± standard error, n = 3).

Similar to the temperature sweep test, the exponential increase of the storage modulus with time
at 37 ◦ C suggests the hardening of IBS samples. The plateau point indicates the curing of the polymeric
chains. The duration of curing decreases effectively with the increase of wt % of the bioceramic
component [50–52].
Figure 9 shows the shear-rate dependent variation of viscosity for IBS samples both at 25 ◦ C and
37 ◦ C.

Figure 9. Shear-rate dependent variation of viscosity for IBS samples measured at (a) 25 ◦ C and
(b) 37 ◦ C, respectively (results expressed as mean ± standard error, n = 3).
Materials 2018, 11, 604 10 of 17

IBS samples were tested for their viscosity variation against change in the shear rate at 25 ◦ C and
37 ◦C to observe whether IBS samples keep their injectability with respect to the increase of wt % of
the bioceramic component. The results reveal that both at 25 ◦ C and 37 ◦ C, all of the samples had
shear thinning properties and P0 had a considerably lower viscosity when compared to P20, P30 and
P50 [24,41]. When the wt % of bioceramic component increased, viscosity also increased. This is due to
the increase of resistance to flow as the number of the cement particles per unit volume increases [49].

3.5. pH Change
Figure 10 shows the pH change of the PBS after incubation of IBS samples at 37 ◦ C.

Figure 10. pH change of the PBS (phosphate buffer saline) that IBS samples incubated at 37 ◦ C. (a) pH
changes of IBS samples until 360 min and (b) pH changes of IBS samples until 21st day (results
expressed as mean ± standard error, n = 3).

The pH profile of biomaterials in PBS is an important indicator of some of their possible biological
responses. The pH response of the samples were measured for 21 days to monitor the pH changes
after setting of IBS samples during the dissolution and re-precipitation. IBS samples had pH values
between 7.89 and 7.39 at the end of day 21. Hence, it can be concluded that the prepared IBS samples
Materials 2018, 11, 604 11 of 17

may not cause any inflammatory reaction under biological conditions due to acidity. The pH of P50
samples was found to be higher than the other IBS samples at the beginning of incubation. P50 samples
had the highest pH value until 60 min after which pH was gradually decreased. For P0, P20 and
P30 IBS samples, the pH value reached a plateau after 3 h until the end of the first day. After day
1, pH values of P0, P20, and P30 started to increase slightly. The increase of pH after the 1st day of
incubation was possibly due to the dissolution and transformation of TTCP into HA as indicated by
Yokoyama et al. [41]. For P30 and P50 samples, the reduction of pH value until day 5 results in PO4 3−
consumption which leads to formation of an apatite-like phase [49].

3.6. In Vitro Degradation

Figure 11 shows % remaining weights of all IBS samples.

Figure 11. Remaining weights of IBS samples in PBS at 37 ◦ C (results expressed as mean ± standard
error, n = 3).

The in vitro degradation behavior of IBS samples was investigated by the measurement of %
weight loss in PBS at 37 ◦ C after the setting of the cement phase. After one week, P0 samples lost 60%
of their weight due to the erosion of MC as Gupta et al. [53] and Tate et al. [54] reported. When the
wt % of bioceramic powder component increased, the weight loss decreased. In vitro degradation
studies were conducted without utilizing any enzymes. Therefore, a faster degradation rate of the IBS
system is expected under in vivo conditions [55–57]. Therefore, the degradation rate of the IBS system
may be further decreased with use of additives.

3.7. XRD Analysis

After the cement phase of the IBS samples was allowed to set, XRD analysis was conducted.
Figure 12 shows the XRD patterns of the IBS samples.
As a result of these analyses, the peaks of powder components, TTCP, HA, DCPD, and CSD were
observed from the XRD spectrum of P20, P30, and P50. The peak intensity of each IBS sample was
increased as the wt % of the bioceramic powder increased. As Thai and Lee [9] concluded, initial XRD
data did not reveal the setting mechanism of P20, P30, and P50 samples. Therefore, the XRD analysis
was also performed after PBS studies to interpret the setting mechanism and mechanical behavior after
incubation for 14 days. Figure 13 shows the XRD analysis after the incubation of IBS samples in PBS at
37 ◦ C.
Materials 2018, 11, 604 12 of 17

Figure 12. XRD analysis results of P20, P30 and P50 samples after setting.

