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NANOCOMPOSITE PASTE FOR BONE REPAIR
FIELD OF THE INVENTION
The present invention relates to nanocomposite composition, more particularly to
nanocomposite composition for bone repair.
BACKGROUND OF THE INVENTION (PROBLEMS IN PRIOR ART)5
The composite bone pastes have been developed from the hydroxyapatite (HA) nanocrystals
(1). HA nanocrystals were prepared by wet chemical method using CaCl2 (Sigma, USA) and
(NH4)2HPO4 (Sigma, USA) as Ca and P precursors, respectively. To synthesis nano HA, 0.3 m aqueous
solution of (NH4)2HPO4was slowly added drop by drop to 0.5 Molar aqueous solution of CaCl2. Then
the mixture was mixed by the stirring process at 60 C at the stirring rate of 1000 rpm and the10
reaction. The minimum pH was adjusted to 10 by adding concentrated NH4OH using an injectable
syringe. The obtained precipitate was aged for 24 h under stirring at the same speed. After aging,
the obtained white precipitate was filtered, washed four to five times with distilled water until
complete removal of ammonium chloride. The prepared nano HA was irradiated using microwave
for 15 min. The final precipitate was centrifuged at 10,000 rpm for 10 min and washed repeatedly15
with de-ionized water followed by drying in a vacuum oven at 60 C. After the preparation of nano
HA, composite bone paste was developed by the combination of nano HA and chitosan using the
stirring method. The method is relatively complicated and takes long time to complete besides the
chemicals used are expensive. In addition, the produced paste bioresorbability has not been tested
in vivo [2]. However, nanocrystalline HA suspension prepared using the same method has been20
assessed in bone defect of in vivo animal model and showed the nanocrystalline HA was not
completely absorbed, and integrated into bone tissue [1].
The chemicals are expensive, long procedure, complexity of the method and finally the paste
of nano HA is not completely bioresorbable, biocompatible and integrals to bone tissue reported by
the previous methods [1, 2].25
The starting materials (nanoHA) are difficult to prepare and very expensive. The process is long,
time consuming, complex and not industrially feasible. Many of the prepared pastes were not
evaluated by in vivostudy. The pastes from nanocrystalline hydroxyapatite are not absorbable,
replaceable and integrals to the bone tissue.30
Hence there is a need for a product for bone healing which is easy to prepare and having
improved absorbability.
SUMMARY OF THE INVENTION
It is an aspect of the present invention to provide a (WILL COMPLETE LASTPOSTCLAIM
VERIFICATION)35
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : illustrates flowchart of method of preparation of nanocomposite of the present
invention.
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Figure 2 : illustrates cleanse and dry cockle shells as raw material of the present invention.
Figure 3 :illustrate ground cockle shells for preparation of nanocomposite of the present
invention.
Figure 4 : illustrates the cockle shells powder, distilled water and BS-12 mixture are stirredusing the mechanical stirrer and magnetic stirrer bar.5
Figure 5 : illustrates the synthsized cockle shells based calcium carbonate are dried in an
oven.
Figure 6 : illustrates the synthesized calcium carbonate nanoparticles (TEM image).
Figure 7 : illustrates the cockle shells based calcium carbonate nanoparticles and chitosan
solutions are mixed and stirred using Multi system Hot plate stirrer and magnetic10
stirrer bar.
Figure 8 : illustrates the surface morphology of the cockle shells based calcium carbonate
nanoparticles (A) and the compsite bone paste (B).
Figure 9 : illustrates the FT-IR of cockle shells based nano calcium carbonate (A), chitosan (B)
and the paste with chitosan solution (C).15
Figure 10 :illustrates XRD of cockle shells based nano calcium carbonate (A) and the paste
with chitosan solution (B).
Figure 11 : illustrates TGA of cockle shells based nano calcium carbonate (A) and the paste
with chitosan solution (B).
Figure 12 : illustrates the rabbit is anesthetized for surgery.20
Figure 13 : illustrates the implantation site is exposed.
Figure 14 : illustrates the bone is drilled to create a bone hole defect.
Figure 15 :illustrates the bone hole defect (indicated by white arrow).
Figure 16 : illustrates the nanocomposite paste was implanting into right bone hole.
Figure 17 : illustrates the nanocomposite bone paste implanted into the bone hole of right25
tibia (A) and the bone without the implant of the left tibia (B) at Day 1 of post-
implantation.
Figure 18 :illustrates the nanocomposite bone paste implanted into the bone hole of right tibia
(A) and the bone hole without the implant of the left tibia (B) at week 7 of post-
implantation.30
Figure 19 illustrates the gross images of right bone hole implanted with the nanocomposite
bone paste (A) and the left bone hole without the implant (B).
