Materials Science and Engineering C 76 (2017) 518–527

Contents lists available at ScienceDirect

Materials Science and Engineering C

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

Investigation of chronic toxicity of hydroxyapatite nanoparticles

administered orally for one year in wistar rats

N.S. Remya 1, S. Syama 1, A. Sabareeswaran, P.V. Mohanan ⁎

Division of Toxicology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura, Thiruvananthapuram 695 012, Kerala, India

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

Article history: Although the toxicity/biocompatibility of hydroxyapatite nanoparticles (nano HA), a prospective nano biomate-
Received 30 September 2016 rial is extensively studied, its interaction on biological systems following chronic exposure is less exploited. In the
Received in revised form 30 December 2016 present study, Wistar rats were given various concentrations of nano HA in the diet to determine the chronic tox-
Accepted 10 March 2017
icity and potential carcinogenicity. Altogether 140 rats were used for the study under various administration dos-
Available online 15 March 2017
ages along with control. The animals were sacrificed after 12 months of controlled continuous dosing. All in-life
Keywords:
parameters, including body weight, food consumption, clinical observations, survival, biochemical and hematol-
Nanomaterials ogy, were unaffected by the chronic exposure of nano HA orally. Similarly, gross and histopathological evaluation
Hydroxyapatite was also unchanged following exposure to nano HA. No evidence of nano HA-related lesions or Nano HA-induced
Chronic toxicity neoplasia was suggested in this rodent bioassay study.
Histopathology © 2017 Elsevier B.V. All rights reserved.

1. Introduction
Nanomaterials as defined as materials with at least one external di-
mension in the size range from approximately 1–100 nm have revolu-
tionized the present era in almost all aspect of human life starting
from nano electronic devices, paints, cosmetic and food industries as
well as health care systems. Owing to the versatile properties of nano
scale materials when compared to their bulk form, they have emerged
as an excellent area of applied research. Hydroxyapatite nanoparticles
(nano HA), a prospective biomaterial is used in various biomedical
areas such as drug delivery, tissue engineering, bone grafts, dental im-
plants and fillings, etc. [1–4].
Hydroxyapatite is a natural constituent of human bone and is bio-
compatible and biodegradable, has got osteo-conductive, osteo-
inductive and osteo-integrative properties and hence it has found
wide application as a bone substitute. Even though the nanoscale
form Hydroxyapatite (nano HA) are documented to be biocompati-
ble [5–7], it is unknown whether they are safe when applied in med-
ical scenarion due to their nano scale particle size. Nano particles
owing to their nano scale size can be internalized by the cells
where they can interact with the biological molecules altering the
⁎ Corresponding author.
E-mail address: mohanpv@sctimst.ac.in (P.V. Mohanan).
1
Equal contribution.
cell response which could affect the cells in a deleterious manner
leading to toxicological response [8]. The acute toxicity studies of
nano HA in vivo [9], sensitization studies [10] in vitro interaction
studies with various cell sources [11], have already been document-
ed in several references, whereas those of chronic toxicity and carci-
nogenicity in vivo were not so thoroughly investigated.
The safety and toxicity of nano HA are of growing concern despite
their promising potential in many biomedical applications [12]. The
biological activity and bio kinetics of the proposed bio nanomaterial
are dependent on many parameters such a size shape, chemistry,
charge and surface modification [13]. Chronic toxicity/carcinogenic-
ity studies are therefore essential in identifying the carcinogenic
properties of a chemical, primarily its potential to induce neoplastic
lesions, and its toxicological responses due to chronic exposure.
Identification of target organs is feasible as nanoparticles are carried
by the bloodstream to be lodged in various target organs that could
elicit cumulative toxicological effects. Chronic toxicity studies are
usually conducted in rodent species where the test compound is ad-
ministered over N90 days, and the animals are observed periodically
[14]. The cumulative/long term effect of a test substance is inferred
from such a study that could be extrapolated to its clinical transla-
tion. A combined chronic toxicity and carcinogenicity study often
provides an added advantage such that the experimental animals
could be monitored for the development of tumors due to the toxic-
ity of test compound under consideration [15].

http://dx.doi.org/10.1016/j.msec.2017.03.076
0928-4931/© 2017 Elsevier B.V. All rights reserved.
 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527 519

Hence the present study provides information on the possible health Table 2
hazards likely to emerge following repeated exposure of nano HA over Common clinical signs and observation.

the majority of lifespan in rodents which could be extrapolated for its Clinical Observed sign Involved system (s)
safe use in humans. observation

