PROTEIN ADHESION ON PROTEIN ADHESION ON BONE BIOCERAMICSBONE BIOCERAMICS

GOPI KRISHNA GIRI12pbt003

DEPARTMENT OF BIOTECHNOLOGY ACHARIYA ARTS & SCIENCE COLLEGE

(Affiliated to Pondicherry University)

VILLIANUR,PUDUCHERRY

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

INTRODUCTIONINTRODUCTIONBiomaterials 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.

INTRODUCTIONINTRODUCTIONCollagens 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.

INTRODUCTIONINTRODUCTION

Structure of Collagen

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.

MECHANISMMECHANISM

PROTEIN ADHESION

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.

AIM & OBJECTIVES OF THE AIM & 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.

MATERIALS & METHODSMATERIALS & METHODS

SYNTHESIS OF CALCIUM SYNTHESIS 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

PREPARATION OF CALCIUM PREPARATION OF CALCIUM SILICATE& TITANIUM DIOXIDE SILICATE& 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

ISOLATION OF COLLAGENISOLATION OF COLLAGENSkins 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.

ISOLATION OF COLLAGENISOLATION OF COLLAGENThen, 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 4oC.

Supernatant was dialyzed against cold distilled water for 3-4

days & the solution was stored at 4oC till further use.

ANALYTICAL TECHNIQUESANALYTICAL TECHNIQUESFTIR 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.

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.

RESULTS & DISCUSSIONRESULTS & DISCUSSION

CD SPECTRA OF CD SPECTRA OF COLLAGENCOLLAGEN

190 200 210 220 230 240 250 260

-45

-30

-15

0

15

Ab

so

rban

ce

Wavelength (nm)

FTIR OF COLLAGENFTIR OF COLLAGEN

(%)

0

90

DEPLETION STUDIES OF DEPLETION STUDIES OF

COLLAGENCOLLAGENTIME 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

DEPLETION STUDIES OF DEPLETION STUDIES OF COLLAGENCOLLAGEN

0 min 2 min 10 min 20 min 45 min0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Ab

so

rban

ce

Time (mins)

Control 0.5% 1% 1.5%

FTIR 0% TITANIUM DIOXIDEFTIR 0% TITANIUM DIOXIDE

(%)

FTIR 1.0% TITANIUM DIOXIDEFTIR 1.0% TITANIUM DIOXIDE

4000 3000 2000 1000

80

0

Tra

nsm

ittan

ce

Wavenumber (cm-1)

FTIR TO ANALYZE PROTEIN FTIR 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

Tra

nsm

itta

nce (

%)

Wavenumber (cm-1)

MINERALIZATION FTIR 1.5% MINERALIZATION FTIR 1.5% TITANIUM DIOXIDETITANIUM DIOXIDE

4000 3500 3000 2500 2000 1500 1000 500

60

0

Tra

nsm

itta

nce (

%)

Wavenumbers (cm-1)

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.

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.

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

biotechnology.1991; 9: 201-208.

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.