Materials Letters 284 (2021) 129013

Contents lists available at ScienceDirect

Materials Letters
journal homepage: www.elsevier.com/locate/mlblue

Green Synthesis of Nano Hydroxyapatite: morphology variation and its

effect on cytotoxicity against fibroblast
Andrea Elizabeth Melgar Aguilar a, Ana Paula Fagundes a, Domingos Lusitâneo Pier Macuvele a,b,
Karina Cesca a, Luismar Porto a, Natan Padoin a, Cíntia Soares a,⇑, Humberto Gracher Riella a,⇑
a
Department of Chemical and Food Engineering, Federal University of Santa Catarina, Florianópolis, SC, Brazil
b
Department Science, Engineering, Technology and Mathematics, University of Rovuma-Extension of Niassa (Extinct Pedagógica University of Mozambique), Lichinga, Mozambique

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

Article history: Nano hydroxyapatite (nHA) was synthesized from different methods. Spherical-like shaped nanoparticles
Received 23 August 2020 of nHA were synthesized by chemical precipitation using Euclea natalensis root extract as a template for
Received in revised form 12 October 2020 the first time. In addition, traditional synthesis was performed, obtaining rod-like nanoparticles. The for-
Accepted 7 November 2020
mation of nHA was confirmed using Fourier Transform Infrared (FTIR), X-Ray Diffraction (XRD), and
Available online 12 November 2020
Transmission Electron Microscopy (TEM). HA from green route presented a spherical-like shape with
smooth surface. However, nanogrooves covered over the surface of nHA obtained without green tem-
Keywords:
plate. Additionally, the cytotoxic effect of the nHA nanoparticles on L929 viability was assessed using
Euclea natalensis
Metabolic activity
MTS assay. The particles exhibited less cellular toxicity at shorter incubation periods. Spherical-like
Biomaterials and rod-like nHA particles interact differently with fibroblast after 24 h of evaluation.
Nanorod Ó 2020 Elsevier B.V. All rights reserved.
Surface patterning

1. Introduction plate. Furthermore, it was compared the cytotoxicity of green

Advances in nanotechnology have allowed improvements in var-
ious domains of study, including the biomedical field. However, for
a nanomaterial to be applicable as a biomaterial it must follow strict
requirements. For this reason, studies that assess the interaction
between a nanomaterial and complex biological systems are extre-
mely relevant. Hydroxyapatite (HA), a bioceramic that is similar to
the mineral part of the bone, has exhibited a versatile nature that
has allowed for its successful application as delivery vehicles for
drugs and genes, bioactive coating, bioimaging, among other appli-
cations [1,2]. However, to date, there are few reported studies con-
cerned with the cellular response to various shapes of nHA [3].
Studies suggest that different shapes can lead to alterations in the
interaction between the cell and the nanoparticles, thus affecting
the cytotoxicity and cell uptake [4]. In view of this, new techniques
intend to manipulate morphology by employing green procedures
that do not require toxic surfactants used in traditional synthesis
[5]. Therefore, the aim of this work is to synthesize nanohydroxya-
patite employing, for the first time, Euclea natalensis roots extract
(mainly composed by naphthoquinones and terpenes) as a tem-
⇑ Corresponding authors.
E-mail addresses: cintia.soares@ufsc.br (C. Soares), humberto.riella@ufsc.br (H.
Gracher Riella).
synthesized nanohydroxyapatite with two other samples obtained
without E. natalensis.
2. Experimental
2.1. Materials
The raw materials for the synthesis were used without any fur-
ther purification. As a calcium source, calcium hydroxide [Ca(OH)2]
95% P.A was purchased from Synth. Phosphoric acid [H3PO4] 85% P.
A and ammonium hydroxide NH4OH 30% P.A were both obtained
from Alphatec. Likewise, Euclea natalensis was received from the
Boane deposit in Mozambique, its roots were grinned and washed
using distilled water.
2.2. Synthesis of non-template-mediated HA
Nanohydroxyapatite particles were synthesized through a sim-
ple chemical precipitation method described previously by Abidi
and Murtaza [6]. As the final product, a white powder was
obtained. The procedure previously referenced corresponds to
Sample 1. Moreover, another sample was synthesized using a more
diluted initial solution, while the other conditions were kept the
same. This sample was denoted as Sample 2.