Respiratory Dyspnea (abdominal breathing, CNS, pulmonary, cardiac

gasping), aponoea, cyanosis,
2. Methodology tachypenea, nostril discharges
Motor Decrease/increase somnolence, CNS, somatomotor,
2.1. Synthesis and characterization of hydroxyapatite nano materials loss of righting, anesthesia, sensory, neuromuscular,
catalepsy, ataxia, unusual automatic respiratory
locomotion, prostration, tremors,
In-house synthesized Nano HA was used for the study [16]. Nano HA fasciculation
was synthesized by wet chemical method where calcium phosphate Convulsion Clonic, tonic, tonic-clonic, CNS, neuromuscular,
was precipitated from the aqueous solution of calcium nitrate asphyxial opisthotonos autonomic, respiratory
tetrahydrate (Ca (NO3)2·4H2O) and ammonium dihydrogen ortho- Reflexes Corneal, righting, myotact, light, CNS, sensory, autonomic,
startle reflex neuromuscular
phosphate (NH4H2PO4). The chemicals were procured from Sigma- Ocular signs Lacrimation, miosis, mydriasis, Autonomic, irritation
Aldrich. After aging, freeze drying and calcination, particles of size exophthalmos, ptosis, opacity,
50 nm was obtained. The particles were characterized for their particle iritis, conjunctivitis,
size by Transmission Electron Microscopy (TEM) [Hitachi H-600]. The chromodacryorrhea, relaxatrion of
nicititating membrane
chemical composition of nano HA particles were compared with stan-
Cardiovsacular Bradycardia, tachycardia, CNS, autonomic, cardiac,
dard material using Nicolet Impact 410 FT-IR spectroscopy (Fourier signs arrhythmia, vasodialation, pulmonary
Transform Infrared Spectrometry) and X-ray diffraction (XRD) spec- vasoconstriction
trum was recorded in a diffractometer (Siemens D5005) for phase Salivation Excessive Autonomic
purity. Piloerection Rough hair Autonomic
Analgesia Decrease reaction CNS, sensory
Muscle tone Hypotonia, hypertonia Autonomic
Gastrointestinal Soft stool, diarrhea, emesis, CNS, sensory, GI motility,
2.2. Experimental animals diuresis, rhinorrhea Kidney
Skin Edema, Erythema Tissue damage and
Healthy Wistar rats weighing 150–180 g were used for the study. irritation
They were maintained in a 12 h light/dark cycle at a constant tem-
perature of 22 ± 3 °C and provided with commercially available
feed and filtered fresh drinking water ad libitum. Individual animals
were identified with picric acid marks. In addition to this, each ani-
mal cage was identified by labels having details such as experiment 2.4. Ante mortem observations
number, name, animal number(s) and date of experiment. All ani-
mals were handled humanely, without causing pain or distress and The animals were observed daily for general appearance, behav-
with due care. The care and management of the animals were in ior, signs of morbidity and mortality (Table 2). Each week, all ani-
compliance with the regulations of the Committee for the Purpose mals were given a detailed physical examination that included
of Control and Supervision of Experiment on Animals (CPCSEA), palpation for the presence of tissue masses. Individual body weights
Govt. of India. The animal experiments were carried out after prior and feed intake were determined biweekly for the duration of the
approval from Institutional Animal Ethics Committee and approved study.
institutional protocol.
2.5. Biochemical and hematological parameters
2.3. Experimental design
Blood from optical plexus was collected in EDTA vials and
analyzed for routine hematological parameters such as Hemoglobin
There were 5 groups and each group consisted of 15 male and 15 fe-
(Hb, g/dl), total count (WBC × 10 3 /mm 3 ), red blood corpuscles
male rats. The animals were exposed to nano HA in rat feed at concen-
count (RBC × 106 /mm3 ), platelet count (PLT × 103 /mm3 ), using
trations of 0, 25, 50 and 100 mg/kg body wt. for 90 weeks. Table 1 shows
automated Vet ABC Animal blood counter (ABX Diagnostics,
corresponding nano HA concentrations. Feed intake was recorded daily.
France). For biochemical analysis blood was collected and the
Animals were weighed initially, and weekly till the end of the studies.
serum was analyzed for biochemical parameters such as urea,
Animals were observed twice daily and clinical findings were recorded
Serum Glutamic Oxaloacetic transaminase (SGOT), Serum Glutamic
at 4-week intervals.
Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP),
After 78 weeks the animals on groups1 to 4 were sacrificed and
Gamma-Glutamyl Transferase (GGT), glucose (GLU), cholesterol,
processed for various analyses, while group 5 animals were again ob-
triglycerides, total protein, albumin, calcium, phosphorus, chloride,
served after providing standard diet without nano HA treatment for a
total bilirubin and creatinine using automated biochemistry analyz-
period of 4 weeks.
er, ERBA Manheim XL 300 (ERBA, Mannheim, Germany).

