Materials Today: Proceedings 23 (2020) 111–118

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Materials Today: Proceedings

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Hydroxyapatite reinforced natural polymer scaffold for bone tissue

regeneration
Sreepadmini Ragunathan ⇑, Gopu Govindasamy, D.R. Raghul, M. Karuppaswamy, R.K. VijayachandraTogo
Department of Biomedical Engineering, Sri Ramakrishna Engineering College, Coimbatore 22, India

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

Article history: Medical Advances had led to an increase in life expectancy. However one of the challenges confronted by
Received 13 March 2019 the orthopaedic surgeon is the repair and restoration of skeletal bone defects resulting from trauma.
Received in revised form 28 July 2019 Nano Hydroxyapatite combined with natural polymer starch can be used as a biomaterial for making
Accepted 30 July 2019
scaffolds for the bone tissue regeneration process. This is used to improve the biocompatibility of the
Available online 22 August 2019
scaffolds for living cells. The materials are synthesized by Wet Chemical Method in liquid phase, dried
to obtain solid particles and solvent casting technique is used for making the scaffolds in the ratio 1:1.
Keywords:
Cytotoxicity test revealed that obtained scaffold materials are highly compatible and non-toxic to the
Biomaterial
Hydroxyapatite
body.
Polymer Ó 2019 Elsevier Ltd.
Scaffold Peer-review under responsibility of the scientific committee of the Advanced Materials for Clean Energy
Biocompatible and Health Applications (AMCEHA).
Cytotoxicity

1. Introduction
A bone is a complex tissue and rigid organ which provides inter-
nal support for higher vertebrates and an active tissue that is com-
posed of different types of bone cells. Bones support and protect
various parts of the body. Bone is not a solid material instead it
is a matrix and the bone tissues are mostly made up of composite
materials containing inorganic mineral calcium phosphate which
is named as calcium hydroxyapatite based on its chemical arrange-
ment. This bone mineral provides rigidity to the bone and collagen,
an elastic protein, provides fracture resistance. Defects and func-
tional bone disorders have become a global health problem and
bone repair has become a major clinical and socio – economical
need with the increasing aging population and social development.
Current treatment for bone injuries largely focuses on replace-
ment of defect bones that are limited by many aspects such as
amount of donor tissue available, complications at the donor site.
The basic idea of tissue regeneration is to take the advantage of
the natural healing potentials of the patients and holds great pro-
mise for the future treatment of large bone defects. The main three
ingredients of tissue engineering and tissue regeneration process
are signals, stem cells and scaffolding technique. Tissue Engineer-
ing has evolved out of the need to repair organs and damaged
⇑ Corresponding author.
E-mail address: srpadmini11@gmail.com (S. Ragunathan).
tissues due to any kind of disease or injury. The concept of tissue
engineering embodies the creation of scaffold structure, which
has an appropriate physical, chemical and mechanical property
inorder to enable the cell penetration and tissue formation in three
dimensions.
Basically, the scaffold structure must be porous and biodegrad-
able. Sometimes the long term retention of the cell scaffold causes
hindrance to natural process of bone regeneration. Hence it is nec-
essary to control the time of scaffold degradation. The mechanical
strength of the scaffold has great impact in the bone tissue regen-
eration process. The scaffolds with a low mechanical strength, gets
deformed readily as it can’t be used to regenerate the bulky tissues.
A highly porous scaffolds gears to form a fully interconnected
structure and contains a lot of open pores for the migration of
the cells and nutrients.
Porosity plays vital role in scaffold that provides capillary
ingrowth, cell – matrix interaction, nutrient supply and waste
removal of cells grown on the scaffolds. The pore structure is nec-
essary for the infiltration of cells into the scaffold, the supply of
oxygen and nutrients to the cells proliferated and to wash out
the cell wastes. Especially hydroxyapatite and calcium phosphate
resembles the mineral phase of the bone and are characterized as
biocompatible, bioactive and osteoconductive in nature. To control
the degradation and resorption rate, composites of slowly degrad-
ing hydroxyapatite was used.

