The document discusses protein adhesion on bone bioceramics. It aims to evaluate how different ratios of calcium silicate and titanium dioxide influence protein adsorption. Collagen was extracted from rat skin and used to study protein interactions. Depletion studies showed that 1% titanium dioxide promoted the most collagen adhesion to calcium silicate composites, while higher concentrations reduced adhesion. FTIR analysis supported that 1% titanium dioxide optimized collagen secondary structure and mineralization on the composites.

1. PROTEIN ADHESION ONPROTEIN ADHESION ON
BONE BIOCERAMICSBONE BIOCERAMICS
GOPI KRISHNA GIRI
12pbt003
DEPARTMENT OF BIOTECHNOLOGY
ACHARIYA ARTS & SCIENCE COLLEGE
(Affiliated to Pondicherry University)
VILLIANUR,PUDUCHERRY

2. LIST OF CONTENTSLIST OF CONTENTS
 I - INTRODUCTION
 II - REVIEW OF LITERATURE
 III - AIM AND OBJECTIVES OF THE STUDY
 IV - MATERIALS AND METHODS
 V - RESULTS AND DISCUSSION
 VI - SUMMARY AND CONCULSION
 VII - REFERENCES
 VIII - ACKNOWLEDGEMENT

3. INTRODUCTIONINTRODUCTION
Biomaterials science forms the cutting edge of current
technological advances due to its role in replacing lost parts.
 A biomaterial is essentially a material that is used and
adapted for a medical application.
Biomaterials are used in dental application, surgery and drug
delivery, joint replacement, dental implants for tooth fixation.

4. INTRODUCTIONINTRODUCTION
Collagens are the major structural proteins of connective
tissues such as skin, tendon, bone, cartilage, blood vessels
and basement membranes.
In humans, collagen comprises one-third of the total protein,
accounts for three-quarters of the dry weight of skin, and is
the most prevalent component of the extra-cellular matrix
(ECM).
Collagen is triple helix proteins, containing rod shaped
domains about 3000 Å long and only 15 Å in diameter.

5. INTRODUCTIONINTRODUCTION
Structure of Collagen

6. INTRODUCTIONINTRODUCTION
For a protein adhesion to occur the protein, transported to the
surface by molecular diffusion or convention.
Adhesion depends upon nature, the charged groups and the
magnitude of the van der wall and hydrophobic interaction
between the protein and the surface.
Analytical techniques like X-ray crystallography, Circular
dichroism (CD spectra), Fourier transform infrared spectroscopy
(FTIR), Depletion etc can be used to measure adsorption of
protein on the surface.

7. MECHANISMMECHANISM
PROTEIN ADHESION

8. REVIEW OF LITERATUREREVIEW OF LITERATURE
Chung et al., (2004) described that the collagens are the major structural
proteins of connective tissues such as skin, tendon, bone, cartilage, blood
vessels and basement membranes.
Wang et al., (2012) -Bioceramics, because of its excellent biocompatible
and mechanical properties, has always been considered as the most
promising materials for hard tissue repair.
Robert et al., (2005) -When a solid material comes in contact with a fluid
that contains soluble proteins, proteins rapidly adsorb onto the surface of
the material.
Hlady et.al., (1999) - Solution depletion is one of the simplest methods
to study protein adsorption.

9. AIM & OBJECTIVES OF THEAIM & OBJECTIVES OF THE
STUDYSTUDY
AIM
To evaluate and analyze the influence of titanium dioxide (TO) on the
protein adsorption/interaction properties of Calcium silicate bioceramics.
OBJECTIVES
To prepare various ratios of Calcium silicate-Titanium dioxide.
Evaluate protein biomaterial interactions between samples and collagen
solution.
Analysis the influence of TO content on the protein interaction.

10. MATERIALS & METHODSMATERIALS & METHODS

11. SYNTHESIS OF CALCIUMSYNTHESIS OF CALCIUM
SILICATESILICATE
 Kept in magnetic stirrer for 3 hours.
 Filter the solution .
 Dried the filtrate at 110 0
c.
Solution Materials Weight(gm) Volume (ml)
A Sodium Silicate 98.02 70.52
Water 429.5 429.5
B Calcium hydroxide 34.73 14.97
33% HCL 27.94 24.00
Water 461.0 461.0
C Slurry ( A + B ) 1051 1000

12. PREPARATION OF CALCIUMPREPARATION OF CALCIUM
SILICATE& TITANIUM DIOXIDESILICATE& TITANIUM DIOXIDE
COMPOSITECOMPOSITE
Calcium silicate (CS) & Titanium dioxide was homogenized.
The pellet was prepared using stainless steel hydraulic press at
50Mpa.
Sample Calcium Silicate (mg) Titanium dioxide (mg) Final (mg)
0% 200 0 200
0.5% 195 5 200
1% 190 10 200
1.5% 185 15 200

13. ISOLATION OF COLLAGENISOLATION OF COLLAGEN
Skins of sacrificed Wistar rats (~200 gm) were obtained from CPCSEA.
Rat skins were cut into small sizes (1inch approx) and soaked in 0.5%
aqueous acetic acid for 3-5 days, till swelling was incipient.
The hair was removed & washed with distilled water.
The skin was soaked in Chloroform: Methanol (3:1) solution for 2 hours.

14. ISOLATION OF COLLAGENISOLATION OF COLLAGEN
Then, the skin was immersed in 10% w/v sodium chloride
solution for 4 days followed by grinding in mixer to homogenize
it.
The mixture was centrifuged at 8000 rpm for 15 minutes at 4o
C.
Supernatant was dialyzed against cold distilled water for 3-4
days & the solution was stored at 4o
C till further use.

