APPLICATION OF FISH SCALE BASED HYDROXYAPATITE-GIC

COMPOUND IN SKULL BONE DEFECT OF LABORATORY RAT

Lai Lai Aye1
1
Zoology Department, University of Medicine 1, Yangon
the disfunctioned and damaged hard
ABSTRACT tissues. HAp is most important to
HAp produced from fish (Notopterus- synthesize nano-composites in order to
notopterus) scales heated at 800°C for have good biocompatibility, high
several hours was added into a mixture of bioactivity and great bonding properties.
glass ionomer powder- glass ionomer HAp promotes faster bone regeneration,
liquid in the ratio of 3:1:8. This freshly and direct bonding to regenerated bones
prepared mixture was substituted at the without intermediate connective tissue
defect of rat skull bone. Four months after (Palanivelu, 2013). Due to the properties
operation, evidences of histological study of hydroxyapatite (Ca10(PO4)6(OH)2) it has
showed this compound solid structure widely been used in biomedical and dental
coalesced into natural skull bone tissue practices due to its similarity to main
and they coexisted well after substitution. mineral components of hard tissues of
human body. Therefore HAp biomaterials
Keywords: F.S.HAp; glass ionomer from different biowastes can widely be
used in human body for tissue repair and
INTRODUCTION
substitution still expand to date.
There has been a growing interest in In bone defect reconstruction caused by a
natural ingredients which are readily benign tumor or trauma, use of autologous
available from animal by-products rather and allograft bone has been popularly used
than using synthetic chemicals to fulfill in clinics. However use of autologous
the needs of human beings. However, lack bone is known to result in secondary
of adequate utilization of technology to trauma and allograft bone induces immune
fully convert such wastes into value-added repulsion. In addition, since bone grafts
products must be seriously addressed are avascular and are dependent on
(FFTC Document Database, 2010). diffusion, the use of autologous and
Many researchers are trying to produce allograft bones are limited by the size of
biomaterials from various biowastes such the defect and viability of the host vascular
as egg shells and its membrane, fish bed. It was reported that graft in large
collagen and fish oil, corals, snail shells, defects were resorbed by the body before
vertebrate bones, oyster shell, shrimp and the completion of osteogenesis. To solve
crab exoskeletons for biomedical these problems, many researches have
applications. Moreover, one of the aquatic focused on the use of artificial bone-like
biowastes, fish scales, are biocomposites materials such as bone cement and
of highly ordered type I collagen fibers bioglass. However as some cements are
and hydroxyapatite (Ca10(PO4)6(OH)2) prepared in the operation room, they are
(Fengxiang Zhang et al.,2011). Apart from therefore susceptible to infection (Oh et
this, Teeth and teleost scales are dermally al., 2006). In an attempt to solve some of
derived and may have evolved from a the problems associated with the use of
common ancestor (Sharpe, 2001). As a autologous bone, allograft and bone
source of hydroxyapatite(HAp.), these cement, the advent of tissue engineering
biowastes are needed to produce has become a major area of medical
biomaterials which can be used to replace research as the search continue to develop
better materials to replace lost or missing hydroxyapatite (F.S.HAp) was done at
tissue from the human body (Oh et al., Universities' Research Centre at Yangon
2006). There are some disadvantages, University, National Laboratory,
however, in the use of autogenous bone Department of Research and Innovation,
grafts, such as (i) the limitation in the and Customer Support Analytical
amount of available bone for laboratory at Amtt Company, Yangon.
