Preparation and characterization of calcium ...

Mansoura University Faculty of Science Physics Department

Preparation and characterization of calcium phosphate ceramics containing some rare earth oxides for using as biomaterials. By Tarek Abbas Ali Elsayed Elkhooly B.Sc. in Biophysics (1999)

A Thesis Submitted in Partial Fulfillment for Requirements of the Degree of M.Sc. in Physics (Biophysics) SUPERVISORS Prof. Dr. W.I. Abdelfattah

Prof. Dr. F.M. Reicha

Professor of ceramics and bioceramics Biomaterial Division National Research Center

Professor of Experimental Solid State Physics Faculty of Science Mansoura University

2007


SUPERVISORS

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" Preparation and characterization of calcium phosphate ceramics containing some rare earth oxides for using as biomaterials." THESIS TITLE :

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RESEARCHER NAME : Tarek

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Abbas Ali Elsayed Elkhooly

SUPERVISORS: No. 1

2

Name

Prof. Dr. F.M. Reicha Prof. Dr. W.I. Abdelfattah

Profession Professor of Experimental Solid State Physics, Faculty of Science Mansoura University Professor of ceramics and bioceramics, Biomaterial Division National Research Centre

Signature

Head of Physics Department

Vice-Dean for Graduate Studies and Research

Dean of Faculty of Science

Prof. Dr. M. A. Madkour

Prof. Dr. E.M. El-Abbasy

Prof. Dr. T.Z Sokkar


REFEREES COMMITTEE DISCUSSION

THESIS TITLE : Preparation and characterization of calcium

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phosphate ceramics containing some rare earth oxides for using as biomaterials. RESEARCHER NAME : Tarek Abbas Ali Elsayed Elkhooly Supervisors Committee: U

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No.

Name

Profession

1


2

Prof. Dr. W.I. Abdelfattah

Professor of Experimental Solid State Physics, Faculty of Science Mansoura University Professor of ceramics and bioceramics, Biomaterial Division National Research Centre

Referees Committee Signatures: Serial

Name

Profession

1


2

Prof. Dr. W.I. Abdelfattah

3

Prof. Dr.

Professor of Experimental Solid State Physics, Faculty of Science Mansoura University Professor of ceramics and bioceramics, Biomaterial Division National Research Centre Professor of Biophysics, Faculty of Science Ain Shams University Professor and Head of Biophysics Department, Faculty of Science Cairo University

A.M. Sallam 4

Prof. Dr. O.W. Guirguis

Head of Physics Department

Vice-Dean for Graduate Studies and Research

Dean of Faculty of Science

Prof. Dr. M. A. Madkour

Prof. Dr. E.M. El-Abbasy

Prof. Dr. T.Z Sokkar

Note

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The present thesis is submitted to Mansoura University in partial fulfillment for the requirements of M.Sc degree in biophysics. Beside this work carried out this thesis; the student has attended and passed successfully the following postgraduate courses during the academic year (May 2001). 1-

Molecular and Atomic Spectrometer.

2-

Laser Applications.

3-

Physics of Biomaterial and Substitutions.

4-

Radiotherapy and Ultrasound Waves.

5-

Biostatistics.

6-

Solid State (Special course)

7-

Molecular Biophysics.

8-

Biophysics of Cells and Membranes.

9-

Ophthalmology.

10-

Oncology and Nuclear Medicine.

11-

Microbiology.

12-

Biomathematics.

13-

English language.

Head of physics department

Prof. Dr. M. Madkour

Contents

Contents

Contents Contents……………………………………………………………………..

i

Acknowledgment……………………………………………………………

viii

Abstract……………………………………………………………………..

xi

Chapter I Introduction and Aim of the Work 1- Introduction………………………………………………………………..

1

2 Aim of the present study……………………………………………………

7

Chapter II Theoretical Background and Literature Survey 1 Theoretical Background……………………………………………………

8

1-1 Prosthesis and biomaterials: two inseparables……………………….

8

1-2 Bioceramics…………………………………………………………

9

1-3 Classification of bioceramics……………………………………….

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1-3.1. Bioinert ceramics…………………………………………….

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1-3.2.Resorbable ceramics………………………………………….

11

1-3.3. Bioactive ceramics…………………………………………..

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1-4 Types of biomaterials fixation……………………………………..

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1-4.1 Morphological fixation……………………………………….

12

1-4.2 Biological fixation…………………………………………….

