European Polymer Journal 113 (2019) 313–320

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European Polymer Journal

journal homepage: www.elsevier.com/locate/europolj

The influence of hydroxyapatite content on properties of poly(L-lactide)/ T

hydroxyapatite porous scaffolds obtained using thermal induced phase
separation technique
Konrad Szustakiewicza, , Małgorzata Gazińskaa, Bartłomiej Kryszaka, Michał Grzymajłoa,
⁎

Jacek Pigłowskia, Rafał J. Wigluszb, Masami Okamotoc

a
Polymer Engineering and Technology Division, Wrocław University of Science and Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland
b
Polish Academy of Sciences, Institute of Low Temperatures and Structure Research, Okólna 2, 50-950 Wrocław, Poland
c
Advanced Polymeric Nanostructured Materials Engineering, Graduate School of Engineering, Toyota Technological Institute, 2-12-1 Hisakata, Tempaku, Nagoya 468-
8511, Japan

ARTICLE INFO ABSTRACT

Keywords: In the research we obtained porous scaffolds based on poly(L-lactide) and synthetic hydroxyapatite (HA) using
Poly(L-lactide) thermal induced phase separation technique (TIPS) supported by salt leaching process (SL). The obtained series
Hydroxyapatite of composite foams differ in hydroxyapatite content (10, 25, 50, 75, 90 wt% of the HA in PLLA/HA systems). The
Thermal induced phase separation investigated scaffolds porosity ranged from 88 to 98%, density from 0.024 to 0.140 g/cm3. Water contact angle
Scaffolds
measurements indicated more hydrophilic scaffold surface with increasing hydroxyapatite content in the com-
Cell proliferation
posites. Thermogravimetric analysis revealed higher thermal stability of all composites comparing to neat PLLA,
indicated presence of ∼3 wt% residual sodium chloride in the scaffolds. Differential scanning calorimetry
analysis showed nucleation effect of hydroxyapatite and higher crystallinity of PLLA in the composites as
compared to the neat PLLA. The above tests were supplemented with compressive strength measurements, which
showed an increase in the Young’s modulus and compressive stresses values parallelly with the increase of the
filler content. Proliferation tests of MC3T3-E1 mouse calvaria pre-osteoblast cells indicated directly proportional
relationship between hydroxyapatite content and cells proliferation rate.

1. Introduction
Tissue engineering is one of the most important field of bio-
technology that has been developing for over two decades [1,2]. One
promising strategy for bone tissue engineering or damaged bone re-
generation is designing and fabrication of binary hybrid materials out
of biodegradable and bioresorbable polymers, i.e. polyglycolide, poly-
lactides, poly (L-lactic acid) [3], poly (D,L-lactic acid), poly-
caprolactone or their copolymers [4]. As polymer fillers, usually in-
organic materials, i.e. tricalcium phosphate [1,5,6] or hydroxyapatite
[7–12] are used. Both minerals have similar chemical composition to
mammal bone, high calcium and phosphorus content and provide
biocompatibility and osteoconductivity [13,14].
One of the most important hybrids for bone tissue engineering is
poly (L-lactide)/hydroxyapatite (PLLA/HA) composite which is widely
investigated by scientists for its special properties, ie. bioresorbability
(input of PLLA) and osteoconductivity (input of hydroxyapatite) [15].
There are few methods of obtaining above mentioned hybrids, in-
cluding extrusion [16], solvent techniques such as solvent casting and
thermally induced phase separation [17], solid free-form fabrication
and microsphere sintering [8]. The solvent and sintering method fa-
cilitate formation of porous composite that can be used as scaffolds in
tissue engineering [18]. However not only porosity but also pore size,
interconnectivity and pores morphology play an important role by the
meaning of cell migration which is important to promote homogeneity
of the developing bone tissue [19].
Thermal induced phase separation method (TIPS) have been de-
veloped to produce microporous membranes in 1978 [20]. Using this
method, it is possible to obtain polymer foams with porosity over 95%
[21] and pore diameter from ∼1 to 100 µm [20]. In this process, pores
appear in the material by phase separation and solvent removal using
freeze-drying method. Nowadays, many variation of TIPS with different
process parameters [22] or different solvents [23] have been devel-
oped. One of TIPS variation is increasing pores size using additional

