Journal of Thermal Analysis and Calorimetry (2018) 134:1521–1530

https://doi.org/10.1007/s10973-018-7498-y(0123456789().,-volV)(0123456789().,-volV)

Thermal, morphological, spectroscopic and biological study

of chitosan, hydroxyapatite and wollastonite biocomposites
Josué da Silva Buriti1 • Maria Eduarda Vasconcelos Barreto1 • Kleilton Oliveira Santos2 • Marcus Vinicius Lia Fook1

Received: 20 December 2017 / Accepted: 22 June 2018 / Published online: 2 July 2018
 Akadémiai Kiadó, Budapest, Hungary 2018

Abstract
The objective of this work was to investigate the thermal, morphological, spectroscopic and cytotoxicity of hydroxyap-
atite–wollastonite powders obtained via sol–gel synthesis and of biocomposites chitosan–hydroxyapatite–wollastonite. A
mixture of wollastonite, calcium nitrate tetrahydrate and ammonium dihydrogen phosphate with a ratio of 1:2:1.2 or
2:2:1.2, respectively, was produced following drying and heat treatment where the final composite was macerated. These
powders were added to a chitosan solution where it was further dried and neutralized. The ceramic loads were used in
various ratios. The materials were characterized by TG, DSC, DRX, MEV, FTIR and cytotoxicity. Based on the studied
properties, it can be said that the sol–gel process proved to be effective in obtaining hydroxyapatite–wollastonite powders.
By TG, it was verified that the thermal stability of the powders increased when a greater percentage of wollastonite was
used. For biocomposites with higher percentages of load, there was increase in thermal stability, probably attributed to the
higher compaction of the biocomposites when compared to the pure. By DSC, there was a tendency of displacement of the
endothermic and exothermic peaks, suggesting that the biocomposite with higher load has greater capacity of retention and
interaction stronger with molecules of water, but also has greater thermal stability. The samples present biomaterial
potential with prospects of endodontic use, which showed cell viability in L929 fibroblast cell culture above 70.00%.

Keywords Biocomposites
Chitosan
Hydroxyapatite
Wollastonite
Sol–gel
Endodontics

Introduction
The search for new devices with optimized properties for
advanced applications and also for improvement of the
ones already commercially available is one of the reasons
for the current technological and scientific evolution. For
biomedical devices, these materials should have desirable
properties and biocompatibility, such as prothesis, drug
delivery devices, artificial organs, wound healing, as well
as in the development of dental materials, such as the
& Josué da Silva Buriti
josueburiti@gmail.com
1 In addition, biopolymers such as chitosan have a struc-
Unidade Acadêmica de Engenharia de Materiais,
Universidade Federal de Campina Grande, Rua Aprı́gio
Veloso, 882, Bodocongó, Campina Grande,
Paraı́ba CEP: 58429-140, Brazil
2
Programa de Pós-Graduação em Quı́mica, Universidade
Estadual do Paraı́ba, Rua Baraúnas, 351, Bodocongó,
Campina Grande, Paraı́ba CEP: 58429-500, Brazil
treatment of lesions and diseases of the pulp and root of the
tooth [1–3].
In the case of biomaterials, bioceramics such as calcium
phosphates and silicates have been had much attention
mainly due to the absence of local or systemic toxicity.
Among calcium phosphates and silicates, hydroxyapatite
and wollastonite are currently used in terms of their great
potential in clinical applications, this occurs because these
ceramics constitutes a major component of the mineral
composition of bones and teeth. One of the most used
techniques to produce these bioceramics is the use of the
sol–gel synthesis, which allows the preparation of materials
at low temperatures with specific properties and high purity
without the need of additional purification steps [4–13].
ture similar to the extracellular matrix of the tissue; this
helps avoid chronic inflammation or immunological reac-
tion and toxicity [14–21]. Thus, the combination of
hydroxyapatite–wollastonite with chitosan has been widely
used due to the characteristics of a good support for cell