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1-4.3 Bioactive fixation………………………………………………

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1-5 Bone Graft…………………………………………………………..

13

1-5.1 Types of bone tissue graft……………………………………..

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1-5.1.A. Autograft……………………………………………….

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1-5.1.B. Allograft……………………………………………….

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1-5.1.C. Xenografts or Heterograft………………………………

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1-5.1.D. Alloplastic or Synthetic bone grafting………………….

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i

Contents

1-5.2. Key factors of an ideal bone graft…………………………….

16

1-5.2.A. Oseoconductive bone graft……………………………

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1-5.2.B. Oseoinductive bone graft……………………………….

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1-5.2.C. Oseogenic bone graft......................................................

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1-5.2.D. Oseoproductive bone graft.............................................

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1-6 Properties primarily required for biomaterials……………………….

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1-6.1 Biocompatibility……………………………………………….

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1-6.2 Bioactivity…………………………………………..................

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1-6.3 Mechanical strength……………………………………………

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1-6.4 Chemical resistance…………………………………………….

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1-6.5 Mechinability…………………………………………………..

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1-6.6 Electrical properties of bone and bioceramics…….....................

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1.6.6.1 Piezoelectricity of bone………………………………………

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1-6.6.2 Medical application of polarized bioceramics………………...

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1-7 Calcium phosphate ceramics…………………………………………

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1-7.1 Classification of calcium phosphate ceramic…………………

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1-7.1.A Hydroxyapatite (HA)……………………………………….

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1-7.1.B Calcium deficient hydroxyapatite (CDHA).........................

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1-7.1.C Beta-tricalcium phosphate (β-TCP)......................................

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1-7.1.D Biphasic calcium phosphate (BCP)…………………………

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1-8 Calcium sulfate based bone substitutes………………………………

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2 Literature survey……………………………………………………………

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2-1 preparation of Hydroxyapatite……………………………………

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2-2 preparation of beta-tricalcium phosphate (β-TCP)……………….

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2-3 preparation of biphasic calcium phosphate (BCP)……………….

36

2-4 Metal ions substitution……………………………………………

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Chapter III Materials preparation and Experimental techniques ii

Contents

1. Materials and preparation techniques…………………………………

40

1.1. β-Tricalcium phosphate (β-TCP)………………………………..

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1.1.A Effect of doping with 2% or 3% SO 4 2- ……………………..

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1.1.B: Effect of microwave curing ……………………………….

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1.2. Hydroxyapatite…………………………………………………

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1.3. Biphasic calcium phosphate ……………....................................

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1.3.A. Preparation of Bµ1D………………………………………

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1.3.B. Preparation of Bµ2D……………………………………….

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1.4 Tantalum hydroxide Ta(OH) 5 doped biphasic Bµ1C……………

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1.5 Niobium hydroxide Nb(OH) 5 doped biphasic Bµ1C……………

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1.6 Nd 2 (SO 4 ) 3 doped biphasic Bµ1C………………………………..

47

B

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B

B

B

B

B

B

PB

P

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B

B

2. Material characterization techniques……………………………….

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2.1 Chemical analysis……………………………………………….

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2.2 Structural assessment…………………………………………….

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2.2.1 X-ray diffraction analysis (XRD)……………………………

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2.2.2 Fourier transformer infrared (FT-IR) analysis……………….

51

2.3 Thermogravimetric analysis……………………………………….

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2.4 Ceramic parameters………………………………………………..

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2.5 Morphological assessment………………………………………….

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2.5.1 Transmission electron microscopy (TEM)……………………….

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2.6 Bioactivity (In-Vitro Test)………………………………………….

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2.6.1 Preparation of Simulated Body Fluid (SBF)………………………

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2.6.2 Biochemical investigation ………………………………………..

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2.6.3 Assessment of biomimetic layer………………………………….

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2.6.3.1 Structural assessment (Diffused reflected FT-IR)………………

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2.6.3.2 Morphological assessment of biolayer formation……………….

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2.7 Bioelectricity…………………………………………………………

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iii

Contents

2.7.1 Measuring device……………………………………

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2.7.2 Cell for measuring A.C. conductivity and dielectric parameters……………………………………………………………..

59

2.7.3 Experimental precautions…………………………..

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2.7.4 Noise consideration…………………………………

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Chapter IV Results and Discussion 1 Beta tricalcium phosphate (β-TCP) ……………………………………….