⁎
Corresponding author.
E-mail address: konrad.szustakiewicz@pwr.edu.pl (K. Szustakiewicz).

https://doi.org/10.1016/j.eurpolymj.2019.01.073
Received 28 September 2018; Received in revised form 22 January 2019; Accepted 31 January 2019
Available online 01 February 2019
0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
K. Szustakiewicz et al.
porogen, that can be removed from the composite after freeze-drying in
leaching process. Usually sodium chloride [17,19,23–26] or sugar
[27,28] are used as porophores to increase the size of pores in scaffolds.
It was found that by increasing pore size using salt or sugar leaching
technique, it is possible to increase cell proliferation in PLLA scaffolds
[29]. In the literature there are three approaches to hydroxyapatite
content in PLLA/HA composites. The first one is up to 10 wt.% percent
[16], the second is up to 20 wt% [30,31], the third approach is
40–50 wt% [32–35] of HA in the system.
In the research, for the first time, we prepared PLLA/hydroxyapatite
composites with HA content ranging from 10 to 90 wt% using combi-
nation of thermal induced phase separation technique supported by salt
leaching process (TIPS-SL). In the article we present the influence of
hydroxyapatite content in PLLA/HA systems on pores structure, den-
sity, porosity, mechanical, thermal properties and cell proliferation.
2. Experimental
2.1. Materials and composites preparation
Poly (L-lactide) Resomer L210s (PLLA) supplied by Evonik
(Germany) and synthetic calcium hydroxyapatite produced in our lab
according to procedure described earlier [36], were used in our re-
search. The average particle size of the filler was ∼35 nm. NaCl salt for
salt-leaching experiments, having size of 200–550 µm was purchased
from Naikai Salt Industries, Japan. The main substrates used for fabri-
cation of the hydroxyapatite with formula of Ca10(PO4)6(OH)2 using co-
precipitation technique, were calcium nitrate tetrahydrate (Ca
(NO3)2·4H2O (99.98% Alfa Aesar)) and diammonium hydrogen phos-
phate ((NH4)2HPO4 (99.99% Sigma Aldrich)) as well as ammonium
hydroxide (NH4OH (99%) Avantor Poland) for pH control. Briefly, to
prepare hydroxyapatite, the stoichiometric amounts of calcium nitrate
Ca(NO3)2·4H2O was dissolved in MQ-water. Subsequently, ammonium
phosphate (NH4)2HPO4 was added to the mixture resulting in a fast
precipitation of the by-product. The solution pH was adjusted to 10
with NH4OH under constant and vigorous stirring at 90 °C for 4 h. Fi-
nally, by-products were dried for 20 h at 90 °C and thermally treated at
500 °C for 3 h resulting in formation of white, fine-grained powders.
The composites with PLLA/HA wt. ratio of 10/90, 25/75, 50/50,
75/25 and 90/10 were obtained in 1,4-dioxane (mixing for 24 h,
magnetic stirrer), with addition of NaCl, then held in a freezer (24 h).
Frozen samples were transferred to freeze-dryer vessel and submitted to
solvent removal process (at −80 °C and vacuum ∼10–20 Pa for 24 h).
Dried samples were then taken out and put in demineralized water to
solve the salt and remove from the scaffolds. The salt leaching process
was supported by slow mixing on magnetic stirrer, took 24 h and water
was changed 3 times (5 l beaker). After leaching, the scaffolds were
dried using air drier (60 °C, 24 h) and vacuum drier (24 h, 72 h).
Reference scaffold sample of neat PLLA was also obtained. Simplified
scheme of composite foams preparation pathway is shown in Fig. 1.
2.2. Scanning electron microscopy (FE-SEM) and EDAX
Field emission scanning electron microscopy was used to evaluate
morphology and porosity of obtained scaffold. For this purpose, FE-SEM
model SU660 by Hitachi Ltd, Japan was used. The microscope operated
at 10 kV and magnification in the range from 60x to 600x. Before
measurements samples were coated with thick layer of gold using Eiko
IB-3 ion coater.
In order to observe the PLLA/HA scaffolds surface morphology,
especially carbon, oxygen, calcium and phosphorus concentration, a
scanning electron microscope equipped with energy dispersive spec-
troscopy FEI Nova NanoSEM 230 equipped with an energy dispersive
spectrometer (EDAX PegasusXM4) with an acceleration voltage of the
range 3.0–15.0 kV and spot 2.5–3.0 has been used. Before observation,
all samples have been sprayed uniformly with a layer of graphite.
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European Polymer Journal 113 (2019) 313–320
2.3. Density and porosity measurements
Porosity was calculated using following equation:
p = 1 sc
b
where:
Φp – porosity, ρsc – scaffold density obtained from buoyancy method
using Shimadzu AUX 220 equipment, ρb - bulk density of the composite
scaffold (measured using buoyancy method on AUX220, Shimadzu Ltd.
Analytical balance).
2.4. Water contact angle
Water surface tension measurements were conducted using PG-X
contact angle goniometer (Testing Machines, Inc.). At least 10 mea-
surements for each sample were performed and results average value as
well as standard deviation were calculated.
2.5. Differential scanning calorimetry
Differential scanning calorimetry (DSC) measurements were per-
formed using the Mettler Toledo DSC1 system, coupled with Huber TC
100 intracooler. The instrument was calibrated using indium
(Tm = 156.6 °C, ΔHm = 28.45 J/g) and zinc (Tm = 419.7 °C,
ΔHm = 107.00 J/g) standards. Samples (∼3.5 mg) were measured in
the 40 mL aluminum pans under a constant nitrogen purge (60 mL/