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Figure 20 : illustrates the histological images of the new bone formation in the nanocomposite
bone paste implanted site of the right bone hole area (A), whereas (B,C and D) show
the defective sites of left bone hole area.
Figure 21 : illustrates the cockle shell based micron size calcium carbonate paste implanted
into the bone hole of right tibia (A) and without the implant on the bone hole of the5
left tibia (B) at Day 1 of post-implantation.
Figure 22 : illustrates the cockle shell based micron size calcium carbonate paste implanted
into the bone hole of right tibia (A) and without the implant on the bone hole of the
left tibia (B) at week 7 of post-implantation.
Figure 23 : illustrates the right bone hole implanted with the cockle shell based micron size10
calcium carbonate paste (A) and the left bone hole without the implant (B) show
that the right bone hole is healed leaving the small remnant of defective area (black
arrow), whereas the left bone hole area is clearly open (green arrow).
Figure 24 : illustratesthe histological images of new immature bone formation in the cockle
shell based micron size calcium carbonate paste implanted site of the right bone15
hole area (A), whereas (B,C and D) the defective sites of left bone hole area.
Figure 25 : illustrates the commercial calcium carbonate paste implanted into the bone hole of
right tibia (A) and without the implant on the bone hole of the left tibia (B) at Day 1
of post-implantation.
Figure 26 : illustratesthe commercial calcium carbonate paste implanted into the bone hole of20
right tibia (A) and without the implant on the bone hole of the left tibia (B) at week 7
of post-implantation.
Figure 27 : illustrates the gross images of bone defect implanted with the commercial calcium
carbonate paste (A) and the left bone hole without the implant (B) show the right
bone hole is healed (black arrow), whereas the left bone hole area is not healed and25
filled by fibrous tissue (green arrow)
Figure 28 : illustrates the histological images of incomplete immature bone formation in the
commercial calcium carbonate paste implanted site of the right bone hole area (A),
whereas (B and C) show the defective sites of left bone hole area.
Figure 29 :illustrates bar graph showing quantification of healing % by radiology among the30
groups. In the group I, the healing is 84.38%, in group II the healing is 71.88% and in
group the healing is 50%.The healing is better in group I significantly (p
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DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to nanocomposite biomaterial bone paste for bone repair
comprises nanoparticle calcium carbonate from cockle shell, dodecyl dimethyl betaine; and chitosan.The calcium carbonate in this invention is in aragonite phase.5
(The particle size is 205 nm in diameter ) Particle sizeranges from.(kindly provide the
information if available)
Figure 1 illustrates the general flowchart of the method of preparation of the
nanocomposite of the present invention. The method comprises the steps of; providing
(nanoparticle calcium carbonate from) fine cockle shells powder; adding water and dodecyl dimethyl10
betaine (BS-12) forming a mixture; stirring the mixture; drying the mixture and mixing( adding)
chitosan solution forming nanopaste and irradiating the bone paste.
In one embodiment, the preferred chitosan solution is chitosan solution which comprises 2%
acetic acid. However, other form of .. (is there alternative to chitosan solution, chitosan solution
preparation and variation of acetic acid percentage) . There is no alternative of chitosan solution15
The drying step may be conducted using .. (general drying method applicable to this
invention).Preferably the drying step is conducted using oven. Alternatively,(to oven) .To dry in
oven is easy and better.
High temperature ranges from __5__ to __10 min___(general)may be used for irradiation.
Preferably the irradiating step is conducted at the temperature of 100
O
C and for a period 10 minutes.20The mentioned irradiating temp and time is ok. We did not find any alternative.
In one embodiment, the nanocomposite of the present invention is usable to fill-up bone
fracture and enhance bone healing process. As biomaterial for healing bone fracture in human or
mammals. We have used to repair for bone fracture of mammals. The investigation is needed to use25
in human.
In another embodiement, the nanocomposite is usable as carrier or medium for drugs
delivery. Example(carrier for ___) as medium
In further embodiment, the nanocomposite bone growth factors to promote bone tissue
healing. Example BMP( bone morphogenic protein)30
In one example of method of preparation of the nanocomposite of the present invention,
the cockle shells were collected, cleaned and dried in an oven. The shells were ground to form
powder using the mechanical blender. The powders were mixed with deionized water to form slurry
and then dodecyl dimethyl betaine (BS-12) was added into the solution. After addition of BS-12 the
mixture was vigorously stirred at 1000 rpm rate at room temperature for 90 minutes using a35
mechanical stirrer. The resulted calcium carbonate nanoparticles were dried in an oven. The calciumcarbonate nanoparticles were mixed with chitosan solution containing 2% acetic acid. For the
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development of the nanocomposite bone paste, the mixture was stirred vigorously at 1000 rpm rate
at room temperature for 6 h. Finally the obtained nanocomposite biomaterial bone paste was
irradiated in a micro oven at 100C for 10 minutes. The prepared paste was characterized using SEM,
FT-IR, XRD, TGA, ICP, EDX and PBS.