transplantation, (ii) the difficulty in Animal study was carried out at Animal
forming a desired shape, and (iii) the Services Division, Department of Medical
requirement for a second surgical Research. Microscopic study was done at
procedure with its associated risks. the Department of Pathology, University
Recently, many kinds of synthetic of Medicine 1 Yangon. This study was
materials have been developed for use as a conducted from December, 2016 to May,
bone substitute. Hydroxyapatite, in 2017.
particular, is considered to be one of the
Fish (Notopterus notopterus) scales
best bioactive ceramics for a bone
Hydroxyapatite heated at 800°C for
substitute because of its superior
several hours, male laboratory rat, Glass
osteoconductivity (Asahinaet al., 1997).
Ionomer, Fuji I (GC, Japan), Ketamin,
Because of the constant and increasing Xyla, Surgical instruments, Bone driller,
demand for reconstruction of hard tissues Betadine and disposable syringe were used
in the treatment of bone defect, autologous in this study.
bone graft options have been designed.
Substitution of F.S.HAp-GIC (F.S.HAp: GI
Though the most widely used are
powder: GI liquid = 3:1:8) in skull bone
allografts and xenografts, synthetic
defect of laboratory rat
alternative known as bone substitutes are
currently being developed. Ideally, these Male rat was selected just to endure the
substitutes must be biocompatible and severity of a surgical trauma. The rat was
possess a structure similar to bone, easy to fasted for 18 hours up to morning of
use and affordable. It is reasonable to operation day. At 9:30 am of operation
believe that combination of the materials day, the fasted rat was given intra-
will provide better results than the peritoneal injection of a mixture of
autologous bone graft (Sesmanet al., Ketamin (0.6 cc) and Xyla (0.4 cc). Five
2013).In conjunction with glass ionomer minutes after injection, under the influence
cement, nano-HAp extracted from fish of anaethesia, it reached to the state of
scales can be used in field of restorative unconsciousness. The rat was placed onto
dentistry and bony substitution as a the operation table and cut the hairs on the
bioceramic material. head with scissor. The scalp was incised
down to the parietal bone. A hole was
The objectives of the present study is an
made on the right side of exposed skull
attempt to assess the feasibility of HAp-
surface (parietal area) with bone driller.
GIC as a substitute for bone defect.
The hole was about 3mm in diameter and
1 mm in depth. This bone defect was
MATERIALS AND METHODS
immediately filled with freshly prepared
Fish scales were collected from F.S.HAp-GIC mixture (3:1:8) (Figure 3).
Mingaladon market. GC Glass Ionomer After the filling of the defect, the scalp
was bought from dental pharmacy, was sutured back with cat-gut and
University of Dental Medicine, Yangon. betadine was applied onto the suture to
Characterization of fish scales prevent infection. About one hour after
operation, the rat regained consciousness. preserved in 10% formalin. And then the
It was treated everyday with clean portion of skull bone including normal
drinking water and normal animal food, skull bone tissue was cut out with a bone
and kept in the mouse cage . The condition cutter and soaked in 10% formalin. After
of rat was observed and recorded every two days, the cut tissue (4 micron in
day. One and half month after operation, thickness) was prepared for microscopic
skull X-ray was taken at Crown Diagnosis study and it was stained with H and E
Center in Yangon. Four months after method. With the help of compound
operation, the sutured skin was reopened microscope (Olympus, Japan),
and the filled up area was observed. histopathological study was carried out at
Subsequently the rat was sacrificed for the Pathology Department, University of
histological study and the rat head was Medicine 1, Yangon.