min) from 0 °C to 200 °C. After the heating cycle, the samples were
thermally equilibrated at 200 °C for 5 min and cooled down to 0 °C.
Second heating scan was also performed. Both heating and cooling rates
were set to 5 °C/min. The glass transition temperature (Tg) from heating
and cooling scans were taken as the inflection point in the heat flux
curve. Crystallinity degree (Xc) of PLLA was calculated according to
following equation:
Hm Hcc
Xc = 100%
w Hm100%
where Hm – melting enthalpy [J/g], Hcc – cold crystallization en-
thalpy [J/g], w – weight fraction of PLLA in composite foams and
Hm100% – melting enthalpy of 100% crystalline PLLA (93.6 J/g) [37].
Experimental data was processed using the generic STARe computer
program. For data presentation, the DSC profiles were exported to
OriginPro 64 (v. 9.0) as ASCII files.
2.6. Thermogravimetry
Thermogravimetric analysis was performed using TGA/DSC1
Mettler Toledo thermobalance. Samples were heated with the rate of
10 °C/min from 25 °C to 900 °C under 30 mL/min of synthetic air flow.
For data presentation, the TGA curves were exported to OriginPro 64
(v. 9.0) as ASCII files.
2.7. Cell culture
In the experiment, mouse calvaria (MC3T3-E1: ATCC, passage four)
cells were used. Cells were cultured in Alpha Minimum Essential
Medium (α-MEM from Gibco, life technologies, Lot no.1836486) sup-
plemented with 10 vol% of fetal bovine serum (FBS, Life Technologies)
and 1 vol% antibiotic-antimycotic mixture (Nacalai Tesque) in 37 °C,
5% CO2 and relative humidity of 95% until reaching 80–100% con-
fluence.
The survivability of cells seeded on composites surface were in-
vestigated using WST-8 (Dojindo) proliferation assay. For the test
composites were first set on microscope slides using spin-coater. Then,
chamber slides were fixed. All tests were performed according to
K. Szustakiewicz et al.
Fig. 1. The diagram showing the procedure for obtaining PLLA/HA scaffolds in TIPS-SL process.
manufacturer‘s instruction. Briefly, 400 µl of α-MEM containing
MC3T3-E1 cells was dripped into each chamber slide on the surface of
the sterilized scaffolds at the cell density of 2.0 × 104 cells/cm2. The
seeded samples were incubated at 37 °C, 5% CO2 and relative humidity
of 95% for 24 h and 72 h. Cell viability was assessed using colorimetric
test that measures activity of intracellular dehydrogenase which is
proportional to living cells (WST-8, Dojindo). Optical density was
measured using Multscan FC microplate reader (Thermo Fischer
Scientific) at 450 nm and 650 nm for the sample absorbance and sub-
strate background absorbance respectively.
2.8. Statistical analysis
Statistical significance between groups was assessed using post-hoc
Tukey-Kramer test. Comparisons are presented as follows: * denotes for
significance p < 0.05 and ** denotes p < 0.01.
3. Results and discussion
3.1. Physical properties of PLLA/HA porous scaffolds
Internal, porous structure of the samples was observed using FE-
SEM microscopy. It was found that for all the materials (including neat
PLLA and the composites), porous structure was obtained (Fig. 2). The
pictures show porous structure of the composite having two size of
pores: (1) with diameter up to 50 µm that origin from sublimation of
1,4-dioxane and (2) bigger, having diameter up to 400 µm, that re-
mained after salt leaching process. It was also found that for sample
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European Polymer Journal 113 (2019) 313–320
having 90 wt% of hydroxyapatite, pores collapse indicating too low
content of polymer in the system. The sample was excluded from fur-
ther research. Closer look shows different morphology of pore walls –
the greater HA content in the system, the higher roughness of the pore
walls (Fig. 2 A‘–F‘).
In addition, size of HA particles in the composites depends mostly
on its content. For the PLLA/HA 90/10 system, the smallest filler par-
ticles, having few micrometers of diameter are observed. With in-
creasing the content of HA in composites, HA starts to aggregate. In the
case of PLLA/HA 25/75, pore wall is evenly covered by hydroxyapatite.
Increasing HA content to 90 wt% (PLLA/HA 10/90) promotes the for-
mation of larger agglomerates, but the walls are cracked due to in-
adequate amount of polymer in the system (Fig. 2 F‘).
EDAX spectra of the PLLA/HA samples show differences in carbon,
oxygen, calcium and phosphorus content in the composites (Table S1,
Fig. S1). It has been shown that for neat PLLA, only carbon (55.86 wt%)
and oxygen (44.14 wt%) can be observed. When increasing the HA
content, calcium and phosphorus content increase, reaching 44.09 and
17.81 wt% respectively for the PLLA/HA 10/90 sample.
For the neat PLLA foam, porosity is on the level of 98.0% (Table 1,
Fig. 3A). No significant changes are observed for the systems having
from 10 to 50 wt% of the filler. Hydroxyapatite content in the compo-
sites affects the porosity of the scaffolds above 50 wt%. However, the
changes are not significant. For the composite foam with 75 wt% of
hydroxyapatite, the porosity is ∼2% lower comparing to neat PLLA
foam. In the case of composite having 90 wt% of the mineral filler,
porosity lowers down to 88%. For this sample also, standard deviation
is highest because the sample is very fragile and brittle. As can be seen,
K. Szustakiewicz et al. European Polymer Journal 113 (2019) 313–320