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1522
growth, biocompatibility, biodegradability, osteoinductiv-
ity and can be used as a resorbable binding agent, which is
responsible for the prevention of particle migration to tis-
sue incorporation. Thus, these biocomposites have many
uses in endodontics, such as direct pulp capping, pulpo-
tomy, opacification, paraendodontic surgery, endodontic
perforations, repair of furcation perforations, lateral root
perforations and bacterial infiltration [22–29].
Thus, the combination of chitosan, hydroxyapatite and
wollastonite has great potential in obtaining compounds
with controlled biological activity, since hydroxyapatite
can act as a crosslinking agent due to the presence of
calcium and phosphorus; wollastonite can act as rein-
forcement of the material and chitosan can offer great
advantages, due to its degradation mechanism which
occurs by the metabolization via specific human enzymes.
In addition, these composites can be a low-cost alternative
compared to materials commercially available [30–36].
Therefore, this work aims to study the thermal, mor-
phological, spectroscopic and biological properties of chi-
tosan polymers loaded with hydroxyapatite–wollastonite
powders obtained via sol–gel synthesis with the application
of endodontic use.
Experimental
The stoichiometric ratio between calcium and phosphorus
(Ca/P) = 1.67 was considered for the synthesis. With this
principle, two molar ratio for the preparation of hydrox-
yapatite–wollastonite powders were used: (A) 1:2:1.2 mol
and (B) 2:2:1.2 mol; for CaSiO3, Ca(NO3)2 and NH4H2-
PO4, respectively.
Wollastonite (CaSiO3; Nyco) was dissolved in deionized
water followed by the addition of a dispersing agent to
obtain a better solubilization. After total solubilization of
wollastonite, calcium nitrate tetrahydrate [Ca(NO3)2;
Sigma-Aldrich)] and ammonium dihydrogen phosphate
(NH4H2PO4; Sigma-Aldrich) were added to the mixture.
After total solubilization, deionized water and ethyl alcohol
were incorporated with a ratio of 6:4 mol and, to adjust the
pH to 9, sodium hydroxide (NaOH; Vetec) was used. The
system was kept under magnetic stirring for 1 h at 70 C
until gelation of the mixture occurred. The gels were fur-
ther submitted to an oven and dried at 120 C for 16 h; the
formed xerogel was heat-treated in a muffle oven at 700 C
(10 C min-1) for 2 h. Subsequently, the heat-treated
xerogel was macerated and passed in mesh 74 lm. The
preparation of chitosan used in this work follows a similar
method of Antonino et al. [37], via deacetylation of chitin
extracted from shrimp shells of Litopenaeus vannamei
Boone. The degree of deacetylation of these chitosan used
was 85% and the viscosity molar mass of 285 kg mol-1.
123
J. S. Buriti et al.
In order to obtain the final biocomposites, the macerated
powders were added in a solution containing 1% chitosan
in 0.1 mol L-1 lactic acid at various concentrations and
maintained under mechanical stirring at 600 rpm for
40 min. The final powder sample concentrations were
added, and labelled, as follows (1) 20% of the load A
(20%A), (2) 40% of the load A (40%A), (3) 60% of the
load A (60%A), (4) 20% of the load B (20%B), (5) 40% of
the load B (40%B), (6) 60% of the load B (60%B). In
addition, the (7) pure chitosan without addition of the
ceramic powder was also studied in this work. Upon
observing complete dissolution, the mixture was added in
an ultrasonic bath without heating for 1 h in order to
eliminate any remaining bubbles. After drying in an oven
for 24 h at 37 C, it was further neutralized in 2%
ammonium hydroxide atmosphere for 72 h (in glass dome).
Afterwards, the materials were submitted the
characterizations.
The materials obtained were characterized by thermo-
gravimetry (TG), differential scanning calorimetry (DSC),
X-ray diffraction (XRD), scanning electron microscopy
(SEM), vibrational absorption with Fourier transform
infrared spectroscopy (FTIR) and cytotoxicity.
The thermogravimetric analysis was performed in a
PerkinElmer thermocouple model Pyris 1 TGA, with air
flow of 20 mL min-1, heating rate 10 C min-1 and tem-
perature range of 30–900 C. The sample mass used was
4.0 ± 1.0 mg. The DSC analysis was obtained in DSCQ20
system of the TA Instruments brand, model Q20, with
nitrogen flow of 50 mL min-1; samples were encapsulated
in sealed aluminium pans, with a heating rate of
10 C min-1, in a temperature range from 30 to 400 C.
The sample mass was 2.0 ± 1.0 mg.
Structural changes were characterized with XRD using a
Shimadzu XRD-7000 equipment, geometry of h–2h from
the scan range of 5–60, 0.6 s step time using Cu-ka radi-
ation, voltage of 40 kV and current 30 mA. The mor-
phology of the studied samples was investigated using the
SEM analysis. The equipment Hitachi TM-1000 model was
used, with a resolution up to 10,0009 and depth of focus of
1 mm, resolution of 30 nm, 15 kV, low vacuum and varied
pressure (1–270 Pa), without metallic coating. Micro-
graphs were obtained with 5009 magnification.
The FTIR characterization was performed on a Perk-
inElmer Spectrum 400 spectrometer. The spectra were
obtained in the absorption region between 4000 and
650 cm-1, with a resolution of 4 cm-1. The cytotoxicity
assays of 60%A, 60%B and pure membrane biocomposites
were performed by the L929 fibroblast cell viability carried
out by the MTT (3-(4,5-dimethylthiazol-2-yl)5-diphenyl-
tetrazolium] according to BS EN ISO 10993-5:2009, being
considered viable the value C 70.00% [38].
Thermal, morphological, spectroscopic and biological study of chitosan, hydroxyapatite and… 1523