https://doi.org/10.1016/j.matpr.2019.07.712
2214-7853/Ó 2019 Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the Advanced Materials for Clean Energy and Health Applications (AMCEHA).
112 S. Ragunathan et al. / Materials Today: Proceedings 23 (2020) 111–118

Ceramics has been used as a bone substitute in bulk, seeded improvement of biodegradable property of the polymer material
intra – operatively with cells from bone marrow. As the Bioceram- and also about the replacement of starch in place of other
ics resembles good biocompatibility and bioactivity, they are used polymers.
mostly for the hard tissue replacements.
Hydroxyapatite has been extensively studied as a replacement
2. Proposed methodology
for the bone material but it lacks sufficient tensile strength capac-
ity in bulk. Though polymers are used as implants, only limited
The following Fig. 1 shows the overall methodology of synthesis
polymers are used for the bone replacement due to limitation of
and scaffolding technique of Biomaterials.
its mechanical properties. The choice of polymer selection is based
on the properties such as: Mechanical properties in case of load
bearing application, Bio-degradation in case implants has to be 2.1. Synthesis of hydroxyapatite
eliminated after certain period of time, ability to bond with the
bone or to induce the bone growth. The scaffold structure thus Hydroxyapatite is a naturally occurring mineral form of bone
formed must resist physiological forces within the implantation calcium apatite. HA is a main component of bone, which is con-
site and also it should have sufficient strength and stiffness to stantly synthesized and degraded naturally in living bone. HA pos-
function for a period until in vivo tissue ingrowth has filled the sess exceptional biocompatibility and bioactivity properties with
scaffold matrix. An equal or excess strength ensures that the scaf- respect to the bone cells and tissues, which is due to its similarity
folds have an equivalent or better load bearing capabilities than with hard tissue bone. Calcium phosphate biomaterial is used
the natural bone. instead in place of HA and used to form the main mineral part of
In 2004, FERRAZ et al. [13], conducted studies to analyze the dif- calcified tissues. Nano HA is being used as a biomaterial in pros-
ferent methodologies adopted for the preparation of HA nanoparti- thetic applications and bone tissue applications, due to its similar-
cle and reported that the wet chemical synthesis methodology ity in size, crystallography and chemical composition with the
offered a bulk yield in large quantity at quicker rate than other human hard tissues and outstanding properties of biocompatibil-
conventional methods. The shape, size and specific surface area ity, bioactivity, osteoconductivity and non-toxicity nature.
of the HA nanoparticles obtained by this method are very sensitive HA is synthesized by wet chemical synthesis method. Wet
to the reactant addition rate, and to the reaction temperature. chemical synthesis is a type of bottom – up approach where the
In 2004, Salgado et al. [14], investigated the various techniques particles in smaller size are increased to obtain the required size.
used for scaffolding in the field of tissue engineering and regener- Here, the synthesis of particles is done in liquid phase and size of
ative medicine. Advantages and disadvantages of various scaffold- the particle can be controlled by capping agents such as charged
ing techniques have been referred from this paper for moulding of molecules, surfactant and polymers which may help in preventing
biomaterials. the agglomeration.
In 2006, Matthias Schieker et al. [12], conducted an experiment Since HA is a form of calcium phosphate, precursors used here
of various biomaterials, for using as scaffolds. It was found that, the includes Calcium Chloride and Disodium Hydrogen Phosphate for
most commonly used raw materials for these RP processes are Calcium and Phosphate respectively. The ratio of calcium to phos-
ceramics, polymers and composites that have already been tested phate (Ca/P) used here is 1.67. Polyethylene Glycol (PEG) is used as