Fig. 2. SEM pictures of scaffolds with magnification of 50x: (A) neat PLLA, (B) PLLA/HA 90/10, (C) PLLA/HA 75/25, (D), PLLA/HA 50/50, (E) PLLA/HA 25/75, (F)
PLLA/HA 10/90 and 1000x: (A‘) neat PLLA, (B‘) PLLA/HA 90/10, (C‘) PLLA/HA 75/25, (D‘), PLLA/HA 50/50, (E‘) PLLA/HA 25/75, (F‘) PLLA/HA 10/90.

for the samples obtained in thermal induced phase separation technique
supported by salt leaching process, the most important by means of
porosity is salt leaching process which, in fact, determines very high
degree of porosity of the materials. In this case, the hydroxyapatite
content in the scaffold is less important.
In contrary, density of the scaffolds rises with content of hydro-
xyapatite in the system which is caused by high density of HA com-
paring to PLLA.
Compression tests of foam scaffold obtained in thermal induced
phase separation depend on several factors, ie. type of material, por-
osity, pore size, density of the material, pore morphology (inter-
connectivity) [38–40]. Fig. 3B contains Young‘s modulus and com-
pressive stress at strain of 40%. The values of Young‘s modulus range
from 37.0 to 56.2 kPa and increase with higher content of hydro-
xyapatite. Similar trend has been observed for compressive stress. The
values range from 17.4 to over 28 kPa (at 40% strain) and from around

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K. Szustakiewicz et al. European Polymer Journal 113 (2019) 313–320

Fig. 2. (continued)

