initial draft_ nano composite paste for bone repair_ dr zuki 7 february

8/12/2019 Initial Draft_ Nano Composite Paste for Bone Repair_ Dr Zuki 7 February

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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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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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5

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

5

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)

initial draft_ nano composite paste for bone repair_ dr zuki 7 february

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