15. ANALYTICAL TECHNIQUESANALYTICAL TECHNIQUES
FTIR ANALYSIS
FTIR Spectroscopy was recorded using Potassium Bromide (KBr) pellet
from 4000-400cm-1
range using Perkin-Elmer FTIR Spectrophotometer
with a resolution of 1 cm-1
.
CIRCULAR DICHROISM STUDIES
CD Spectra were made using Jasco J815 CD Spectrometer from 190 to
260 nm at 0.5 nm resolution and a protein concentration of ~0.5 mg/ml.
DEPLETION STUDIES
The sample was placed in ~0.5 mg/ml solution of salt soluble collagen. At
definite time interval, i.e. (0, 2, 10, 20, 45 minutes), Absorbance in
UV-Vis spectrophotometer was recorded at 280nm.

16. MINERALIZATION STUDIESMINERALIZATION STUDIES
The solution was prepared by dissolving Potassium dihydrogen
orthophosphate: 122.4mg, Calcium chloride: 166.5mg,
Tris (Hydroxy Methyl aminomethane): 6.057g and 33% Hydrochloric acid
(1M):40ml in 1Litre of distilled water.
Sample pellets were immersed in buffer which was changed once in a 3
days till 21 days. Subsequently, samples were taken and dried in hot air
oven at 1000
C for 1 hr. FTIR Spectroscopy was recorded for the surface of
the samples.

17. RESULTS & DISCUSSIONRESULTS & DISCUSSION

18. CD SPECTRA OFCD SPECTRA OF
COLLAGENCOLLAGEN
190 200 210 220 230 240 250 260
-45
-30
-15
0
15
Absorbance
Wavelength (nm)

19. FTIR OF COLLAGENFTIR OF COLLAGEN
(%)
0
90

20. DEPLETION STUDIES OFDEPLETION STUDIES OF
COLLAGENCOLLAGEN
TIME 0% 0.5% 1% 1.5%
0 min 0.499 0.499 0.499 0.498
2 min 0.483 0.379 0.457 0.485
10 min 0.406 0.294 0.339 0.294
20 min 0.405 0.268 0.287 0.284
45 min 0.233 0.11 0.056 0.1

21. DEPLETION STUDIES OFDEPLETION STUDIES OF
COLLAGENCOLLAGEN
0 min 2 min 10 min 20 min 45 min
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
Absorbance
Time (mins)
Control
0.5%
1%
1.5%

22. FTIR 0% TITANIUM DIOXIDEFTIR 0% TITANIUM DIOXIDE
(%)

23. FTIR 1.0% TITANIUM DIOXIDEFTIR 1.0% TITANIUM DIOXIDE
4000 3000 2000 1000
80
0
Transmittance
Wavenumber (cm
-1
)

24. FTIR TO ANALYZE PROTEINFTIR TO ANALYZE PROTEIN
SECONDRY STRUCTURE ( 1.5%)SECONDRY STRUCTURE ( 1.5%)
1700 1680 1660 1640 1620 1600
20
30
40
50
60
70
80
90
100
110
Transmittance(%)
Wavenumber (cm
-1
)

25. MINERALIZATION FTIR 1.5%MINERALIZATION FTIR 1.5%
TITANIUM DIOXIDETITANIUM DIOXIDE
4000 3500 3000 2500 2000 1500 1000 500
60
0
Transmittance(%)
Wavenumbers (cm
-1
)

26. SUMMARY & CONCLUSIONSUMMARY & CONCLUSION
 The salt soluble collagen was successfully extracted from skin of
sacrificed rats.
 Calcium Silicate was synthesized and CSTO composites were
successfully prepared.
 The present study has thrown valuable light on the influence of TO on
the protein adsorption of CS.

27. SUMMARY & CONCLUSIONSUMMARY & CONCLUSION
 The studies have proved that the protein adhesion on the CSTO
composites was a direct function of TO content.
 At lowest concentration range, TO promoted protein adhesion. As the
content of TO increased beyond 1%, the protein adhesion was reduced
 Hence, 1% TO added to CS was determined to be the optimal
concentration.

28. REFERENCESREFERENCES
 Bhattacharjee A and Bansal M. Collagen structure: the madras triple helix and the
current scenario. IUBMB Life. 2005; 57: 161 – 172.
 Chnandrakasan G, Dennis A, Torchia, and Piez K.A. Preparation of intact monomeric
collagen from rat tail tendon and skin and the structure of the non-helical ends in
solution. Biological Chemistry.1976; 251: 6062 - 6067.
 Wassell DT, Ebbery G. Adsorption of bovine serum albumin on to titanium powder.
Biomaterials.1996; 9: 859 - 864.
 Zeng H, Chittur KK, Lacefied WR. Analysis of bovine serum albumin adsorption on
calcium phosphate and titanium surfaces. Biomaterials.1999; 20: 377 - 84.
 Wahlgren M and Arnebrant T. Protein adsorption to solid surfaces. Trends in

29. ACKNOWLEDGEMENTACKNOWLEDGEMENT
 I felt extremely honored for getting the opportunity to work under the guidance of Dr. T. P. Sastry,
Senior Principle Scientist and Head, Bioproduct Laboratory, CSIR-Central leather Research
Institute, Chennai, for his meticulous guidance, insightful suggestions and encouragement
throughout the progress of my project.
 It is my great privilege to express my sincere gratitude to my research guide Dr. Salamun DE,
Head of the Department, Department of Biotechnology, Achariya Arts and Science College,
Puducherry, for his suggestion, guidance & discussion throughout the course of study.
 With profound gratitude, I express my sincere thanks to my college staff & my friends for their
support and encouragement which served as a beacon to reach this destination.