in vivo. The combination of individual custom-made scaffolds with surfactant that forms as a protective layer among the particles in
stem cells might offer new therapeutic options for the treatment of solvent and controls the size and prevents of agglomeration of
bone defects by tissue engineering strategies in the near future. the particles. Sodium Hydroxide is used as a reducing agent that
In 2006, Hoi-Yan Cheung et al. [11], the integration of compos- reduces the precursors of calcium and phosphate to form
ite techniques to fabricate high strength and durable biodegrad- hydroxyapatite.
able polymer-based composites and Properly applying right Calcium Chloride and Disodium Hydrogen Phosphate are taken
polymer with the considerations of porous size, degradation rate at a molarity of 1.5 M and 0.9 M respectively. Precursors are held
and surface morphology, as a parent material with mixing with for continuous stirring for about 30 min and surfactant is added
high-strength fibers can indeed provide a good alternative for to the stirring solution, drop by drop. Reducing agent is added to
existing polymer and metallic based biocompatible materials for the stirrer that forms the hydroxyapatite in drop wise and then
scaffold applications and the fundamental information for the
development of Bio- Engineered scaffolds has been provided in this
paper.
In 2008, Ryan K. Roeder et al. [9], conducted an experiment for
the reinforcement of hydroxyapatite with polymer for the ortho-
paedic implant fixation, synthetic bone graft substitutes and tissue
engineering scaffolds. HA-reinforced polymers will remain fruitful
and an active area of biomaterials research for the foreseeable
future. Many aspects of the composite structure can be tailored
in order to design for specific mechanical, biological, and surgical
functions.
In 2009, Yasuhiko Tabata [8], investigated the biomaterial tech-
nology plays an important role in the creation of the cell environ-
ment for the new therapeutic strategy and it is indispensable to
provide cells with a local environment that enhances and regulates
their proliferation and differentiation for cell-based tissue
regeneration.
In 2009, Isabelle Vroman et al. [10], studied on various
biodegradable polymers for tissue engineering applications. This
paper reviews about the concept and need for biodegradable poly-
mers tissue engineering applications and technology developed in Fig.1. Overall Methodology.
 S. Ragunathan et al. / Materials Today: Proceedings 23 (2020) 111–118
stirred for half an hour, it forms white precipitate which is cen-
trifuged, washed, cleaned and kept for drying in hot air oven.
2.2. Synthesis of natural polymer
Starch is a carbohydrate consisting of large number of glucose
units joined by glycoside bonds. Starch can be oxidised and
reduced, and may precipitate in the formation of hydrogen bonds.
Starch granules exhibit hydrophilic properties and string inter-
molecular association via hydrogen bonding formed by the hydro-
xyl groups on the granule surface. The hydrophilicity of starch can
be used to improve the degradation rate of some degradable
hydrophobic polymers. Starch is totally degradable in a wide vari-
ety of environments and can be hydrolysed into glucose by
microorganism or enzymes. Starch is not used directly, since it is
poor in dimensional stability and mechanical properties. Starch
polymer is prepared by taking urea and starch in a beaker at ratio
1:1 respectively. Samples are dispersed in 110 ml of distilled water
and held for stirring continuously at temperature of about 95 °C.
Then, the obtained solution is cleaned, washed and kept for drying.
2.3. Scaffolding technique
2.3.1. Solvent casting
Solvent Casting is a type of technique used to make scaffolds
that is used for bone tissue applications. Solvent Casting, otherwise
known as Particulate Leaching, is probably the best known and
widely used method for preparation of bone tissue engineering
scaffolds. This method was first described by Mikos et al. which
consists of dispersing calibrated mineral or organic particles in a
polymer solution. This technique allows for an increase in pore