Table 1 100 0,20

Density and porosity of neat PLLA and the composites.
A)
98
Sample PLLA/HA mass ratio porosity Φp [%] density ρsc [g·cm−3] 0,16
96

PLLA 100/0 98.0 ± 0.1 0.0238 ± 0.001 94

density [g⋅cm-3]
PLLA/HA 90/10 90/10 98.2 ± 0.2 0.0240 ± 0.002 0,12
porosity [%]

PLLA/HA 75/25 75/25 98.0 ± 0.4 0.0307 ± 0.001 92

PLLA/HA 50/50 50/50 97.6 ± 0.1 0.0431 ± 0.001
PLLA/HA 25/75 25/75 96.0 ± 0.4 0.0861 ± 0.008 90
0,08
PLLA/HA 10/90 10/90 88.1 ± 1.2 0.1406 ± 0.028
88

86 0,04
100 kPa to over 280 kPa at 80% strain (see also Table S5). The highest
values have been observed for PLLA/HA 25/75 confirming the hydro-
xyapatite act as an efficient reinforcement. Standard deviation for the 0 0,00
0 20 40 60 80 100
PLLA/HA 25/75 for both, Young‘s modulus as well as for compressive
hydroxyapatite content [wt.%]
stress is very high, suggesting that 75 wt% of the HA in the scaffold
60
might be inhomogeneous. For the PLLA/HA 10/90 scaffolds, the sam- B) Compressive stress at 40% strain [kPa]
ples were to brittle to conduct the measurements. 60 50
Water contact angle measurements are presented in Fig. 4. The re-
sults revealed decreasing of contact angle with increasing the amount of
Young`s Modulus [kPa]

40
hydroxyapatite in the system. For neat PLLA the contact angle for water
was 73° ± 1.0° and decrease to 55° ± 2.7° for PLLA/HA 25/75. This 40
30
represents an increase in hydrophilicity. The effect is related to hy-
droxyapatite which is known to be hydrophilic material with water
20
contact angle of ∼10° [41,42]. The reason of improving wettability of 20
PLLA/HA systems is fact that pore walls are covered with a layer of
hydroxyapatite. This can be clearly observed on SEM images (Fig. 2). 10

0 0
0 20 40 60 80 100
3.2. Thermal properties of PLLA/HA porous scaffolds
hydroxyapatite content [wt.%]

The first heating DSC curves of PLLA and PLLA/HA composites are
shown in Fig. 5. The estimated thermal parameters are presented in
Table S2. In the all presented DSC curves there are three characteristic
thermal effects: glass transition with Tg at ∼67 °C, cold crystallization
at temperature range of 90° ÷ 160 °C and melting at temperatures over
160 °C with peak temperature (Tm) at 182 °C.
Fig. 3. (A) Porosity-density dependence and (B) Compression of PLLA/HA
scaffolds with different concentration of HA.
The strongest differences between samples concern endotherm of
melting, enthalpy of this effect for PLLA/HA 90/10 composite is higher
than for PLLA and deceases with decreasing content of PLLA in

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K. Szustakiewicz et al. European Polymer Journal 113 (2019) 313–320

0.05W/g T1c T2c

endo
Tg
PLLA

80

Heat flow/sample mass [W/g]

Water contact angle (°)

PLLA/HA 90/10

PLLA/HA 75/25
60
PLLA/HA 50/50

PLLA/HA 25/75

0
PLLA PLLA/HA 90/10 PLLA/HA 75/25 PLLA/HA 50/50 PLLA/HA 25/75 175 150 125 100 75 50 25
ο
Fig. 4. Water contact angle for PLLA/HA scaffolds with different concentration Temperature [ C]
of HA.
Fig. 6. The cooling DSC curves of PLLA and PLLA/HA foams.