Figure 1. SEM image of (N. notopterus)scalepowder heated at 800°C

GI liquid (8drops)

Drops into

Mixture of F.S.HAp.-GI powder (3:1)

Stirs with small glass paddle

Adds into

Skull bone defect of rat

Figure 2. Flow chart showing substitution of F.S.HAp-GIC compound into rat skull bone
defect
Figure 3 Filling of F.S.HAp-GIC into skull bone defect of rat

area at the right side of parietal bone was

RESULTS
seen in X-ray.
Even though the rat suffered from severity
The rat was subsequently sacrificed for
of the surgical trauma, it was healthy,
histopathological study of the skull bone
actively moving, eating well every day and
where F.S.HAp-GIC was substituted on
growing normally during the whole
the right side of the parietal bone of the
observation period of four months. One
skull in the defect area. Aggregates of
and half month after operation, X-ray was
particles could be easily seen with the
taken .The opaque image of the filled up
naked eye (Figure 4).

F.S.HAp-GIC
compound in
skull bone

Figure 4. Re-examination of the scalp by exposing the skull bone defect area (after four
months)
Under the view of compound microscope, tissue. A view at magnification of 100
the aggregate of HAp composite was shows that there was direct contact
surrounded by connective tissue with giant between the F.S.HAp-GIC and normal
(inflammatory) cells in inflammatory bone tissue (Figure 5). As normal bone
reaction areas. A significant finding is that tissue, osteocytes and Haversian systems
the microscopic study shows the were observed in the skull bone without
coexistence of F.S.HAp-GIC and normal substitution of F.S.HAp-GIC compound.
bone tissue in the rat skull bone. The There is no inflammatory cells in this
composite was coalesced in natural bone tissue.
Figure 5. Microscopic image of F.S.HAp-GIC in bone defect (100)
minimal pore diameter necessary for bone
DISCUSSION
ingrowth was considered to be
According to Venkatesan and Kim (2010), approximately 100 µm. The diameter of
suitable pore diameters in the substituted the smallest capillary is 3 to 4 µm
tissue are very important for nutrient flow (Capillaries, https:// www.
and cell to cell connection. In present histology.leeds.ac.uk >capill.). Based on
study, to achieve retention of F.S.HAp in the record stated above, for reconstruction
bone structure, the N. notopterus F.S.HAp of hard tissues (teeth and bone), it is
was mixed with GIC which has adhesive impossible to get nutrient supplementation
property for chemical adhesion to bone by capillaries into the substituted
structure. In this research work, in F.S.HAp-GIC of the present study. It is
repairing the bone defect, the nano-sized seemed to be difference in diameter of
F.S.HAp crystallites and particles may pore and capillary may lead to a barrier for
enter into bone tissue in a certain period of nutrient supplementation and cell to cell
time. But to get a functional status of the connection into the substituted tissue. So
solid structure organ like tooth and bone in there is a very little chance of capillary
a very short period of time, the help of the penetration into defect area with use of
GIC is absolutely necessary. F.S.HAp-GIC.
Rahman (2013) stated that adding of glass Four months after substitution in the
ionomer cement into hydroxyapatite-silica present study, inflammatory cells were
nanopowder composite makes still present in the histological
enhancement of hardness. So many efforts preparations of the skull bone tissue. It
were done in order to increase hardness. In was likely to be an immunologic response.
previous study, to perform hardness test, It is noteworthy that this response
glass ionomer cements with different obviously did not prevent reconstruction
ratios were combined with the F.S.HAp. of the damaged bone tissue with F.S.HAp
The highest hardness value of 274 HV was in the present work.
obtained by mixing F.S.HAp powder and
This research is an attempt to produce
GI powder at 3:1 into liquid of GI at
mass HAp from the N. notopterus fish
8 (3:1:8).
scales, which is a new product from this
Pore diameters in the F.S.HAp-GIC experimental research, with particular
composite ranges from 26 nm-1402 nm. attention to use commonly in restorative
Majority of pores in the composite was dentistry and reparation of bone defects.
nano-sized. Mour et al. (2010) stated that This process of extracting F.S.HAp from
fish scales also helps as a way in produced easily and it can be reinforced in
environmental sanitation and waste GIC manufacturally or by user easily.
material recycling.
This study revealed that the potential use
F.S.HAp of the present work is therefore, of nano-sized F.S.HAp in natural bone