interconnectivity and salt particles produce a porous polymer
matrix. The main disadvantage of solvent casting method is usage
of toxic solvents and limitation to produce thin wafers.
Starch polymer and HA are taken in the ratio of 1:1. This ratio
mixture is dispersed in 5 ml distilled water at 1:1 ratio and kept
for stirring about few minutes. Poly Vinyl Alcohol (PVA) is taken
at 10% weight of mixture sample and mixed with stirring beaker.
Then Sodium Chloride salt is added to stirring solution at required
weight which is used for obtaining pores in the scaffold. After stir-
ring solution is poured into the mould and kept for air drying for
about 2–3 days. Fig. 2 Shows the moulded scaffold by Solvent Cast-
ing Technique.
2.4. Cytotoxicity test – MTT assay
MTT Assay is a colorimetric assay for assessing cell metabolic
activity. NAD(P)H – dependent cellular oxidoreductase enzymes
Fig. 2. Scaffold Biomaterial.
113
are capable reducing tetrazolium dye MTT into its insoluble for-
mazan, which is purple colour. Tetrazolium dyes can also be used
for measuring cytotoxicity (loss of viable cells) or cytostatic activ-
ity (shift from proliferation to quiescence).
MTT Assay is a type of cytotoxicity test of cells which is a qual-
ity of being toxic to cells. Yellow MTT is reduced to purple for-
mazan in mitochondria of living cells. The absorbance of this
coloured solution can be quantified by measuring at certain wave-
length (usually between 500 and 6000 nm) by spectrophotometry.
The absorption max is dependent on solvent employed. This reduc-
tion takes only when mitochondrial reductase enzymes are active
and therefore, reduction can be directly related to the number of
viable (living) cells.
The culture medium from L292 monolayer is replaced with
fresh medium. Test sample in triplicates are added on the cells
and after incubation at 37 ± 1 °C for 18 h, MTT are added in all
the wells and incubated for 4 h. The culture medium is supple-
mented with foetal bovine serum. After incubation DMSO is added
in the wells and read at 570 nm using photometer. Cytotoxicity
and cell viability are calculated by following formula
Cytotoxicity ¼ ½ðControl  TreatedÞ=Control  100
Cell Viability ¼ Treated=Control  100
3. Results and discussions
3.1. Characterisation of HA
3.1.1. Dynamic light scattering (DLS)
Dynamic Light Scattering (DLS) is a non – invasive, well estab-
lished technique for measuring the size and size distribution of
molecules and particles typically in a submicron region. Fig. 3 dis-
plays the Debye plot of KC/R vs. concentration and size distribution
of the particles. The average size distribution of the particles in the
dispersion medium is 174.2 ± 14.1 nm.
3.1.2. Fourier transform infrared spectroscopy (FTIR)
Fourier Transform Infrared Spectroscopy (FTIR) is a technique
used to obtain an infrared spectrum of absorption or emission of
a solid, liquid and gas and indicates the presence of functional
groups or side chains. Fig. 4 displays the graph Transmittance (%)
vs. Wavenumber (cm1) of HA which is characterised using FTIR
that resembles peaks at 3337.76 cm1, 1635.53 cm1,
1025.27 cm1, 873.25 cm-1, 633.15 cm1. In the spectrum of
hydoxyapatite, the band at 600–633 cm1 is due to characteristic
bands of (PO3 4 ). In phosphate network, bending and stretching
modes of P-O vibrations are present as bands around 600 cm1
and 1025 cm1, respectively. The characteristic bands of the
1
(HPO24 ) groups is observed at 873 cm
3.1.3. X – Ray diffraction
X – Ray diffraction is a tool for identifying atomic and molecular
structure of crystal, in which the crystalline atoms cause beam of
incident X – rays to diffract into many specific directions. The
peaks are indexed according to the standard pattern JCPDS card
no: 89-6438. The XRD pattern of nano hydroxyapatite shows shar-
per peaks which indicate better crystallinity. The peak positions
are in good agreement with the JCPDS (89-6438). Fig. 5 shows that
prepared nanoparticle is found to be hydroxyapatite which has
been confirmed from the following XRD graph.
114 S. Ragunathan et al. / Materials Today: Proceedings 23 (2020) 111–118