Tg Tm
endo

0.1W/g
Tg Tm

endo
0.1W/g

PLLA
Heat flow/sample mass [W/g]

Heat flow/sample mass [W/g]

PLLA

PLLA/HA 90/10 Tα'-α

Tα'
PLLA/HA 75/25
PLLA/HA 90/10
PLLA/HA 50/50
Tα'
PLLA/HA 25/75
PLLA/HA 75/25
PLLA/HA 50/50
25 50 75 100 125 150 175 200 PLLA/HA 25/75
ο
Temperature [ C]
25 50 75 100 125 150 175 200
Fig. 5. The 1st heating DSC curves of PLLA and PLLA/HA foams. ο
Temperature [ C]

composites. Crystallinity degree (Xc) of PLLA in all the composites is
higher than for pure PLLA due to nucleating activity of HA particles,
wherein the highest nucleating effectiveness is in case of 10 wt% HA
content. With increasing HA content Xc of PLLA in composite foams
decreases, but it does not reach a value lower than for neat PLLA foam.
On the cooling DSC curves presented in Fig. 6 melt crystallization
and glass transition are visible. For PLLA foam and composite PLLA/HA
foams with 10, 25 and 50% of HA the exotherm of melt crystallization
is characterized by bimodal peak with distinguishable two maxima
denoted as Tc1 and Tc2 at ∼108 °C and ∼115 °C respectively (see also
Table S3). Melt crystallization enthalpy is lower in composites than for
neat PLLA and decreases with increase of HA content. Both maxima of
melt crystallization exotherm are at slightly higher temperatures than
for neat PLLA foam. There is general tendency of diminishing of higher
temperature maximum of exotherm with increasing HA content. For the
composite with the lowest HA content only one maximum at higher
temperature (119.6 °C) is present. These results can indicate on influ-
ence of HA content on nucleation of melt crystallization of different
PLLA crystal forms. It is known that during melt crystallization at
temperatures higher than 120 °C highly ordered α-crystals are formed,
whereas at temperatures lower than 120 °C crystals are developed much
faster and are referred in the literature as α’ [43]. The α’ form crystals
are not perfect and are considered as conformationally disordered α-
crystals with slightly increased lattice spacing [43].
On the second heating DSC curves (Fig. 7) prior to the melting peak
Fig. 7. The 2nd heating DSC curves of PLLA and PLLA/HA foams.
small exothermic effect is present for PLLA with maximum at 163 °C.
This peak indicates phase transition of crystalline α' phase to thermo-
dynamically favored α form. The area of this exothermic peak decreases
with increasing HA content in composites with 10, 25 and 50% of HA
(Table S4) and an additional small endothermic peak (denoted as Tα’ on
Fig. 7) is observed at slightly lower temperature. Presence of Tα’ peak
and reorganization of α’-crystals into α-crystals was explained by R.
Androsch et al. [44] as reorganization proceeding via melting of α’-
crystals followed by recrystallization. In the composite with the highest
HA content, reorganisation of α’-α is not present. This is in agreement
with disappearance of low temperature contribution of melt crystal-
lization during cooling.
This leads to conclusion that PLLA in neat PLLA foam and in almost
all composite foams after cooling consists of mixture of α’ and α phase
except of the composite foam with the highest HA content. In PLLA/HA
25/75 sample PLLA crystallize on cooling in α form.
3.3. Thermogravimetric analysis
Based on TGA analysis (Fig. 8), hydroxyapatite content in composite
foams was calculated from residue at 550 °C with taking account of
0.58% mass loss of hydroxyapatite and constant content of 3.25 wt% of
NaCl. NaCl content was determined from residue values at 550 °C of
PLLA foams manufactured without salt and reference PLLA foam

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K. Szustakiewicz et al. European Polymer Journal 113 (2019) 313–320

24h
HA 1400
100 72h
**
1200 **
80 * *

proliferation rate [%]

1000
PLLA/HA 25/75
Mass [%]

60 800
PLLA/HA 50/50
600
40

PLLA/HA 75/25 400

20
PLLA/HA 90/10 200
PLLA
0 0
PLLA PLLA/HA 90/10 PLLA/HA 75/25 PLLA/HA 50/50 PLLA/HA 25/75
50 100 150 200 250 300 350 400 450 500 550

Temperature [ C]ο Fig. 9. MC3T3-E1 proliferation on scaffolds (*p < 0.01 = 5, **p < 0.01).

Fig. 8. TGA curves of PLLA and PLLA/HA foams.