Results and discussion 100 (a) A (TG)

B (TG) 1
A (DTG)
B (DTG)
Thermogravimetry (TG) 90

Derivative mass /% min–1

0

80
Figure 1 shows the thermogravimetric (TG) curves of A –1

Mass/%
and B powders and the 20%A, 40%A, 60%A, 20%B, 70
–2
40%B, 60%B biocomposites and pure membrane.
Table 1 presents the results of the thermogravimetric 60
–3
decomposition of A and B powders and 20%A, 40%A,
50
60%A, 20%B, 40%B, 60%B biocomposites and pure –4
membrane. 40
The TG profiles and values of the studied powders and –5
0 200 400 600 800 1000
biocomposites, as shown in Fig. 1 and Table 1, exhibit
Temperature/°C
three thermal decomposition steps which were observed for
all samples. The pure ceramic powders increased its ther-
20% A
mal stability when a higher molar ratio of wollastonite was 100
(b) 40% A
60% A
used in the powder synthesis. Sample with increased wol- Pure
lastonite content (B) exhibited slightly increased values of 80
initial decomposition temperatures than lower wollastonite
content (A); also, the mass loss was lower than sample A
with a value of 49.5% compared to 57%. Mass/% 60

For samples A and B, the first decomposition step was

assigned to the water loss, as well as the evolution of 40

organic compounds still present in the gel, which were

added as precursors; the second step could be related to the 20
formation of hydroxyapatite and calcium carbonate in the
form of calcite (CaCO3). The third step can correspond to 0
the conversion of calcium carbonate to calcium oxide. 0 100 200 300 400 500 600 700
These attributions corroborate with works of Encinas- Temperature/°C
Romero et al. [30], Tõnsuaadu et al. [36], Encinas-Romero
20% B
et al. [26], Skwarek [39], Neamtu et al. [32], 100
(c) 40% B
60% B
Gonzalez et al. [40], in similar searches. No mass loss was Pure
observed for wollastonite in the temperature range studied. 80
For the biocomposites TG curves, as shown in Fig. 1b,
c, it can be observed that an increasing in ceramic load
Mass/%