Fig. 3. DLS of Hydroxyapatite.

Fig. 4. FTIR of Hydroxyapatite.

3.2. Characterisation of natural polymer
3.2.1. Fourier transform infrared spectroscopy (FTIR)
Fourier Transform Infrared Spectroscopy (FTIR) technique is
mainly used where spectra and low concentration of sample is
required and also in case of obtaining quick spectrum. Fig. 6. dis-
plays the FTIR result Transmittance (%) vs. Wavenumber (cm1)
of polymer which resembles peaks at 3858.80 cm1,
3746.36 cm1, 3674.33 cm1, 3648.36 cm1, 3587.89 cm1,
3441.02 cm1, 3244.02 cm1, 2635.76 cm1, 1745.45 cm1,
 S. Ragunathan et al. / Materials Today: Proceedings 23 (2020) 111–118 115

Fig. 5. XRD of Hydroxyapatite.

Fig. 6. FTIR of Natural Polymer (Starch).

1698.70 cm1, 1648.23 cm1, 1514.48 cm1, 947.41 cm1,
1 1 1
803.61 cm , 772.35 cm , 655.12 cm .The O–H stretching for
the natural polymer starch occurred at 3746.36 cm1 .The peaks
at 3674.36 cm1 was observed as a result of C–H bondstretching.
The peaks at 1514 cm1 and 1530 cm1 were assigned as the C–
O bond stretching. This indicates that compounds belonging to
hydroxyl group, hydrocarbon and aldehydes group are present in
the natural polymer starch.
3.3. Characterisation of scaffold
3.3.1. X – Ray diffraction
X – Ray Diffraction is a common technique use for the study of
crystal structure and atomic spacing. This technique is based on
the principle of Bragg’s Law (nk = 2d sin h). The peaks are indexed
according to the standard pattern ICDD 09-432 standard. The
peak positions are in good agreement with the ICDD 09-432
standard.
Fig. 7 shows the XRD result of scaffold biomaterial and resem-
bles the atomic arrangement of lattices and crystal arrangement.
3.3.2. Fourier transform infrared spectroscopy (FTIR)
Fourier Transform Infrared Spectroscopy (FTIR) is a technique
that could also be used to verify the identity and specification of
raw materials and products and identification of unknown sam-
ples. Fig. 8 shows the graph of hydroxyapatite with polymer Trans-
mittance (%) vs. Wavenumber (cm1) in the ratio 1:1 and peaks are
obtained at 3858.80 cm-1, 3746.36 cm1, 3674.33 cm1,
3648.36 cm1, 3337.76 cm1, 1745.45 cm1, 1698.70 cm1,
1648.23 cm1, 1635.53 cm1, 1514.48 cm1, 1025.27 cm-1,
947.41 cm1, 873.25 cm1, 633.15 cm1.In the spectrum O–H bond
stretching for the natural polymer starch occurred at
116 S. Ragunathan et al. / Materials Today: Proceedings 23 (2020) 111–118

Fig. 7. XRD of Scaffold.

Fig. 8. FTIR of Scaffold.

3746.36 cm1.The absorption bands at 1745.45 cm1 suggests the
presence of CO2
3 in hydroxyapatite structure. The characteristic
1
bands of the (PO3 4 ) is observed at 1514.48 cm and (HPO24 )
1
groups is observed at 1025.27 cm respectively indicating the
presence of both hydroxyapatite and natural polymer starch in
the obtained scaffold material.
3.3.3. Scanning electron microscope (SEM)
SEM is a type of electron microscope that produces images of a
sample by scanning with a focussed beam of electrons. The elec-
trons interact with atoms present in the sample, producing various
signals that containing information about the sample’s surface
topology and composition. The electron beam is generally scanned
in a raster scan pattern and specimens can be observed in high vac-
uum, low vacuum, in wet conditions and at a wide range of cryo-
genic and elevated temperatures. Figs. 9 and 10 show images of
scaffolds obtained at different magnification.
The SEM images show that the pores are formed with size of
16.3 mm. The surface topology of the scaffold shows that the dense
structure is obtained due to high density characteristic of PVA. The
obtained pore size is compared with the reference paper Gunawan
et al. [1], in which the morphology of sample was observed using
SEM and the porosity size obtained was about 17.5 lm and
12.5 lm.
3.3.4. Cytotoxicity test
MTT Assay is a colorimetric assay for assessing cell metabolic
activity. NAD (P) H – dependent cellular oxidoreductase enzymes
are capable reducing tetrazolium dye MTT into its insoluble for-
mazan, which is purple colour. Tetrazolium dyes can also be used
 S. Ragunathan et al. / Materials Today: Proceedings 23 (2020) 111–118 117

Fig. 9. SEM of Scaffold at Magnification 2.33 K X.