For comparison, we prepared the pastes of micron- sized cockle shell based calcium5
carbonate and commercial calcium carbonate respectively by the same method. This invented paste
was evaluated in the treatment of bone defect using the rabbit model. For the in vivo study, 12
rabbits were used and divided them into three groups comprised of 4 rabbits in each group. The
surgery was done in surgical room maintaining aseptic environment. A round bone defect with a
diameter of 5 mm was made into each left and right tibia respectively. The left one used as the10
negative control and the right one used as the treatment the defect. The x-rays were taken on the
day of surgery and up to seven week of post operation. The animals were anesthetized by general
anesthesia prior to sacrifice at 7 weeks of post surgery. The samples were collected for gross
examination and then preserved in the 10% buffered formalin for histological examination. The
samples were decalcified, processed, sectioned and stained by H&E and Massionstrichrome. The15
slides were examined using image analyzer and evaluated the new bone formation in the implanted
sites.
The present invention will be described in more detail below with reference to the following
examples, but the present invention is not restricted to these specific examples at all.
EXAMPLES20
Raw material preparation
The cockle shells were collected, cleaned and dried in an oven (Fig.2) The shells were ground
to form powder using the mechanical blender (Fig.3). The powders were mixed with deionized water
to form slurry and then dodecyl dimethyl betaine (BS-12) was added into the solution. After addition
of BS-12 the mixture was vigorously stirred at 1000rpm rate at room temperature for 90 minutes25
using a mechanical stirrer (Fig.4). The resulted calcium carbonate nanoparticles were dried in an
oven (Fig.5).
Synthesis of nanocomposite
The calcium carbonate nanoparticles (Fig.6) were mixed with chitosan solution containing 2%
acetic acid. For the synthesis of the nanocomposite bone paste, the mixture was stirred vigorously at30
1000 rpm rate at room temperature for 6 h (Fig.7). Finally the obtained nanocomposite biomaterial
bone paste was irradiated in a micro oven at 100C for 10 minutes. The prepared pastes were
characterized using SEM( Fig.8), , FT-IR( Fig.9) XRD( Fig.10) TGA( Fig.11), EDXA (Table 1).
Table 1: The elemental contents of cockle shells based nano calcium carbonate (A) and the paste
with chitosan solution (B).35
A (Cockle shells based nano calcium carbonate)
Spectrum In stats C O Ca Total
Spectrum 1 Yes 22.97 26.03 51.00 100.00
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Spectrum 2 Yes 20.88 27.04 52.08 100.00
Spectrum 3 Yes 19.71 28.15 52.14 100.00
Mean 21.19 27.07 51.74 100.00
Std.deviationn
1.65 1.06 0.64
Max. 22.97 28.15 52.14
Min. 19.71 26.03 51.00
B ( Paste with chitosan solution)
Spectrum In stats C O Ca Total
Spectrum 1 Yes 24.20 30.92 44.88 100.00
Spectrum 2 Yes 25.57 31.73 42.70 100.00
Spectrum 3 Yes 23.78 29.07 47.15 100.00
Mean 24.51 30.58 44.91 100.00
Std. deviation 0.94 1.36 2.23
Max. 25.57 31.73 47.15
Min. 23.78 29.07 42.70
This invented paste was used as implant in the treatment of bone defect by in vivostudy
using the rabbit model.5
In vivo study
For in vivostudy, 12 rabbits were chosen and divided into three groups as 4 rabbits in each
group GoupI, group II and group III were treated with nano paste, micron paste and commercial
paste respectively to compare the effectiveness of the pastes. The surgery was done in the surgical
room under incentive care (Fig.12). The bone holes area was exposed (Fig. 13). The bone holes were10
made using drill beat (Fig.14). The bone hole was created (Fig.15). The paste was implanted into the
right bone hole (Fig.16). The x-ray was taken on day of the surgery and upto seven week. The bone
formation was determined by the radio-opaque, whereas the radiolucent were indicated as fibrous
tissue formation. The animals were anesthetized by general anesthesia and sacrificed by slaughter
after seven week. The samples were collected under strictly sterilized condition for gross15
examination. The bone defective areas were observed under the stereomicroscope. Then the
samples were preserved in the 10% buffered formalin for seven days for histological examination.