60
leads to an increase in thermal stability. This is further
evidenced in the second degradation stage compared to the
raw powders. Similarly, for the biocomposites with higher 40
ceramic loadings, a decrease in mass loss occurs. The first
degradation step curve is attributed to the water–polymer 120
interactions strongly bounded to the structure and also to
water retention capacity of the material [14, 16, 18, 41–45]. 0
The second step can be attributed to thermal degradation 0 100 200 300 400 500 600 700
resulted from the disruption of the polymer chitosan chains, Temperature/°C
more specifically, decomposition of the pyranose ring
Fig. 1 TG curves of A and B powders (a) and 20%A, 40%A, 60%A
occurs through dehydration, following deamination and biocomposites and pure membrane (b); 20%B, 40%B, 60%B and pure
ring breaking reactions causing degradation of the sac- membrane (c); under heating rate of 10 C min-1, with air flow
charide structure of these molecules [46–49]. The third
degradation step can be attributed to the carbonization of
the material. Differential scanning calorimetry (DSC)

Figure 2 shows the DSC curves of 20%A, 40%A, 60%A,

20%B, 40%B, 60%B biocomposites and pure membrane.

123
1524 J. S. Buriti et al.

Table 1 Results of the

Samples Loss stage Temperature range/C Mass/% Total mass loss/%
thermogravimetric
decomposition of the A and B A 1st 30–157 9.8 57.0
powders and 20%A, 40%A,
60%A, 20%B, 40%B, 60%B 2nd 157–360 32.3
biocomposites and pure 3rd 357–800 14.9
membrane B 1st 30–172 9.2 49.5
2nd 172–357 26.1
3rd 360–800 14.2
20%A 1st 30–207 17.4 82.0
2nd 207–467 35.5
3rd 467–677 29.1
40%A 1st 30–264 22.1 80.3
2nd 264–469 28.3
3rd 469–679 29.9
60%A 1st 30–240 18.3 72.9
2nd 240–442 26.5
3rd 442–680 28.1
20%B 1st 30–218 16.0 81.5
2nd 218–457 34.0
3rd 457–680 31.5
40%B 1st 30–214 16.7 78.6
2nd 214–432 28.6
3rd 432–679 33.3
60%B 1st 30–221 16.6 69.0
2nd 221–452 25.5
3rd 452–680 26.9
Pure 1st 30–200 14.0 83.0
2nd 200–487 41.0
3rd 487–678 28.0

The DSC curves (Fig. 2) of the studied samples present
at lower temperatures, endothermic peaks, between 85 and
126 C, which are attributed to the evaporation of volatile
substances such as water bound via hydrogen bonds in the
hydroxyl groups of chitosan. With increasing temperature,
endothermic peaks around 192 C are due to the inorganic
phase of the biocomposites which is not observed in the
pure sample. Increasing the temperature leads to a shifting
of the baseline in the exothermic direction at 305 C in the
pure chitosan, related mainly to polymer decomposition or
the ability of the biocomposite to retain remaining water
particles. Researches by Guinesi and Cavalheiro [50],
Blachnio et al. [42], Budnyak et al. [41], Corazzari et al.
[51], Geetha et al. [52], Budnyak et al. [18], Lino et al.
[53], Topcu et al. [54] show the results of the thermal
events.
It can be observed that when the ceramic content is
increased, a tendency of peak change from endothermic to
exothermic occurs, suggesting that higher ceramic loading
improves the crosslinking density of the material,
increasing the number of inter–intramolecular bonds and
consequently improving the polymeric network density
[55, 56].
X-ray diffraction (XRD)
Figure 3 shows the X-ray diffractograms of A and B
powders (a) and 20%A, 40%A, 60%A, 20%B, 40%B,
60%B biocomposites and pure membrane (b).
The ceramic powder diffractograms (Fig. 3a) presented
phases of hydroxyapatite (JCPDS: 01-072-1243) and wol-
lastonite (JCPDS: 00-043-1460). The presence of crys-
talline phases of hydroxyapatite resulted in 78.2% (sample
A) and 57.9% (sample B); on the other hand, crystalline
phases of wollastonite resulted in 21.8% (sample A) and
41.2% (sample B).
For the diffractograms of biocomposites (Fig. 3b), an
increase in crystallinity occurs as the percentage of charge
increases; this can be attributed to the crystalline phases of
hydroxyapatite and wollastonite powders that crosslinked
in between the biocomposites as well as the aggregates of
these ceramics in between the chitosan polymer