Fig. 10. SEM of Scaffold at Magnification 3.05 K X.

for measuring cytotoxicity (loss of viable cells) or cytostatic activ- Table 1

ity (shift from proliferation to quiescence). Observation of MTT Assay Test.

The culture medium from L292 monolayer is replaced with Sample Particulars Cytotoxicity Cell Viability Cytotoxic
fresh medium. Test sample in triplicates are added on the cells (%) (%) Reactivity
and after incubation at 37 ± 1 °C for 18 h, MTT are added in all Hydroxyapatite 50 ml 6.519 93.48 None
the wells and incubated for 4 h. The culture medium is supple- Hydroxyapatite 75 ml 8.97 91.02 None
mented with foetal bovine serum. After incubation DMSO is added Hydroxyapatite 9.05 90.94 None
100 ml
in the wells and read at 570 nm using photometer. Table. 1 shows
the observation result of MTT Assay test of scaffold biomaterial.
Cytotoxicity and cell variability are calculated by following
formula
The sample hydroxyapatite reinforced with polymer at a ratio of
Cytotoxicity ¼ ½ðControl  TreatedÞ=Control  100 1:1 showed none cytotoxic reactivity to mammalian cell line that
the culture medium supplemented with foetal bovine serum after
Cell Viability ¼ Treated=Control  100 24 h contact. Control gave none cytotoxic reactivity as expected.
118 S. Ragunathan et al. / Materials Today: Proceedings 23 (2020) 111–118
4. Conclusion
Wet Chemical Synthesis method is used to produce good yield
of the materials in large quantity. Solvent Casting of scaffolding
technique is commonly used to produce good mechanical strength
and 90% of pores are produces with this Solvent Casting technique
with pore range varies from 14 mm to 17 mm. The size of pores is
dependent on the size of salt used which is responsible for pore
formation. Poly Vinyl Alcohol exhibits high density property and
hence it helps to form high density scaffold material that suits
for load bearing of regeneration of bone. This helps to increase
the mechanical strength of the scaffold for load bearing capacity.
The scaffold formed is tested for Cytotoxicity which results in
non-toxicity to mammalian cells. Hence, this scaffold material is
safe to be used as implant and do not show any toxicity to
recipient.
5. Future scope
Carbon Nano Tubes can be used for increasing mechanical
strength of the scaffold due to its high load bearing property. Scaf-
folds can also be used for drug delivery applications such as dia-
betes, cancer, etc. These are used for controlled release of drug
after implantation over period of time. The cytotoxicity of the scaf-
fold material can be further reduced by using different ratio com-
bination and methodology in the synthesis of material such as
natural synthesis and materials obtained from naturally. Iron
Oxide nanoparticles can be integrated with biomaterials and used
for anti – microbial applications. Fibres are used for increasing the
degradability rate of starch polymer. Sisal fibre is used for stability
and mechanical strength of the biomaterial. Pore size of the scaf-
fold can be controlled by optimising the size of salts used for for-
mation of pores in Solvent Casting. Solvent Casting scaffolding
method can be replaced with Phase Inversion method that has
advantage of formation of pores with variable size. Fibre bonding
technique can also be used for scaffolding since three dimensional
scaffold can be formed with this method that provide large surface
area to bond with. Synthesis of hexagonal shaped HA provides bet-
ter results since natural HA in bone has shape of hexagon and
enables easy bonding with bone after implantation.
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Junjie Deng, Yiyuan Cao, Cijun Shuai, Shuping Peng, Current progress in
bioactive ceramic scaffolds for bone repair and regeneration, Int. J. Mol. Sci. 15
(2014) 4714–4732.
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Brizuela, Javier Aragón, A Comparative study of hydroxyapatite nanoparticles
synthesized by different routes, Quim. Nova 35 (9) (2012) 1724–1727.
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[18] Minna KellomaK ki, Henna Niiranen, Katja Puumanen, Nureddin Ashammakhi,
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