2.6. Postmortem examination

Table 1
Experimental groups.
Groups Animals
1 Control (15♂ + 15♀)
2 Low (15♂ + 15♀)
3 Medium (15♂ + 15♀)
4 High (15♂ + 15♀)
5 High recovery (10♂ + 10♀)
All animals were euthanized using carbon dioxide chamber using
Average daily ingested dose (mg/kg) accepted protocol and subjected to a postmortem examination. At
0 necropsy, all organs and tissues were examined for grossly visible le-
25 sions, and the required tissues were fixed and preserved in 10% neu-
50 tral buffered formalin, for microscopic examination. For all paired
100 organs (e.g., adrenal gland, kidney, ovary, lung, eyes), samples from
100
each organ were examined. The entire gastrointestinal tract was
520 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527
Fig. 1. Physicochemical characterization of hydroxy apatite nanoparticles (A) TEM image showing the particle size b50 nm, (B) XRD spectrum of nano HA, (C) FTIR spectrum of nano HA
and (D) EDS spectrum of nano HA.
opened and potential lesions were collected for microscopic evalua-
tion. Reproductive organs, nervous tissue, thyroid and parathyroid
glands, skin, muscle, urinary bladder tissue etc. were also histopath-
ologically examined.
2.7. Histopathological analysis
Tissues to be examined were trimmed, processed, embedded in par-
affin, sectioned to a thickness of 4–6 μm, and stained with hematoxylin
and eosin (H&E) and examined under a light microscope by a veterinary
pathologist. The observations were assigned a semi-quantitative rank-
ing of the severity grade: normal, minimal, (mild or slight); moderate,
marked, or severe. Neoplastic lesions were generally not assigned a
grade. All tumors and potential target organs were examined
thoroughly.
2.8. Preparation of liver homogenate and anti-oxidant assays
The experimental animals were sacrificed by cervical dislocation
and their liver was rapidly excised, washed in normal saline
and were collected. 10% of tissue homogenate was prepared in phos-
phate buffer (0.1 M, pH 7.4) and was used for the estimation of total
protein, Lipid peroxidation, Glutathione reductase, reduced glutathi-
one, Glutathione peroxidase and Superoxide dismutase assay using
standard protocols with slight modifications.
2.9. Total protein
Total protein in rat liver homogenate was estimated by the method
of Lowry et al. [17] using bovine serum albumin as standard.
2.10. Lipid peroxidation (LPO)
The extent of LPO in rat liver homogenate was determined as the con-
centration of malondialdehyde (MDA) generated by the thiobarbituric
acid reactive substances (TBARS), as described by Ohkawa et al. [18].
The amount of malondialdehyde (MDA) formed was measured spectro-
photometrically at 532 nm and expressed in nmol/mg protein.
2.11. Glutathione reductase (GR)
GR activity in rat liver homogenate was determined by measuring
the reduction of GSSG in the presence of NADPH [19]. Briefly, this
assay measures the rate of NADPH oxidation to NADP +, which is
 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527 521

Fig. 2. (A) and (B) Mean Body weights of experimental animals of male and Female respectively (C) and (D) represents feed intake of experimental animals both male and female
respectively.

accompanied by a decrease in absorbance at 340 nm and can be moni-
tored spectrophotometrically and expressed as units/mg protein. One
GR unit is defined as the reduction of 1 μM of GSSG per minute at 25
°C and pH 7.6.
2.12. Reduced glutathione (GSH)
The level of GSH in rat liver homogenate was determined by the
method of Moron et al. [19], with slight modifications in which Ellman's
reagent or DTNB (5,5′-dithiobis-(2-nitrobenzoic acid), reacts with GSH
Table 3
Clinical observations of experimental group. “N” denotes normal.
to form a spectrophotometrically detectable product at 412 nm. The
change in absorbance is a linear function of the GSH concentration in
the reaction mixture and is based on the reaction of GSH with DTNB
to give a compound that absorbs at 412 nm. The amount of GSH was
expressed as nmol/mg protein.
2.13. Glutathione peroxidase (GPx)
Activity of GPx was assayed using a protocol described by Rotruck
[20]. The remaining GSH after the enzyme catalyzed reaction was com-
plexed with 5,5′dithiobis 2-nitrobenzoic acid (DTNB) that absorbs at
maximum wavelength of 412 nm. Enzyme activity was expressed as
units/mg protein.

Experimental animals 2.14. Superoxide dismutase assay (SOD)

Control Low Medium High High
recovery
Assay of SOD in rat liver homogenate was done using modified pyro-
Sex ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ gallol auto oxidation method [21], which can be spectrophotometrically
No of animals 15 15 15 15 15 15 15 15 10 10 measured at 420 nm and is expressed in units/mg protein.
Respiratory N N N N N N N N N N
Motor N N N N N N N N N N
Convulsion N N N N N N N N N N 3. Results and discussion
Reflexes N N N N N N N N N N
Ocular signs N N N N N N N N N N 3.1. Synthesis and physico chemical characterization of hydroxy apatite
Cardiovascular N N N N N N N N N N
nano materials
Salivation N N N N N N N N N N
Piloerection N N N N N N N N N N
Analgesia N N N N N N N N N N Nano HA was synthesized by wet chemical method and was charac-
Muscle tone N N N N N N N N N N terized by Transmission electron microscopy; X-ray diffraction analysis
Gastrointestinal N N N N N N N N N N and Fourier transform infrared spectral analysis. TEM images showed
Skin N N N N N N N N N N
that the particles were having a rod like structure with particle size
522 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527

Table 4
Hematology data of the experimental group animals.

Control Low dose Medium dose High dose High recovery

Male
No. of animals examined 6 6 6 6 6
Hb (g/dL) 15.15 ± 1.05 15.51 ± 0.72 15.3 ± 0.25 15.35 ± 0.48 15.4 ± 0.55
RBC (×106mm3) 8.92 ± 0.63 9.04 ± 0.33 8.83 ± 0.25 8.91 ± 0.38 9.08 ± 0.23
WBC (×103mm3) 8.5 ± 2.83 7.38 ± 0.96 5.9 ± 2.1 7.58 ± 1.58 6.95 ± 2.4
Platelets (×103mm3) 8.38 ± 1.11 8.54 ± 0.82 8.9 ± 1.3 8.16 ± 1.11 9.33 ± 0.87