123
Thermal, morphological, spectroscopic and biological study of chitosan, hydroxyapatite and… 1525

298 °C
(a) Hidroxiapatita
Wollastonita
Pure

119 °C
20%A 282 °C
B

Intensity/a.u
94 °C
Exo

193 °C
132 °C
40%A 302 °C
Endo

82 °C
192 °C
122 °C
60%A 309 °C
A
Heat flow/W g–1

83 °C 190 °C
133 °C
10 20 30 40 50 60
20%B 297 °C
2θ /°

83 °C (b)
194 °C
123 °C Pure
40%B
302 °C
20%A

85 °C 194 °C
116 °C Intensity/a.u 40%A

60%B 307 °C
60%A

84 °C 187 °C
132 °C 20%B

0 50 100 150 200 250 300 350 400 450 40%B

Temperature/°C

Fig. 2 DSC curves of 20%A, 40%A, 60%A, 20%B, 40%B, 60%B
biocomposites and pure membrane at 10 C min-1, under nitrogen
atmosphere
[26, 30, 40, 57–62]. The values of the pure chitosan
membrane presented crystallinity of 35%, while the sam-
ples with ceramic loadings 20%A, 40%A, 60%A, 20%B,
40%B and 60%B had crystallinity of 53, 64, 74, 49, 62 and
71%, respectively. This evidences the improvement in
crystallinity resulted from the molecular bonding in
between the polymer.
Scanning electron microscopy (SEM)
Figure 4 shows the SEM images of A and B powders (a, b)
and 20%A, 40%A, 60%A, 20%B, 40%B, 60%B biocom-
posites and pure membrane (c, d).
A heterogeneous surface morphology is observed in
Fig. 4a, b with round shape for hydroxyapatite and a fibre
mesh surface for wollastonite; this is similar to other author
findings [63]. The direct interaction between the ceramics
particles has a major significance to the biological prop-
erties; also, it is possible to notice the effect of the sol–gel
synthesis in obtaining a controlled morphology.
The morphology of the hydroxyapatite–wollastonite–
chitosan biocomposites, in Fig. 4c–i, evidences that the
60%B
10 20 30 40 50 60
2θ /°
Fig. 3 Diffractograms of A and B powders of the 20%A, 40%A,
60%A, 20%B, 40%B, 60%B biocomposites and pure membrane
presence of hydroxyapatite–wollastonite powder was well
distributed within the chitosan matrix. Comparing with the
pure chitosan, it was verified that there was a greater
aggregation of these particles by increasing the percentage
of the powder obtained by the sol–gel process. With
increasing loading of the ceramic powder, an increase in
aggregation of these ceramics occurs [54, 64, 65].
Vibrational absorption with Fourier transform
infrared spectroscopy (FTIR)
Figure 5 shows the absorption spectra in the infrared region
(FTIR) of 20%A, 40%A, 60%A, 20%B, 40%B, 60%B
biocomposites and pure membrane. Table 2 shows the
results concerning the main bands observed in FTIR
spectra.
Figure 5 exhibits the infrared (FTIR) absorption spectra
of the studied samples. Samples presented absorption

123
 1526

membrane

123
magnification
membrane, with 9500
biocomposites and pure
powders and 20%A, 40%A,
60%A, 20%B, 40%B, 60%B