Figure 13. XRD analysis results of P20, P30 and P50 samples incubated in PBS at 37 ◦ C for 14 days.

The deposition of an apatite layer on bone substitutes in the biological environment is an essential
phenomenon as it indicates the osseointegration ability of implants [56]. The XRD results of the
incubated IBS samples showed HA peaks which indicated the formation of an apatite layer on the
samples. CSD peaks were also observed for P50 which is possibly due to a higher wt % of CSD present
in P50 samples.

3.8. Compressive Strength Measurements

Figure 14 shows the compressive strength results of IBS samples in 100% humidity at 37 ◦ C.
Figure 14 shows the compressive strength values of the samples after incubation in 100% humidity
at 37 ◦ C for 14 days. According to the results, P0 and P20 had almost the same compressive
strength values.
Materials 2018, 11, 604 13 of 17

Figure 14. Log compressive strength of IBS samples vs. incubation time in 100% humidity at 37 ◦ C
(results expressed as mean ± standard error, n = 6).

Until day 7, the compressive strength of P30 samples had a similar trend with P0 and P20.
However, interestingly, P30 had a significant increase in compressive strength on day 7. This increase
was correlated with XRD results which indicated the phase transformation of TTCP into HA [41]. P50
samples had a much earlier rise in compressive strength than P30 samples; however, both P30 and P50
reached a plateau on day 7 indicating the completion of their phase transformation into HA. On day 14,
P50 samples had approximately 7 times higher compressive strength when compared to P0, P20, and
P30 samples. The compressive strength of cancellous bone varies between 0.22 to 10.44 MPa [4,58,59].
Compared to human cancellous bone, the compressive strength of IBS was found to be lower. One
way to improve the mechanical properties of IBS is to increase the wt % of the bioceramic component.
In this study, wt % of the bioceramic component could not be increased further due to the inability to
inject IBS with higher wt % of the bioceramic component. The mechanical properties of IBS may be
improved with the addition of carbon-based nanomaterials, such as carbon nanotubes and graphene
oxide [60–62].

4. Conclusions
In this study, novel IBS were prepared by incorporation of different wt % of CaP/CS-based
bioceramic powder into an MC-based solution. The rheological studies revealed that all of the samples
had shear thinning properties; therefore, they had a high degree of injectability. This study showed
that the incorporation of the bioceramic powder into MC-based polymeric matrices may improve the
rheological, mechanical and degradation properties of IBS. In the future, it would be worthwhile to
analyze the biocompatibility and biological responses of the developed IBS. Overall, the prepared IBS
samples are promising candidates for the treatment of bone defects for non-load bearing applications.

Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/11/4/604/s1,

Figure S1: TTCP phase identified by XRD with JCPDS No. 25-1137, No. 09-0432 and No. 09-0080 files, Figure
S2: Extrusion videos of IBS samples through syringe with 18-gauge needle. in PBS at 37 ◦ C (a) extrusion of P0,
(b) extrusion of P20, (c) extrusion of P30 and (d) extrusion of P50., Figure S3: Injectability of P0, P20, P30 and
P50 samples.
Acknowledgments: The support of Boğaziçi University Research fund (Project No.: 12240) and TUBITAK (Project
No.: 117M231) are kindly acknowledged.
Author Contributions: Öznur Demir Oğuz and Duygu Ege conceived and designed the experiments; Öznur
Demir Oğuz performed the experiments; Öznur Demir Oğuz and Duygu Ege analyzed the data and wrote
the paper.
Materials 2018, 11, 604 14 of 17

Conflicts of Interest: The authors declare that they have no conflict of interest.

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