Differential leukocyte count

Neutrophils 25.6 ± 12.8 21.83 ± 6.3 29.8 ± 17.0 29 ± 18.38 28.28 ± 9.17
Lymphocytes 69.6 ± 12.1 74 ± 8.4 66.8 ± 17.6 69.33 ± 18.97 73.42 ± 9.2
Eosinophils 1.2 ± 0.44 1.3 ± 1.03 1.6 ± 0.54 1.66 ± 0.577 1.14 ± 0.3
Monocytes 5.2 ± 3.8 3 ± 3.5 3 ± 2.16 1 ± 0.6 2.0 ± 1.0
Erythrocyte sedimentation rate 1.00 ± 0.75 1.16 ± 0.75 1.00 + 0.2 1.00 ± 0.1 1.2 ± 0.48
PCV 47.03 ± 3.86 49.33 ± 2.40 47.8 ± 0.85 48.51 ± 1.93 48.48 ± 2.08
MCV 52.66 ± 1.37 54.55 ± 1.32 54.13 ± 1.55 54.48 ± 1.22 53.37 ± 1.58
MCH 16.9 ± 0.35 17.13 ± 0.33 17.35 ± 0.49 17.25 ± 0.45 17.14 ± 0.45
MCHC 32.25 ± 0.45 31.4 ± 0.33 32.03 ± 0.25 31.65 ± 0.51 31.72 ± 0.34

Female
No. of animals examined 6 6 6 6 6
Hb (g/dL) 14.6 ± 0.61 14.95 ± 0.46 14.68 ± 0.37 14.71 ± 0.39 14.33 ± 0.37
RBC (×106mm3) 7.7 ± 0.3 8.05 ± 0.26 7.9 ± 0.15 7.9 ± 0.36 7.8 ± 0.34
WBC (×103mm3) 5.8 ± 1.7 4.9 ± 1.2 4.0 ± 1.3 4.8 ± 0.39 4.08 ± 1.0
Platelets (×103mm3) 7.6 ± 0.67 9.05 ± 0.66 9.3 ± 0.35 8.27 ± 1.24 10.04 ± 1.2

Differential leukocyte count

Neutrophils 31.16 ± 13.6 22.66 ± 8.6 28.16 ± 6.43 21.33 ± 12.4 16 ± 6.60
Lymphocytes 63.6 ± 13.0 74.0 ± 10.54 68.8 ± 6.7 75.66 ± 13.2 81.16 ± 6.27
Eosinophils 1.4 ± 0.5 0.66 ± 0.51 2.1 ± 2.4 1.5 ± 0.54 1.83 ± 0.98
Monocytes 4.1 ± 2.7 3.8 ± 2.1 1.3 ± 0.5 1.8 ± 0.4 1.83 ± 0.98
Erythrocyte sedimentation rate 0.83 ± 0.75 0.16 ± 0.4 0.3 ± 0.5 0.16 ± 0.40 0.6 ± 0.8
PCV 44.4 ± 1.69 46.36 ± 1.33 44.9 ± 1.4 45.15 ± 1.66 43.6 ± 1.14
MCV 57.16 ± 0.99 57.58 ± 0.73 56.7 ± 1.7 57.2 ± 1.67 55.95 ± 1.25
MCH 18.85 ± 0.27 18.6 ± 0.23 18.5 ± 0.47 18.65 ± 0.59 18.36 ± 0.39
MCHC 32.98 ± 0.54 32.2 ± 0.11 32.68 ± 0.41 32.6 ± 0.45 32.83 ± 0.12

below 50 nm. The X-ray diffraction pattern of the Nano HA matches with
the standard pattern of the Hydroxyapatite (ICDD NO-PDF00-009-0432)
demonstrating the phase purity of Nano HA material. The FTIR spectral
analysis of Nano HA showed that the hydroxyl stretching was observed
at 3568 cm−1 and the different phosphate vibrations were observed at
1090, 1036, 962 and 476 cm−1 and thus the presence of phosphate and
hydroxyl group confirms the chemical nature of hydroxyapatite (Fig. 1).
3.2. Antemortem evaluations
3.2.1. Body weight and food consumption
Body weight gain and feed intake remained unaffected in both sexes
at all doses studied during and recovery period. Mean body weights
were comparable with that of control in male and female rats studied
(Fig. 2A and B). The mean values for feed intake/animal were slightly
increased in male group compared to female group. However no signif-
icant change were observed in treated group animals compared to con-
trol group animals (Fig. 2C and D).
3.2.2. Clinical observations and mortality
Survival of male and female rats was unaffected by the exposure.
There was no evidence of cannibalism, autolysis in any of the animal
groups. No morbidity or mortality was observed throughout the exper-
imental period along with clinical findings attributable to chronic oral
exposure to nano HA was not observed. Table 2 gives the common clin-
ical signs and observation considered for determining toxicity at the
systemic level following oral exposure to nano HA. Table 3 summarizes
the clinical observations of experimental group animals. The animals
were found to be normal devoid of the treated clinical signs throughout
the experimental period.

Table 5
Biochemical parameters analyzed.

