Fabrication and Characterization

of Chitosan / Hydroxyapetite
Nano Composites for Tissue
Engineering Applications
Supervisor: Dr. S.U Adikary
Hewa Wedage N.A 040409X
Thilawala K.G.N 080505B

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 Bionanocomposites
Form a fascinating interdisciplinary
area.

 Brings together biology, materials

science, and nanotechnology.

2
 Bionanocomposites
Cont’d…
Bionanocomposites are combination of
Polymers and Inorganic/Organic Fillers
at the nanometer scale.

3
 Biopolymers used

Existing biopolymers include, but are not

limited to, polysaccharides, aliphatic
polyesters, polypeptides and proteins, and
polynucleic acids.

4
 Why include fillers into the
matrix?
The interaction between filler components of
nanocomposites at the nanometer scale enables them to act
as molecular bridges in the polymer matrix.

 This is the basis for enhanced mechanical properties of the

nanocomposite as compared to conventional micro
composites.

Fillers include clays, hydroxyapatite, and metal nano

particles.
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What should be satisfied by a
polymer nanocomposite?
A polymer nanocomposite implant must
meet certain design and functional criteria.

Including; biocompatibility,
biodegradability, mechanical properties and
in some cases, aesthetic demands.

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 Why use HAP ?
Hydroxyapatite (HAP)–polymer Nano composites
have been used as a biocompatible and
osteoconductive substitute for bone repair and
implantation.

As the main inorganic component of hard tissue,

HAP [Ca10(PO4)6(OH)2] has long been used in
orthopedic surgery.

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 Why not use HAP only?
HAP is difficult to shape because of its
brittleness and lack of flexibility.

HAP powders can migrate from implanted

sites, thus making them inappropriate for use.

Moreover, these powders do not disperse

well and agglomerate easily.

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 Why not use HAP only?
Cont’d…
Therefore, the incorporation of HAP in polymeric
nanocomposites to overcome processing and
dispersion challenges is of great interest to the
biomedical community.

Consequently, a desirable material for use in

clinical orthopedics would be a biodegradable
structure that induces and promotes new bone
formation at the required site.
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 Why use chitosan as the
polymer matrix?
The matrix used for this study, chitosan (a
cationic, biodegradable polysaccharide) is
flexible and has a high resistance upon
heating.

 During heating intra molecular hydrogen

bonds formed between the hydroxyl and
amino groups. 10
 How to overcome HAP
aggregation?

The traditional problem of HAP aggregation

was overcome by precipitation of the apatite
crystals within a polymer solution.

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 The method used to make
the composite
Therefore the resulting nanocomposite is
prepared by the co-precipitation method.

It is mechanically flexible and can be formed

into any desired shape.

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 Methodology
Chitin Extraction

Chitosan Extraction

Composite Fabrication

Characterization
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Chitin/Chitosan Extraction
•Preconditioning with commercial grade acetic acid.

•Deproteination with commercial grade NaOH.

•Demineralization with commercial grade HCl.

•Deacetylation 25M commercial grade NaOH.

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Chitosan Extraction Cont’d…
Chitin Chitosan

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Chitosan Extraction Cont’d…

Extracted Chitosan

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 Preparation of composite
 Preparation of the composite was carried out as to
have a final CS/HA ratio of 70/30.
 3g of chitosan was mixed with 100ml of 2% acetic acid
using a magnetic stirrer until it dissolve completely.
 Chitosan, Ca(OH)2, H3PO4, acetic acid, NaOH and
deionized water were used to prepare the composite.
 76.5ml of 0.1M H3PO4 was mixed with chitosan
solution and stirred thoroughly.
 A solution of Ca(OH)2 was prepared by adding
0.9512 g of Ca(OH)2 into 250ml of deionized water.

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 Preparation of Composite
cont’d...
 Chitosan/H3PO4 solution was slowly added to Ca(OH)2
solution while it's being thoroughly stirred. The rate of
addition was kept to 3ml per minute.

 After every addition of 3ml ,the resulting solution was

ultrasonically treated and the pH variation was recorded.

 These steps were repeated until all the chitosan/H3PO4

solution was mixed with Ca(OH)2.

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pH variation of Ca(OH)2 solution
with CS/H3PO4 addition
pH value of the solution at 300C

CS/H3PO4 in ml

19
 Preparation of Composite
cont’d...
 The resulting solution was quite transparent and had a
stable pH value.

 To precipitate the composite, 0.5M NaOH was slowly

added at a rate of 3ml per minute while the resulting
solution being stirred and after every 3ml, the solution was
ultrasonically treated and pH variation was measured.

 These steps were repeated until the resulting solution

reached a pH value a little above 10.

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 Preparation of Composite
cont’d...
 Refluxing of the composite was done at 700C for 24 hours. To
compare the effect of refluxing temperature on the composite,
samples of composites were refluxed at two different
temperatures; 700C and 900C.

 Finally ,the prepared composites were aged at least for 2 days

before drying.

 After drying the composites, samples of composites were

characterized under FTIR and TGA-DTA.