https://doi.org/10.1016/j.matlet.2020.129013
0167-577X/Ó 2020 Elsevier B.V. All rights reserved.
Andrea Elizabeth Melgar Aguilar, Ana Paula Fagundes, Domingos Lusitâneo Pier Macuvele et al.
2.3. Synthesis of green-template-mediated HA
For the synthesis of Sample 3, the roots extract was used as a
green template to possibly alter the nanoparticles’ morphology.
Mixture of grinned Euclea natalensis and distilled water were
mixed with Ca(OH)2. The following procedures are as described
in the previous section. Once the precipitate was dried and
grinded, the resultant product was sintered in a muffle furnace
(Quimis) at 800 °C with residence time of 180 min in order to
remove the remaining organic material.
2.4. Characterization
To identify the functional groups present in the samples, a Four-
ier Transform Infrared (FTIR) spectroscopy was performed using
the AGILENT Technologies-Cary 600 Series FTIR spectrometer in
the region of 400–4000 cm 1. The powder obtained was character-
ized by X-Ray Diffraction on a diffractometer (MiniFlex 600 DRX
Rigaku). The particles’ size and morphology were observed under
a Transmission Electron Microscopy (TEM, JEM-1011, JEOL).
2.5. Cytotoxicity assay
The cytotoxicity of nHA nanoparticles was evaluated by measur-
ing metabolic activity of L929 cell (BCRJ, Brazil) following the ISO
10993-5 (2009) by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox
ymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] colorimetric
assay (Promega, Brasil). Cells were cultured in DMEM, supple-
mented with 10% FBS and 1% penicillin/streptomycin. Cells were
placed in 96-well plates at a density of 1
104 cells/well and cultured
for 24 h. The nanoparticles were sterilized in an autoclave (121 ℃
and 1 atm) for 20 min and resuspended in DMEM medium before
analysis. Subsequently, 200 mL of the solutions of the nHA nanopar-
ticles in different concentrations were added and incubated for 24 h
and 48 h. The control wells containing culture medium only. After
incubation, 100 lL each well was transferred to a 96-well plate
for optical density (OD) measurement by spectrophotometry
(490 nm, Molecular Devices). The cell viability (%) was determined
concerning the control set up as 100%. A more delineated procedure
can be found in the Supplementary Information.
3. Results and discussion
3.1. Characterization
The FTIR patterns are presented in Fig. 1 (A). The spectra pos-
sessed an OH group in the region of 3.443 cm 1. Typical bands of
Fig. 1. FTIR spectra (A) and DRX diffractograms (B) for Sample 1 (traditional synthesis), Sample 2 (synthesized using a more diluted initial solution) and Sample 3 (green
synthesis). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2
Materials Letters 284 (2021) 129013
the material were identified, the band at 1038 cm 1, which is
attributed to phosphate vibration (PO4)3 , and the band at
1448 cm 1, which is attributed to carbonate (CO3)2. A weak band
of (HPO4)2 was detected in the region around 756 cm 1, OH in
608 cm 1 and (PO4)3 in 545 cm 1 [7]. The presence of sharp peaks
in the characteristic bands of OH 1 and (PO4)3 confirms the for-
mation of HA [6]. Fig. 1 (B) shows the diffractograms obtained
for the synthesized samples. All samples exhibited the characteris-
tic peaks of hydroxyapatite. However, the green synthesized
nanohydroxyapatite (Sample 3) displayed the sharpest peaks when
compared to the other samples (Sample 1 and Sample 2).
Fig. 2 shows the TEM micrographs of all samples of hydroxyap-
atite synthesized from different methods. All the samples present
size less than 100 nm. The Sample 1 [Fig. 2 (A)] presents the rod
like structure. The Sample 2 [Fig. 2 (B)] also has a similar shape
to the previous one, but presented larger particle size. Nano-
grooves covered over the surface of nHA can be observed in TEM
image for methodologies that have not used the green extract.
Fig. 2 (D,E) shows in detail these changes in the nanoparticles sur-
face. This hierarchical nanorod had already been found by Ji et al.
(2020). They obtained the material synthesized via a surfactant-
free route, promoted, induced and stabilized by tetrahydrofuran,
resulting in a surface covered with labyrinthine nanogrooves [8].
The morphology of the sample that used the plant extract as a tem-
plate presented different shape [Fig. 2 (C)]. The particles do not
have a rod-like shape, but a spherical agglomerate. In addition,
the Sample 3 presented smooth surface compared to others.
3.2. Cytotoxicity
The cytotoxic effect of the nHA nanoparticles on L929 viability
was assessed with five different concentrations (10, 25, 50, 75
and 100 mg/mL) at 24 h and 48 h using MTS assay. It was noticed
that Sample 1 and Sample 2 [Fig. 3 (A,B)] displayed insignificant
toxicity on L929 at all concentrations after 24 h incubation. Consid-
ering the same incubation period, Sample 3 [Fig. 3 (C)], with a
sphere-like morphology, presented a much more stable metabolic
activity at all concentrations. After a 48 h incubation period, Sam-
ples 1 and 2, with a rod-like shape, exhibited minimal toxicity.
On the other hand, Sample 3, in the concentrations of 50, 75, and
100 mg/mL was at the limit of cytotoxicity.
It is interesting to note that in every sample, at both the incuba-
tion periods, metabolic activity decreased with the increase of con-
centration of nHA. This result is congruent with the study
performed by Zhang et al. [9]. Based on other similar reports, we
hypothesize that a slightly higher level of toxicity in Sample 3
might be due to higher oxidative stress that might induce cell
Andrea Elizabeth Melgar Aguilar, Ana Paula Fagundes, Domingos Lusitâneo Pier Macuvele et al. Materials Letters 284 (2021) 129013