Parameters Control Low Medium High High recovery

Total protein (g/dL) 8.27 ± 0.87 8.87 ± 0.27 8.35 ± 0.33 9.42 ± 0.75 8.25 ± 0.27
Creatinine (mg/dL) 0.74 ± 0.06 0.76 ± 0.09 0.72 ± 0.07 0.78 ± 0.12 0.71 ± 0.04
Urea (mg/dL) 38.85 ± 5.35 35.72 ± 1.24 34.07 ± 2.93 35.92 ± 2.27 31.47 ± 4.25
SGPT (U/L) 162.13 ± 80.78 148.75 ± 36.66 132.75 ± 15.26 132.62 ± 29.84 108.90 ± 17.21
SGOT (U/L) 150.22 ± 40.99 140.03 ± 36.91 124.58 ± 10.82 135.77 ± 22.96 139.88 ± 27.03
Alkaline phosphatase (U/L) 274.17 ± 44.26 275.67 ± 42.38 289.33 ± 26.15 337.33 ± 27.37 261.17 ± 64.13
Glucose (mg/dL) 124.95 ± 27.18 100.38 ± 9.00 100.97 ± 13.21 118.57 ± 13.07 100.43 ± 8.46
Cholesterol (mg/dL) 56.17 ± 11.36 75.33 ± 13.95 64.20 ± 13.92 59.17 ± 14.96 62.50 ± 9.35
Triglycerides (mg/dL) 171.33 ± 76.79 160.00 ± 23.62 140.17 ± 32.29 154.00 ± 33.02 137.67 ± 24.28
Bilirubin total (mg/dL) 0.13 ± 0.03 0.16 ± 0.03 0.15 ± 0.02 0.14 ± 0.03 0.14 ± 0.02
Albumin (BCG) (g/dL) 4.72 ± 0.22 4.86 ± 0.17 4.82 ± 0.38 4.92 ± 0.23 4.65 ± 0.24
Phosphorus (mg/dL) 8.68 ± 2.63 8.38 ± 1.76 7.18 ± 0.82 7.64 ± 1.92 6.63 ± 0.86
Chlorides (mEq/L) 103.37 ± 26.75 111.83 ± 1.84 112.00 ± 1.66 117.85 ± 6.48 111.62 ± 1.96
GGT (U/L) 2.90 ± 0.69 1.17 ± 0.59* 1.93 ± 0.46 1.50 ± 0.34 1.63 ± 0.70
Calcium (mg/dL) 12.27 ± 3.67 14.07 ± 0.41 13.82 ± 0.58 14.18 ± 0.96 11.25 ± 0.43
 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527 523

Table 6
Incidence of non-neoplastic histopathologic findings.

Total incidence
Males Females

Dose level Control Low High Medium High recovery Control Low High Medium High recovery
No of animals 15 10 15 10 10 15 10 15 10 10

Lung
a. BALT proliferation 13 0 15 0 0 13 0 15 0 0
b. Abscess 2 0 0 0 0 1 0 0 0 0

Liver
a. Cyst, fibrosis 1 0 0 0 0 0 0 0 0 0
b. Focal MNC infiltration 0 0 1 0 0 0 0 0 0 0

Kidney
a. Cystic, tubular degeneration and fibrosis 1 0 1 0 0 0 0 0 0 0
b. Focal interstitial MNC infiltration 1 0 4 0 0 0 0 0 0 0

Spleen
a. Hemosiderosis 12 0 9 0 0 12 0 10 0 0

Trachea
MNC infiltration 11 0 10 0 0 2 0 7 0 0

Other organ lesions

a. Follicular hyperplasia of mammary gland – – – – – 0 0 1 0 0
b. Abscess in prepuce 0 0 1 0 0 – – – – –
c. Ovary, cystic – – – – – 0 0 1 0 0

Table 7
Incidence of neoplastic findings in carcinogenicity study.

Total incidence
Males Females

Dose level Control Low High Medium High recovery Control Low High Medium High recovery
No of animals 15 10 15 10 10 15 10 15 10 10

Organs examined
Eyes 0 0 0 0 0 0 0 0 0 0
Esophagus 0 0 0 0 0 0 0 0 0 0
Trachea 0 0 0 0 0 0 0 0 0 0
Pharynx 0 0 0 0 0 0 0 0 0 0
Lungs 0 0 0 0 0 0 0 0 0 0
Heart 0 0 0 0 0 0 0 0 0 0
Liver 0 0 0 0 0 0 0 0 0 0
Kidney 0 0 0 0 0 0 0 0 0 0
Spleen 0 0 0 0 0 0 0 0 0 0
Aorta 0 0 0 0 0 0 0 0 0 0
Thymus 0 0 0 0 0 0 0 0 0 0
Adrenal 0 0 0 0 0 0 0 0 0 0
Pancreas 0 0 0 0 0 0 0 0 0 0
Stomach 0 0 0 0 0 0 0 0 0 0
Duodenum 0 0 0 0 0 0 0 0 0 0
jejunum 0 0 0 0 0 0 0 0 0 0
Ileum 0 0 0 0 0 0 0 0 0 0
Caecum 0 0 0 0 0 0 0 0 0 0
Rectum 0 0 0 0 0 0 0 0 0 0
Lymph nodes 0 0 0 0 0 0 0 0 0 0
Urinary bladder 0 0 0 0 0 0 0 0 0 0
Ovary, FT 0 0 0 0 0 0 0 0 0 0
Uterus, Uterine horns 0 0 0 0 0 0 0 0 0 0
Cervix 0 0 0 0 0 0 0 0 0 0
Vagina, Vulva 0 0 0 0 0 0 0 0 0 0
Skin 0 0 0 0 0 0 0 0 0 0
Muscle 0 0 0 0 0 0 0 0 0 0
Sciatic nerve 0 0 0 0 0 0 0 0 0 0
Cerebrum 0 0 0 0 0 0 0 0 0 0
Cerebellum, pons 0 0 0 0 0 0 0 0 0 0
Spinal cord 0 0 0 0 0 0 0 0 0 0
Pituitary 0 0 0 0 0 0 0 0 0 0
Thyroid 0 0 0 0 0 0 0 0 0 0
Parathyroid glands 0 0 0 0 0 0 0 0 0 0
Salivary glands 0 0 0 0 0 0 0 0 0 0
524 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527

Fig. 3. Histopathology of organs examined (male).[a–f] control heart, liver kidney, spleen, testes and lung and [g–l] High dose test animals heart, liver kidney, spleen, testes and lungs.