 Some samples of composites were prepared for characterization

by using XRD analysis and SEM analysis.
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Preparation of Composite
cont’d...
Photograph of the composite
Agglomerates of HA Particles

Chitosan Matrix

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 Characterization
 FTIR Analysis

 TGA/DTA Analysis

 XRD Analysis

 SEM Analysis

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Absorbance unit
FTIR Analysis

Wavenumber cm-1
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FTIR for chitosan/S1 used in Composite
CS-HA/T70/S1 (Refluxed at 700C)
Wave number Corresponding
3440.97

2882.96

1646.29
1598.19

1422.52
1380.52
1328.23

1254.87

1152.89
1090.13
1033.60
(cm-1) Bond(s)

3440 O-H, N-H

stretching
0.30

2882 C-H stretching

0.25

1646 amide I(C=O) , H2O

0.20
Absorbance Units

1598 amine (NH2)

0.15

1422,1380 C-H deformation

0.10

1380,1328,1254 C-N
0.05

1152,1090,1033 saccharides
0.00

3500 3000 2500 2000 1500 1000

Wavenumber cm-1

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 FTIR for chitosan/S4 used in composite
CS-HA/T90/S4 (Refluxed at 900C) and composite
CS-HA/-/S5 (Aged without precipitation by NaOH)
3442.96

2884.07

2371.68
2338.36

1644.79
1597.74

1422.04
1378.65
1327.85
1253.56

1151.27
1088.47
1033.49

895.79

662.63
598.64
Wave number (cm-1) Corresponding
Bond(s)

3442 O-H, N-H

stretching
0.35

2884 C-H stretching

0.30

1644 amide I(C=O),

0.15 0.20 0.25

H2O
Absorbance Units

1597 amine (NH2)

1422,1378 C-H deformation

0.10

1378,1327,1253 C-N
0.05

1151,1088,1033,895 saccharides
0.00

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

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 FTIR for Composite Sample
CS-HA/T70/S1(Refluxed at 700C)
3377.50

2923.32

1568.21

1417.03

1256.00

1150.87

1033.23

603.65
565.46
Wave number Corresponding
(cm-1) Bond(s)
1.4

3377 O-H,N-H
stretching
1.2

2923 C-H stretching

1.0

1568 amine (NH2),

Absorbance Units

amide II(NH)
0.6 0.8

1417 CO32-
1256 C-N
0.4

1150,1033 saccharides
0.2

1033,603,565 PO43-
0.0

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

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C:\Program Files\OPUS_65\MEAS\Composite 1 15812.4 Composite 1 15812 Instrument type and / or accessory 15/08/2012
 FTIR for composite sample
CS-HA/T90/S4(Refluxed at 900C)
3469.48
3399.68

2925.89

1643.74

1561.47

1416.43

1150.16

1038.90

896.13

656.61
604.76
567.49
Wave number Corresponding
(cm-1) Bond(s)

3469 ,3399 O-H,N-H

stretching
3.0

2925 C-H stretching

2.5

1643 amide I(C=O)

,H2O
Absorbance Units
2.0

1561 amine
(NH2),amide
1.5

II(NH)
1416 CO32-
1.0

1150,1038,896 saccharides
0.5

656
1038,604,567 PO43-
0.0

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

C:\Program Files\OPUS_65\MEAS\Compo Sam 4 201212.0 Compo Sam 4 201212 Instrument type and / or accessory 20/12/2012
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 FTIR for composite sample CS-HA/-/S5(Aged
without precipitation by NaOH)
3426.31

2926.44
2857.63

1636.88

1454.46
1421.49

1038.89

871.08

604.26
567.36
Wave number Corresponding
(cm-1) Bond(s)

3426 O-H,N-H
stretching
2.0

2926,2857 C-H stretching

1.5
Absorbance Units

1636 amide I(C=O) ,

H2O,NH3+
1.0

1454,1421,871 CO32-
0.5

1038,604,567 PO43-
0.0

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

C:\Program Files\OPUS_65\MEAS\Composite S 5 030113.0 Composite S 5 030113 Sample Compartment 03/01/2013

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Page 1/1
 TGA/DTA ANALYSIS
Composite Sample CS-HA/T70/S1(Refluxed at 700C)

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XRD analysis
 The crystallite size of HA nanoparticles in the
composite was determined using the diffraction
peak of the (0 0 2) plane and the Scherrer
equation.
D=Kλ / βcosθ

D -average crystallite size

β -FWHM (in radians)
λ -the wavelength of X-ray radiation (0.1540562 nm)
K is a constant related to the crystallite shape and is approximately 1

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XRD analysis cont’d...
 Lattice parameters (a and c) can calculated from peaks
(2 0 0) and (0 0 2), respectively, using the standard
HCP unit cell plane spacing relationship.

d -distance between adjacent planes in the set of Miller

indices (h k l).

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XRD analysis cont’d...
• XRD analysis was carried out using software called
fityk version 0.9.8. FWHM of (002) plane was
calculated after fitting the curve using the
Gaussian function.
• Shape factor was assumed to be 1 in this
calculation.
• The direction of the crystal growth can be
measured by relative intensity of (002) diffraction
plane [I002/I211].

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XRD analysis cont’d...
 Two broad peaks were seen around 2θ values of 100
and 200 which are attributed to chitosan crystalline
phase.

 Characteristic peaks of HA at 25.860 and around

320 were attributed to (002) plane and the
overlapping of (211), (112) and (300) respectively.

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XRD pattern for Composite Sample
CS-HA/T70/S1 (Refluxed at 700C)

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XRD pattern for Composite Sample
CS-HA/T90/S4 (Refluxed at 900C)

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XRD pattern for both Composite Samples
CS-HA/T70/S1 and CS-HA/T90/S4

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Mean crystallites sizes of HA in Composite
Samples CS-HA/T70/S1 and CS-HA/T90/S4

Refluxing temperature of Mean crystallite size

composites

700C 25.76 nm

900C 44.06 nm

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SEM analysis of the composite(cross
section)
HA particles
Composite Sample CS-HA/T70/S1

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THANK YOU

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QUESTIONS

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