Fig. 2. TEM micrographs of the Sample 1 (A), Sample 2 (B) and Sample 3 (C). Magnification of TEM micrographs to show the difference of nanogrooves: Sample 1 (D) and
Sample 2 (E).

Fig. 3. Metabolic activity of L929 treated with nHA nanoparticles. A. Sample 1B. Sample 2, and C. Sample 3 at different concentrations for 24 h and 48 h. Data is represented as
mean of percent cell growth ± SD. Three replicates were set up for each treatment group (n = 3) with experiments repeated in duplicate. * Means are significantly different
from the control at p less than 0.05.

apoptosis as the incubation time increases [10]. Regarding a [3]. Therefore, these findings suggest that spherical-like and rod-
shorter incubation period, rod-like morphology might have caused like nHA particles interact differently with fibroblast in most
higher mechanical stress on the cell, which might lead to apoptosis aspects.

3
Andrea Elizabeth Melgar Aguilar, Ana Paula Fagundes, Domingos Lusitâneo Pier Macuvele et al.
4. Concluding remarks
In this study, nanohydroxyapatite was synthesized via chemical
precipitation method using Euclea natalensis roots extracts as tem-
plate for the first time. The particles obtained from this green pro-
cedure displayed a different morphology and surface topography
when compared to a traditional synthesis. The results obtained
indicated that the influence of nHA on cell cytotoxicity depended
on nanoparticle shape, concentration of nHA, and incubation time.
The particles produced with the green template exhibited less cel-
lular toxicity at shorter incubation periods. Overall, the experi-
ments indicate minimal in vitro cytotoxicity. The results
presented in this short communication are preliminary and addi-
tional experiments are still necessary to deep understanding the
interaction of nHA with different shapes and cells.
CRediT authorship contribution statement
Andrea Elizabeth Melgar Aguilar: Conceptualization, Method-
ology, Data curation, Investigation, Writing - original draft, Writing
- review & editing. Ana Paula Fagundes: Conceptualization,
Methodology, Investigation, Writing - original draft, Writing -
review & editing. Domingos Lusitâneo Pier Macuvele: Conceptu-
alization, Methodology, Investigation, Writing - original draft,
Writing - review & editing. Karina Cesca: Methodology, Data cura-
tion, Investigation, Writing - original draft, Writing - review & edit-
ing. Luismar Porto: Resources, Methodology, Writing - original
draft, Writing - review & editing. Natan Padoin: Supervision,
Resources, Methodology, Writing - original draft, Writing - review
& editing. Cíntia Soares: Supervision, Resources, Methodology,
Writing - original draft, Writing - review & editing. Humberto Gra-
cher Riella: Supervision, Resources, Writing - original draft, Writ-