3.2.3. Biochemical and hematological parameters
Hematology data of the experimental group animals are summarized
in Table 4. In both sexes, there were no significant differences in the he-
matological data of all the groups treated with respect to that of control
animals. Hematological parameters evaluation is required to rule out he-
matological malignancies and also gives an idea on the health status of the
experimental animals. All the white blood cell types are given as a per-
centage in differentiated leukocyte count and as an absolute number
per cubic mm in total WBC count. The number of red cells is represented
as an absolute number per cubic mm. Malignant cells present in blood
smear is a valid tool for diagnosis of hematological neoplasms [22]. In
the present study, the hematological parameters evaluated (Table 4)
were comparable to that of normal reference range and no significant
pathological changes were noted in the blood smear of all the experimen-
tal animals. Similarly the results of biochemical data provided no sugges-
tion of a compound-related effect in either sex at any dose studied. The
results are summarized in Table 5. Biochemical analysis of serum often
serves as a marker or specific indicator of the health status of tissues or or-
gans. As liver is the primary site of detoxification and a major site of me-
tabolism, it is vulnerable to adverse consequences as a result of exposure
to the toxins of extrinsic as well as intrinsic forms. When the liver cells are
damaged, liver enzymes are increased in liver and released in the blood-
stream. Commonly high level of liver enzymes in the serum is an indica-
tion of damage to liver cells (extensive hepatic necrosis) and even liver
cell death [23]. But often elevations of these enzymes are unexpectedly
encountered on routine blood screening study in healthy individuals as
well. In the present study, there was no significant rise in liver enzymes
in any of the dose studied compared to control. On the other hand elevat-
ed alkaline phosphatase enzyme in high group were observed when com-
pared to control and is of particular interest as it shows the incidence of
active bone formation, since ALP is a byproduct of osteoblast activity.
This enhancement of osteoblast activity may be attributed by the orally

Fig. 4. Histopathology of organs examined (female). [a–f] control heart, liver kidney, spleen, testes and lung and [g–l] High dose test animals heart, liver kidney, spleen, testes and lungs.
 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527 525

Fig. 5. Antioxidant enzymes analyzed. 5a and 5d for Glutathione reductase 5b and 5e for Glutathione peroxidase and 5c and 5f for super oxide dismutase of male and female experimental
animals respectively. (All values are expressed as mean ± standard deviation from three different replicates.* denotes significance of p b 0.05).

administered nano HA as it forms the natural component of bone. It was
also noted that the elevation of ALP levels were transient as the high re-
covery groups maintained the same levels of ALP expression as that of
control. All other biochemical parameters did not show much variation,
suggesting the normal health condition of experimental animals.
3.2.4. Histopathological analysis
Gross examinations of the carcasses of the treated groups (low, me-
dium, high and high recovery groups) and control rats did not reveal
any gross abnormality in the organs examined. However the gross ex-
aminations of high dose group did not reveal any abnormality in the or-
gans except one rat showed a fibrous fluctuating mass at the base of the
left ear and was considered as not pertaining to the Nano HA material.
Incidence of non-neoplastic and neoplastic histopathologic findings
are summarized in Tables 6 and 7. Histopathological examinations
of carcasses of all groups showed mild to moderate bronchial
associated lymphoid tissue proliferation (BALT), proliferation in lungs,
heamosiderosis in spleen and mononuclear cells infiltration in trachea.
One case of control and high dose showed follicular hyperplasia of the
mammary gland. Hyperplasia of C cells of thyroid gland and congestion
of spleen was observed each in one control group animal. Lungs of one
control animal showed pneumonic lesions, pleurisy and consolidated
alveoli whereas abscess was observed in two animals. Liver in one con-
trol group animal showed a cyst and appeared fibrosed. Kidney in one
each case of control and high dose groups showed cystic tubular degen-
eration and fibrosis whereas mono nuclear cell collection was observed
in four high dose group animals. Ovaries were found to be cystic in one
animal of control as well as high dose groups. An abscess was observed
in prepuce in one high dose group animal. Three cases of sub mucosal
mono nuclear cell infiltration in intestinal tract were observed in high
dose group. Bronchus-associated lymphoid tissue (BALT) proliferation
a non-neoplasmic lesion often reflects the immune status of respiratory
system in connection with viral induced respiratory infections. This was
evident in some of the groups examined and could not be statistically
correlated and was considered as unrelated to the compound tested.
The histopathology images are represented in Fig. 3 and 4 for male
and female respectively.
3.2.5. Measurement of oxidative stress level
Reactive oxygen species are chemically reactive molecules contain-
ing oxygen which are formed as a natural byproduct of the normal oxy-
gen metabolism in the body and have important roles in maintaining
homeostasis. However, excess reactive oxygen species production as
in the case of environmental stress like UV or heat exposure or toxic
chemicals, can lead to self-destructive phenomenon called oxidative
stress. This may result in significant damage to cell structures which
can ultimately impact on the health of an individual. Under normal con-
ditions, the cells counteract oxidative stress by activating the cellular
antioxidant defense system mediated by cellular antioxidant enzymes.
Hence measurement of the levels of antioxidant enzymes reflects the
oxidative status of the tissues or organs. Liver, the major metabolic
site is often targeted for various anti-oxidants studies [24]. Liver
526 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527