40%A, 60%A, 20%B, 40%B,

Fig. 5 FTIR spectra of 20%A,

60%B biocomposites and pure

Fig. 4 SEM images of A and B

Absorbance

4000
Pure

60%A

60%B
40%B
40%A

20%B
20%A

3500
3302 cm–1

3145 cm–1 3124 cm–1 3168 cm–1 3168 cm–1

3126 cm–1 3137 cm–1

2977 cm–1 2980 cm–1 2979 cm–1 2977 cm–1 2984 cm–1 2978 cm–1

3000
2979 cm–1

2500
Wave number/cm–1
2000
1690 cm–1 1688 cm–1 1676 cm–1 1686 cm–1 1672 cm–1 1665 cm–1 1647 cm–1

1566 cm–1 –1 –1 1574 cm–1

1567 cm–1 1563 cm–1 1563 cm–1 1563 cm 1567 cm

1500
1418 cm–1 1402 cm–1 1403 cm–1 1400 cm–1 1420 cm–1 1415 cm–1 1418 cm–1
1374 cm–1 1374 cm–1 1358 cm–1 1364 cm–1 1382 cm–1 1377 cm–1 1378 cm–1
1313 cm–1 1313 cm–1 1314 cm–1 1314 cm–1 1315 cm–1 1313 cm–1 1317 cm–1
1124 cm–1

1149 cm–1
1123 cm–1 1121 cm–1 1123 cm–1 1120 cm–1 1122 cm–1
–1 –1 –1
1047 cm–1 1048 cm 1044 cm 1047 cm–1 1040 cm 1025 cm–1

1000
1029 cm–1
858 cm–1 856 cm–1 858 cm–1 860 cm–1 857 cm–1 856 cm–1 852 cm–1
–1 –1 –1 –1 –1 –1
778 cm 777 cm 776 cm 778 cm 774 cm 778 cm
J. S. Buriti et al.
Thermal, morphological, spectroscopic and biological study of chitosan, hydroxyapatite and… 1527

Table 2 Results regarding to

Sample O–H C–H C=O N–H –CN CH3 –CN CHO CHO CHO CO2- 3-
3 |PO4
the main bands observed in the
FTIR spectra of the 20%A, Pure 3302 2879 1647 1574 1418 1378 1317 1149 1029 857 ND
40%A, 60%A, 20%B, 40%B,
60%B biocomposites and pure 20%A 3168 2978 1665 1567 1415 1377 1313 1122 1025 856 778
membrane 40%A 3168 2984 1672 1563 1420 1382 1315 1120 1040 857 774
60%A 3124 2977 1686 1563 1400 1364 1314 1123 1047 860 778
20%B 3145 2979 1676 1563 1403 1358 1314 1121 1044 858 776
40%B 3137 2980 1688 1566 1402 1374 1313 1123 1048 856 777
60%B 3126 2977 1690 1567 1418 1374 1313 1124 1074 858 778

bands related to the fundamental vibrations, and stretching

of chitosan due to the carbonate and phosphate groups 100.00 ± 12.38%
100 97.95% ± 10.46%
92.67 ± 8.11%
occurs. These bands occur around 3167, 2965, 1675, 1566,
1411, 1372, 1314, 1126, 1044, 857 and 777 cm-1, and it is 80 77.51% ± 7.25%
further described in Table 1. The main bands observed

Cell viability/%
were axial stretching band of O–H (between 3124 and
60
3302 cm-1), which appears superimposed on the N–H
stretch band; C–H shifts (between 2879 and 2984 cm-1);
axial deformation of C=O amide I (between 1647 and 40

1690 cm-1); angular deformation of N–H (between 1563

and 1574 cm-1); axial deformation of amide –CN (be- 20
tween 1402 and 1420 cm-1); symmetrical angular defor-
mation of CH3 (between 1358 and 1382 cm-1); axial 0
deformation of –CN of amino groups (between 1317 and Control 60%A 60%B Pure