4. Conclusions

Table 2 In the article we have presented the influence of hydroxyapatite

Mass loss at 550 °C, calculated HA content and temperature corresponding to content in PLLA/HA system on porous structure, pore walls mor-
3 wt% mass loss from TGA curves.
phology, and supramolecular structure, thermal properties and com-
sample Mass loss at 550 °C [%] HA wt.% content T-5% [°C] pressive strength of the investigated materials. The research revealed
that using TIPS-SL technique, it is possible to obtain PLLA/HA com-
PLLA 95.38 – 312.1
PLLA/HA 90/10 89.23 7.02 320.5
posites with up to 75 wt% of hydroxyapatite with stable porous struc-
PLLA/HA 75/25 73.92 22.47 331.0 ture. For the PLLA/HA material with higher (90 wt%) of HA, pores
PLLA/HA 50/50 50.58 46.17 320.2 collapsed.
PLLA/HA 25/75 34.41 62.65 313.5 Due to incorporation of sodium chloride as an additional porogen in
HA 0.58 100.00 –
salt-leaching process, high porosity materials (96–98%) were obtained
for the composites with up to 75 wt% of HA.
Thermal analysis (TGA) revealed higher thermal stability of PLLA/
prepared according to the procedure described in experimental section
HA compared to neat PLLA but also residue of sodium chloride in the
(see also Fig. S2 in Supporting Info). TGA results indicated good
composites walls.
agreement between the estimated and processing filler contents
Compared to PLLA foams, the PLLA/HA systems exhibit higher
(Table 2). Moreover, to investigate the influence of hydroxyapatite on
temperatures of crystallization indicating nucleating effect of HA in the
thermal stability of foams temperature corresponding to 5 wt% loss of
system. With higher filling level, the degree of PLLA crystallinity de-
mass (T-5%) was determined. TGA analysis revealed that thermal sta-
creases. Moreover, it was found that for the foams having up to 50 wt%
bility of all composites is higher than for neat PLLA, the highest in-
of HA, PLLA consists of mixture of α’ and α phase except in the PLLA/
crease of T-5% of ∼19 °C compared to neat PLLA is for composite with
HA 25/75 sample PLLA crystallize on cooling in α form. Compression
25 wt% of HA.
tests revealed that the highest values for Young‘s modulus as well as for
compression stress were obtained for the PLLA/HA 25/75 scaffold. The
research confirmed also that the proliferation rate of osteoblast cells is
3.4. Cellular proliferation
proportional to the HA content in the foam, reaching 3–4 times higher
rate for PLLA/HA 25/75 comparing to neat PLLA.
Fig. 9 illustrates proliferation rate of MC3T3-E1 osteoblast cells. The
measurements were conducted after 24 and 72 h. It was earlier de-
Acknowledgement
scribed that the addition of hydroxyapatite stimulate cell adhesion and
proliferation [45] on scaffolds. However, in previous articles the effect
This work was supported by Polish Ministry of Higher Education for
was observed for the composites with lower hydroxyapatite content, ie.
Department of Chemistry, Wroclaw University of Science Technology,
up to 20 wt% [30]. The results indicate that there is a difference for the
statuary funds no. 0401/0125/18 as well as Polish National Research
cell proliferation rates in all the five systems at the first day of culture.
Center no. 2015/19/B/ST5/01330. KS would like to thank prof.
The proliferation rate is significantly higher for the composites having
Masami Okamoto from Toyota Technological Institute (Nagoya, Japan)
higher content of hydroxyapatite. The effect is observed after 24 h, with
and Matsumae International Foundation (Tokyo, Japan) for hospitality
one exception (PLLA/HA 25/75). For the measurements conducted
and financial support of the research.
after 72 h, the effect is even stronger. Obtained values of proliferation
rate after 72 h ranged from 307% for the PLLA foam (as compared to
Appendix A. Supplementary material
the same sample after 24 h) to 1168% for the PLLA/HA 25/75.
Statistical analysis shows significant difference (p < 0.01) for most
Supplementary data to this article can be found online at https://
of the samples within the group of samples measured after 24 h. The
doi.org/10.1016/j.eurpolymj.2019.01.073.
same situation is observed for group of samples after 72 h. The only
exceptions are differences after 24 h for PLLA/HA 90/10 vs. PLLA/HA
References
75/25 and after 72 h for PLLA/HA 50/50 vs. PLLA/HA 25/75 where the
significant difference is p < 0.05.
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