Fig. 6. Antioxidant parameters analyzed.6a and 6b; concentration of malondialdehyde measured in liver of both male and female experimental animals. 6c and 6d is the amount of reduced
glutathione measured in males and female experimental animals respectively. (All values are expressed as mean ± standard deviation from three different replicates.* denotes significance
of p b 0.05).

antioxidant status was assessed by estimating the antioxidant parame- liver examined suggesting that ROS generation and ROS mediated
ters from liver homogenate. Data is summarized in Figs. 5 and 6. oxidative stress is an unrelated event to the chronic oral administration
GPx or glutathione peroxidases is an enzyme that catalyzes the reduc- of nano HA.
tion of hydroperoxides using glutathione (GSH), which is one of the most
essential antioxidative defense mechanism and its increased levels in
4. Conclusion
human body represent oxidative stress. GPx levels were lower in low, me-
dium and high recovery group when compared to control, while a slight
The in house synthesized nano HA having a nano scale particle size
increase was observed in high group in case of males. However, the levels
of b50 nm is found to be non-toxic/biocompatible as evidenced from
were lower in all the groups in females when compared to control (Fig. 5b
the chronic toxicity studies. No adverse toxic effects or mortality were
and e). Glutathione reductase is another antioxidant enzyme that cata-
found associated with the chronic oral administration of nano HA at
lyzes the reduction of glutathione disulfide (GSSG) to glutathione
the concentration specified. Even though indirect measurements of ox-
(GSH), which is an important molecule in resisting oxidative stress.
idative stress level indicates the generation of ROS by nano HA, no sig-
There was no significant increase in glutathione reductase activity in all
nificant effects associated with ROS mediated cellular damage was
the groups when compared to control reflecting the well maintained ox-
evident suggesting the levels of ROS generated is not crossing the
idative homeostasis (Fig. 5a and d). Super oxide dismutase enzyme
threshold level which the system could manage. No evidence on carci-
dismutate O2− thereby inactivating oxygen derived free radicals and
nogenic potential of the Nano HA material could be obtained highlight-
hence forms a first line of defense in combating against oxidative stress
ing the safety concern in using it clinically. The chronic toxicity and
experienced by the tissue/organ. From the data analyzed, it is evident
combined carcinogenicity studies of nano HA in rats suggests the nano
that nano HA orally administration at high dose led to increased SOD ac-
HA is non-toxic and biocompatible at the concentrations specified and
tivity in liver when compared to control in both males and females. How-
could be exploited for clinical use.
ever the activity was lower than the control in the high recovery dose
animals suggesting the transient increase was due to the adaptive re-
sponse of high dose treated experimental animals to the non-significant Acknowledgements
increase in reactive oxygen species generated in the system (Fig. 5c and
f). Furthermore Lipids form the major components of cellular mem- The authors are grateful to the Director of Sree Chitra Tirunal
branes; oxidative degradation of lipids by the ROS has received deliberate Institute for Medical Sciences and Technology and Head of Biomedical
attention as a potential toxicological hazard. The end products of lipid Technology Wing for their support. The authors acknowledge the
peroxidation are reactive aldehydes, such as malondialdehyde (MDA) financial support provided by Department of Science and Technology,
and hence its level in the tissues/organs attends to an indirect biomarker India (Grant No. SR/NM/NS-90/2008).
for ROS mediated cellular damage. Nano HA administration however re-
sulted in a non-significant increase in MDA concentration in the liver References
treated group when compared to their respective control groups in both
[1] G. Gao, A.F. Schilling, T. Yonezawa, J. Wang, G. Dai, X. Cui, Bioactive nanoparticles
male and female groups (Fig. 6a and b). But no cellular damage or associ- stimulate bone tissue formation in bioprinted three-dimensional scaffold and
ated pathologies were observed in the histopathological findings of the human mesenchymal stem cells, Biotechnol. J. 9 (10) (2014 Oct) 1304–1311.
 N.S. Remya et al. / Materials Science and Engineering C 76 (2017) 518–527
[2] S. Ghanaati, J. Lorenz, K. Obreja, J. Choukroun, C. Landes, R.A. Sader, Nanocrystalline
hydroxyapatite-based material already contributes to implant stability after 3