1313 cm-1); (between 856 and 1149 cm-1), as well as Samples

bands corresponding to the stretching modes of the CO2- 3 Fig. 6 Cell viability of 20%A, 60%B biocomposites and pure
and PO3- 4 groups (between 774 and 778 cm-1). These
attributions corroborate with works of Fraga et al. [64],
Blachnio et al. [42], Budnyak et al. [41], João et al. [60],
Kalaycioglu et al. [10], Dang et al. [66], in similar
searches.
For all biocomposites with ceramic loadings, a shift in
the vibration bands of O–H and C–H groups occurs, and
this evidences the internal bonding between chitosan
polymers. Furthermore, with increase in loading of the
ceramic powder, an increase in shifting of the bands
occurs; this suggests that the presence of silicate, carbonate
and phosphate changes the polymeric chain inducing a
more preferably bond between these ceramics in between
the chitosan polymer structure [18, 46, 54].
Although some of the bands of hydroxyapatite and
wollastonite are occluded by the presence of chitosan
bands, some distinct bands such as O–H (between 3124 and
3302 cm-1), phosphates (between 774 and 778 cm-1) and
carbonates (between 1402 and 1420 cm-1) are related to
hydroxyapatite, as well as the silicate bands (between 856
and 1149 cm-1) corresponding to wollastonite.
The presence of a new band formed in the biocompos-
ites containing ceramic powders is verified around
777 cm-1. This band, corresponding to the phosphate and
carbonate groups, increases with ceramic loading. This is
justified by the preferential path, which is connected
membrane
primarily with the hydroxyapatite phosphate, to which this
vibration is associated Encinas-Romero et al. [30].
Cytotoxicity
Figure 6 shows the cytotoxicity results of the 20%A, 60%B
biocomposites and pure membrane.
It is verified that the samples submitted to the analysis of
in vitro cytotoxicity presented cellular viability in cell
culture of L929 fibroblasts above 70.00% as specified in
BS EN ISO 10993-5:2009. However, the percentage of cell
viability from the biocomposite 60% B was slightly below
60% A. This effect may be due to the influence of wol-
lastonite particles agglomeration in higher percentage.
Cells are very sensitive to surface, chemical, and mor-
phological energy, including those of ceramic–phase
agglomerations, which may slow their proliferation and
differentiation. Nonetheless, these results still remain
above the minimum specified by the standard. Research on
biocomposites based on chitosan and hydroxyapatite
Shakir et al. [67], Dumont et al. [68], Iqbal et al. [69]
shows that by cytotoxicity assay, cell viability was satis-
factory, corroborating the results obtained by this study.

123
1528
Conclusions
The sol–gel process proved to be effective in obtaining
hydroxyapatite–wollastonite powders. Based on the ther-
mogravimetric curves, it was verified that the thermal
stability of the powders increased when a greater percent-
age of wollastonite was used. For biocomposites with
higher percentages of load, there were a decrease in total
mass loss and increase in thermal stability, probably
attributed to the higher compaction of the biocomposites
when compared to the pure membrane. By the differential
scanning calorimetry, there was a tendency of displacement
of the endothermic and exothermic peaks, suggesting that
the biocomposite with higher load has greater capacity of
retention and interaction stronger with molecules of water,
but also has greater thermal stability.
By X-ray diffraction, an increase in crystallinity can be
observed as the percentage of inorganic filler increases,
which may be attributed to the crystalline phase of the
hydroxyapatite and wollastonite powders that have
remained in the biocomposites. It was observed the pres-
ence of hydroxyapatite–wollastonite aggregates distributed
in the chitosan matrix, verifying greater aggregation of the
particles by increasing the percentage of the powder
obtained by the sol–gel process.
By Fourier transform infrared spectroscopy, absorptions
related to the vibrations or fundamental stretches of chi-
tosan and carbonate and phosphate groups were observed.
It can be noticed that with the increase in load, there are
changes in the intensities of vibrations or appearance of
new bands, suggesting new connections related to silicate,
carbonate and phosphate, characterized by the presence of
hydroxyapatite and wollastonite. By the in vitro cytotoxi-
city test, the 60%A and 60%B samples showed cellular
viability in L929 fibroblast cell culture above 70.00%, for
the use of bone regeneration and in endodontic treatments.
Acknowledgements The National Postdoctoral Program of the
Coordination of Improvement of Higher Education Personnel
(PNPD), the Coordination of Improvement of Higher Education
Personnel (CAPES), the Laboratory of Evaluation and Development
of Biomaterials of the Northeast (CERTBIO) and the Federal
University of Campina Grande (UFCG).
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