After fixation, the samples were decalcified by 5% formic acid for five days. The samples were thenprocessed by the automatic tissue processor.The sample were blocked by the melted paraffin .The
samples were sectioned transversely by the microtome machine as 6m thickness. The slides were20
dried in an oven at 37C for overnight. Then the slides were stained by the hematoxylene and
eosin .The samples were observed under the image analyzer to check the bone formation. The
radiographic images, gross images and histological images of group I are shown in Figs 17, 18, 19 and
20 respectively. For group II are shown in Figs 21, 22, 23 and 24. For group III, are shown in Figs 25,
26, 27 and 28. The healing is quantified by measuring the radiological images (Figure 29).The overall25
findings are shown in table 2.
Table 2 Overall observation of the result of radiology, gross and histology
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Conclusion: The radiological, gross and histological results revealed that the nanocomposite
biomaterial bone paste showed the better healing significantly (p
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CLAIMS
1. Nanocomposite biomaterial bone paste for bone repair comprises of cockle shell-based5calcium carbonate nanoparticles from cockle shell, dodecyl dimethyl betaine; and chitosan.
2. Nanocomposite biomaterial bone paste for bone repair according to Claim 1, characterizedin that the calcium carbonate is in aragonite phase.
3. Nanocomposite biomaterial bone paste for bone repair according to Claim 1, characterisedin that the nanocomposite biomaterial has following characteristic;10
Calcium content ranges from 42-47%
Carbon 23-25%
Oxygen 29-31%
4. Nanocomposite biomaterial bone paste for bone repair according to Claim 1, characterisedin that the nanocomposite biomaterial particle size ranges fromThere is no separate15
particle which can be measured. All particles are agglomerated by chitosan solution.
5. Use of nanocomposite biomaterial bone paste for bone repair according to Claim 1 as bonefiller thereby enhance bone healing process.
6. Use of nanocomposite biomaterial bone paste for bone repair according to Claim 1 as acarrier for drugs delivery for bone healing process.20
7. Use of nanocomposite biomaterial bone paste for bone repair according to Claim 1 as bonegrowth factors to promote bone tissue healing.
8. Method of preparation of the nanocomposite for bone repair comprises the steps of;a) providing (nanoparticle calcium carbonate from) fine cockle shells powder;b) adding water and dodecyl dimethyl betaine (BS-12) while stirring forming a mixture;25c) drying the mixture;
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d) mixing( adding) chitosan solution forming nanopaste; ande) irradiating the nanopaste.
9. Method of preparation of nanocomposite for bone repair according to Claim 6;characterizedin that the step d.the chitosan solution comprises 2% acetic acid.
10.Method of preparation of nanocomposite for bone repair according to Claim 6;5characterized in that the drying step c. is using oven.
11.Method of preparation of nanocomposite for bone repair according to Claim 6;characterised in that irradiating step e. is at 100
OC and for a period 10 minutes.
10
15
20
25
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ABSTRACT5
NANO COMPOSITE PASTE FOR BONE REPAIR
The present invention relates to nanocomposite bone paste for bone repair and to a method
for preparation of nanocomposite of the present invention.
Most illustrative Figure: Figure 2910
15
20
25
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5
10
and
15
Figure 1
providing (nanoparticle calcium carbonate from)
fine cockle shells powder
adding water and dodecyl dimethyl betaine (BS-12)
while stirring forming a mixture;
drying the mixture
mixing( adding) chitosan solution
formin nano aste
irradiating the nanopaste
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Figure 9
Figure 10
0
100
200
300
100020003000
(C)
(B)
(A)
6598571028
14391549
553
1017
13751576
1649
712
857
1082
1455
1794
2872
3341
Wavenumbers / cm-1
Transmittance/%
0
200
400
600
20 30 40 50 60
(B)
(A)
2/ degree
intensity/arbitrary
unit
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Figure11
5
Figure12
Figure1310
30
45
60
75
90
105
200 400 600 800
BA
Temperature(C)
weightloss/%
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Figure14
10
Figure 15
15
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Figure 16
5
Figure 17
Day 1
Nano ImplantControl
(A) (B)
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Figure 18
5
10
Figure 19
15
(A) (B
(C) (D)
(A) (B)
Week 7
Nano implant
Control
(A) (B)
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Figure 20
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Figure 21
Figure 2210
Day 1
Micron implantControl
(A) (B)
Week 7
Micron implant
Control
(A) (B)
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Figure 235
Figure 2410
(D)
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Figure 25
5
Figure 26
Week 7
Control
(A) (B)
Day 1
Control
(A) (B)
Commer implant
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Figure 27
Figure 28
5
Figure 29
(A) (C)(B)
(A) (B)