months: a clinical and radiologic 3-year follow-up investigation, The Journal of
oral implantology. 40 (1) (2014 Feb) 103–109.
[3] L. Li, H. Pan, J. Tao, X. Xu, C. Mao, X. Gu, et al., Repair of enamel by using hydroxyap-
atite nanoparticles as the building blocks, Journal of Materials Chemistry 18 (34)
(2008) 4079–4084, http://dx.doi.org/10.1039/B806090H.
[4] F. Ye, H. Guo, H. Zhang, X. He, Polymeric micelle-templated synthesis of hydroxyap-
atite hollow nanoparticles for a drug delivery system, Acta Biomater. 6 (6) (2010
Jun) 2212–2218.
[5] S. Barkarmo, A. Wennerberg, M. Hoffman, P. Kjellin, K. Breding, P. Handa, et al.,
Nano-hydroxyapatite-coated PEEK implants: a pilot study in rabbit bone, J. Biomed.
Mater. Res. A 101 (2) (2013 Feb) 465–471.
[6] J. Huang, Y.W. Lin, X.W. Fu, S.M. Best, R.A. Brooks, N. Rushton, et al., Development of
nano-sized hydroxyapatite reinforced composites for tissue engineering scaffolds,
Journal of materials science Materials in medicine. 18 (11) (2007 Nov) 2151–2157.
[7] N.S. Remya, S. Syama, V. Gayathri, H.K. Varma, P.V. Mohanan, An in vitro study on
the interaction of hydroxyapatite nanoparticles and bone marrow mesenchymal
stem cells for assessing the toxicological behaviour, Colloids Surf B Biointerfaces.
117 (2014 May 1) 389–397.
[8] M.-A. Shahbazi, B. Herranz, H.A. Santos, Nanostructured porous Si-based nanoparti-
cles for targeted drug delivery, Biomatter. 2 (4) (2012) 296–312.
[9] L. Liu, Z. Xiao, Y. Xiao, Z. Wang, F. Li, M. Li, et al., Potential enhancement of intrave-
nous nano-hydroxyapatite in high-intensity focused ultrasound ablation for treating
hepatocellular carcinoma in a rabbit model, Oncol. Lett. 7 (5) (2014) 1485–1492.
[10] Y. Xiong, C. Ren, B. Zhang, H. Yang, Y. Lang, L. Min, et al., Analyzing the behavior of a
porous nano-hydroxyapatite/polyamide 66 (n-HA/PA66) composite for healing of
bone defects, Int. J. Nanomedicine 9 (2014) 485–494.
[11] S.C. Rodrigues, C.L. Salgado, A. Sahu, M.P. Garcia, M.H. Fernandes, F.J. Monteiro, Prep-
aration and characterization of collagen-nanohydroxyapatite biocomposite scaffolds
by cryogelation method for bone tissue engineering applications, J. Biomed. Mater.
Res. A 101 (4) (2013 Apr) 1080–1094.
[12] F. Xiaoli, C. Aijie, Z. Yanli, W. Jianfeng, S. Ongquan, W. Limin, Application of dental
nanomaterials: potential toxicity to the central nervous system, International Jour-
nal of Nanomedicine 10 (2015) 3547–3565.
527
[13] E.N. Andre, M. Lutz, V. Darrell, X. Tian, M.V. Eric, Hoek, S. Ponisseril, K. Fred, Vince Ca,
T. Mike, Understanding biophysicochemical interactions at the nano–bio interface,
Nature Materials 8 (2009) 543–557.
[14] S. Parasuraman, Toxicological screening, Journal of Pharmacology and Pharmaco-
therapy 2 (2) (2011) 74–79.
[15] L.C. Kathryn, H. Henry, E.B. Lauren, B. Marilyn, C. Gary, C. Christine, C. Jessica, et al.,
Pharmaceutical toxicology: designing studies to reduce animal use, while maximiz-
ing human translation, Regulatory Toxicology and Pharmacology 66 (1) (2013)
88–103.
[16] C.S. Geetha, N.S. Remya, K.B. Leji, S. Syama, S.C. Reshma, P.J. Sreekanth, et al., Cells-
nano interactions and molecular toxicity after delayed hypersensitivity, in guinea
pigs on exposure to hydroxyapatite nanoparticles, Colloids Surf B Biointerfaces.
112 (2013 Dec 1) 204–212.
[17] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the
Folin phenol reagent, J. Biol. Chem. 193 (1) (1951 Nov) 265–275.
[18] A. Okado-Matsumoto, I. Fridovich, Subcellular distribution of superoxide dismutases
(SOD) in rat liver: Cu,Zn-SOD in mitochondria, Journal of Biological Chemistry 276
(42) (2001) 2001 October 19. (38388-93).
[19] M.S. Moron, J.W. Depierre, B. Mannervik, Levels of glutathione, glutathione reduc-
tase and glutathione S-transferase activities in rat lung and liver, Biochim. Biophys.
Acta 582 (1) (1979 Jan 4) 67–78.
[20] J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman, W.G. Hoekstra, Se-
lenium: biochemical role as a component of glutathione peroxidase, Science (New
York, NY) 179 (4073) (1973 Feb 9) 588–590.
[21] A. Nandi, I.B. Chatterjee, Assay of superoxide dismutase activity in animal tissues, J
Biosci. 13 (3) (1988) 305–315 1988/09/01.
[22] A.J. Mach, O.B. Adeyiga, D. Di Carlo, Microfluidic sample preparation for diagnostic
cytopathology, Lab Chip 13 (6) (2013) 1011–1026.
[23] A.M. Alvarez, D. Mukherjee, Liver abnormalities in cardiac diseases and heart failure,
The International Journal of Angiology 20 (3) (2011) 135–142 (Official Publication
of the International College of Angiology, Inc.).
[24] K. Rahman, Studies on free radicals, antioxidants, and co-factors, Clin. Interv. Aging 2
(2) (2007) 219–236.