ing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Council for Scientific
and Technological Development (CNPq, Brazil) and the
Materials Letters 284 (2021) 129013
Coordination for the Improvement of Higher Education Personnel
(CAPES, Brazil). We also thank the LCME/UFSC for TEM analysis
and EQA/UFSC for FTIR analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.matlet.2020.129013.
References
[1] A.P. Fagundes, D.L.P. Macuvele, N. Padoin, C. Soares, H. Gracher Riella, A novel
ultrahigh-molecular-weight polyethylene-based nanocomposite for
contaminants adsorption in aqueous systems, Mater. Lett. 240 (2019) 197–
200.
[2] R. Chen, J. Shen, The synthesis of hydroxyapatite crystals with various
morphologies via the solvothermal method using double surfactants, Mater.
Lett. 259 (2020) 126881, https://doi.org/10.1016/j.matlet.2019.126881.
[3] Z. Xu, C. Liu, J. Wei, J. Sun, Effects of four types of hydroxyapatite nanoparticles
with different nanocrystal morphologies and sizes on apoptosis in rat
osteoblasts: Effects of hydroxyapatite nanoparticles on rat osteoblasts, J.
Appl. Toxicol. 32 (6) (2012) 429–435.
[4] A. Albanese, P.S. Tang, W.C.W. Chan, The Effect of Nanoparticle Size, Shape, and
Surface Chemistry on Biological Systems, Annu. Rev. Biomed. Eng. 14 (1)
(2012) 1–16.
[5] X. Ji, Z. Fang, X. Fan, J. Deng, R. Shan, Q. Li, C. Liu, Novel sodium acetate
promoted, induced and stabilized mesoporous nanorods: Hydroxyapatite
nanocaterpillars, Mater. Lett. 271 (2020) 127857, https://doi.org/10.1016/
j.matlet.2020.127857.
[6] S.S.A. Abidi, Q. Murtaza, Synthesis and Characterization of Nano-
hydroxyapatite Powder Using Wet Chemical Precipitation Reaction, J. Mater.
Sci. Technol. 30 (4) (2014) 307–310.
[7] R.A. Youness, M.A. Taha, H. Elhaes, M. Ibrahim, Molecular modeling, FTIR
spectral characterization and mechanical properties of carbonated-
hydroxyapatite prepared by mechanochemical synthesis, Mater. Chem. Phys.
190 (2017) 209–218.
[8] X. Ji, Q. Li, C. Liu, F. Qi, Y. Zhao, Q. Chen, C. Liu, H. Gao, A novel surface
patterning nanorod: Hydroxyapatite nano labyrinth, promoted, induced and
stabilized by tetrahydrofuran, Microporous Mesoporous Mater. 294 (2020)
109904, https://doi.org/10.1016/j.micromeso.2019.109904.
[9] H. Zhang, F. Qing, H. Zhao, H. Fan, M. Liu, X. Zhang, Cellular internalization of
rod-like nano hydroxyapatite particles and their size and dose-dependent
effects on pre-osteoblasts, J. Mater. Chem. B 5 (6) (2017) 1205–1217.
[10] S. Pujari-palmer, S. Chen, S. Rubino, H. Weng, W. Xia, H. Engqvist, L. Tang, M.
Karlsson, Biomaterials In vivo and in vitro evaluation of hydroxyapatite
nanoparticle morphology on the acute in flammatory response, 90 (2016) 1–
11. doi:10.1016/j.biomaterials.2016.02.039.