considered a promising additive for GIC healing process is believed to penetrate
used as restorative materials in bone into the pores of dentine tissue and bone
grafting. Within limitation of this study, tissue.
nano-F.S.HAp-GIC complex compound
gave promising results which could lead to CONCLUSION
increase in scientific research on the Fish scales, once thought to be a waste in
subject. Further researches are necessary fishing industry and fishery market, and a
for more application of calcium based nuisance to the environment and human
materials, which are optimized by beings, are now can be regarded as an
specifying their geometry, dimension, environmentally friendly and cost
density, pore size, mechanical strength, effective source of an invaluable
purity and chemical phase. biomaterial known as hydroxyapatite
The materials in current use for (HAp). This HAp which has been
reconstruction of bone and teeth are extracted from the N. notopterus scales
mostly metals (gold, steel, platinum, may be a new biomaterial for future hard
titanium), plastic (polyethylene, tissue engineering technology as it can be
polytetrafluoethylene) and ceramics. applied as an alternative source in
Instead of using these expensive ready- reconstruction of hard tissues, needing not
made synthetic substitutes, it is a better to regard race and religion of the
alternative way of using affordable recipients.
biomaterials in bone and teeth
ACKNOWLEDGEMENTS
reconstruction. There are many advantages
of using fish scales HAp-GIC in I would also like to express my thanks to Dr. Aye
reconstruction of bone and teeth. It can be Win Oo (Head, Animal Services Division,
Department of Medical Research, Yangon) and his
used to mould easily to desired shape. It is colleagues for their invaluable help in substitution
also easily available and may be cheaper of F.S.HAp-GIC compound in skull bone defect.
than the materials already in use in clinical Most importantly, I would like to thank Professor
practice. It can be used as restorative Dr. Myint Myint Nyein (Pro-rector, University of
Medicine -1) and Dr. Kay Thwe Oo (Lecturer,
material in dentistry and restoration of
Pathology Department, University of Medicine-1)
bone defect area which are in non-load for their helps in Microscopic study.
bearing regions like skull bone,
maxillofacial, cleft palate, vertebrae REFERENCES
deformity, ribs and digits.
1. Asahina, I., Watanabe, M., Sakurai,
This preliminary pilot study showed that N., Mori, M. and Enomoto, S. 1997.
the implantation of fish scale HAp, with Repair of bone defect in primate
the aid of GC Glass Ionomer, induced mandible using a bone morphogenetic
successful reconstruction of hard tissues. It protein (BMP)-Hydroxyapatite-
is very much hopeful that the mass Collagen Composite. J Med Dent Sci
production of HAp from N. notopterus 1997; 44; 63-70.
scales may fulfill the need of HAp 2. Fengxiang Zhang, Anning Wang,
materials for hard tissue engineering in our Zhihua Li, Shengwen He, and Lijun
country. Moreover, F.S.HAp can be Shao, 2011. Preparation and
 characterization of collagen from from tuna bone. Materials, 3(10),
freshwater fish scales. Food and 4761-4772.
Nutrition Sciences,2011,2,818-823. 10. Website :Capillaries, https:// www.
3. FFTC document data base, 2010. histology.leeds.ac.uk >capill.
Improved utilization of fishery by-
products as potential nutraceuticals
and functional food .Food and
fertilizer technology center for the
Asian and pacific region, copy right ©
1998-2018.
4. Mour, M., Das, D., Winkler, T.,
Hoenig, E., Mieke, G., Michael M.
Morlock, M.M., and Schilling, A.F.,
2010.Advances in porous biomaterials
for dental and orthopaedic
applications. Materials, 3, 29472974;
10.3390 /ma 3052947.
5. Oh, S., Oh, N., Appleford, M. and
Ong, J.L. 2006. Bioceramics for tissue
engineering application: A Review,
American Journal of Biochemistry and
Biotechnology 2(2):49-56.
6. Palanivelu. R….,2013, “Synthesis and
Spectroscopic characterization of
Hydroxyapatite by Sol-Gel Method”,
Research, School of Advanced
Sciences, VIT University, India,.. ,
International Journal of Chem Tech
Vol.5, No.6, pp 2965-2969.
7. Rahman, I.A., Masudi, S.M., Luddin,
N. and Shiekh, R.A., 2013. One-pot
synthesis of Hydroxyapatite-silica
nanopowder composite for hardness
enhancement of glass ionomercement.
Bull.Mater. Sci., Vol.37, No.2, pp.
213-219.
8. Sesman, A., Ruvalcaba, E., Herrera,
A., Sanchez, G.S., Lecona, H.., Baena-
Ocampo, L., Solis, L., Avila. H.,
Delapuente, G. S., Vargas, B.,
Guerrero, X., and Velasquillo, C.,
2013.Morphological study of bone
cranial in athymic mice”, Int. J.
Morphol., 31(I); 321-328.
9. Venkatesan, J. and Kim, S.K. 2010.
Effect of temperature on isolation and
characterization of hydroxyapatite