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1-1 Dried samples………………………………………………………….

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1-1-1 Chemical analysis……………………………………………….

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1-1-2 Structural analysis……………………………………………….

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1-1-2-A X-ray diffraction (XRD)…………………………………

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1-1-2-B FT-IR results......................................................................

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1-2 Calcination.............................................................................................

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1-2-1 Chemical analysis.........................................................................

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1-2-2 Structural analysis..........................................................................

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1-2-2-A X-ray diffraction (XRD).......................................................

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1-2-2-B FT-IR results........................................................................

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1-3 Thermal Analysis (TGA/DTG)..............................................................

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1-4 Morphological analysis, transmission electron microscope................

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2- Hydroxyapatite (HA)………………………………………………………

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2-1Dried samples..........................................................................................

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2-1-1 Chemical analysis..........................................................................

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2-1-2 Structural analysis………………………………………………..

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2-1-2-A X-ray diffraction (XRD)…………………………………..

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2-1-2-B FT-IR analysis…………………………………………….

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2-2 Calcination………………………………………………………………

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2-2-1 Chemical analysis…………………………………………………

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Contents


88

2-2-2-A X-ray diffraction (XRD)………………………………….

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2-2-2-B FT-IR Analysis……………………………………………

90

2-3 Thermogravimetric analysis…………………………………………

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2-4 Morphological analysis, transmission electron microscope…….. ….

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3- Biphasic calcium phosphate (BCP) ………………………………………..

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3-1 Biphasic calcium phosphate (BCP) with different Ca/P molar ratio.......

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3-1-1 Chemical analysis………………………………………………..

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3-1-2-A X-ray diffraction (XRD)…………………………………..

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3-1-2-B FT-IR results........................................................................

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3-1-3 Thermal Analysis (TGA/DTG)..........................................................

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3-1-4 Morphological analysis, transmission electron Microscope.........

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3-2 Biphasic calcium phosphate (Bµ1D) doped with tantalum hydroxide…

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3-2-1 Chemical analysis………………………………………………..

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3-2-2 Structural analysis……………………………………………….

109

3-2-2-A X-ray diffraction (XRD)…………………………………

109

3-2-2-B FT-IR results......................................................................

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120

3-2-4 Morphological analysis, transmission electron microscope............

122

3-3 Biphasic calcium phosphate (Bµ1D) doped with niobium hydroxide…..

124

3-3-1 Chemical analysis………………………………………………….

124

3-3-2 Structural analysis…………………………………………………

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3-3-2-B FT-IR results.......................................................................

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135

3-3-4 Morphological analysis, transmission electron microscope............

136

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Contents

3-4 Biphasic calcium phosphate (Bµ1D ) doped with neodymium sulfate

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3-4-1 Chemical analysis…………………………………………………

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3-4-2 Structural analysis…………………………………………………

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3-4-2-B FT-IR results........................................................................

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152

3-4-4 Morphological analysis, transmission electron microscope...........

154

4-Bioactivity (In-vitro Test)………………………………………....................

155

4-1 β-TCP samples………………………………………………………….

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4-1-1 Biochemical analysis………………………………………………

155

4-1-2 Surface analysis……………………………………………………

159

4-1-2-a Diffused reflected Fourier transformer infrared (DRFT-IR)

159

4-1-2-b Scanning electron microscope……………………………

163

4-2 Hydroxyapatite (HA) …………………………………………………...

167

4-2-1 Biochemical Analyses…………………………………………….

167

4-2-2 Surface Analyses………………………………………………….

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4-2-2-b Scanning electron microscope……………………………

171

4-3 Biphasic calcium phosphate (B1µC) doped with various metals………

172

4-3-1 Biochemical analyses……………………………………………..

172

4-3-2 Surface analyses…………………………………………………

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4-3-2-b Scanning electron microscope…………………………..

184

5- Electricity………………………………………………………………..

190

5-1 Dielectric and A.C. conductivity of pure calcium phosphate samples……… 190 5-1-1 A.C. conductivity measurement of pure calcium phosphate samples……

190

5-1-1-a Frequency dependence of A.C. conductivity………………………….

190

vi

Contents

5-1-1-b Temperature dependence of A.C. conductivity………………………

201

5-1-2 A.C. conductivity measurement of biphasic calcium phosphate (BCP) doped with various metals…………………………………………………….

203

5-1-2 Frequency dependence of A.C. conductivity……………………………

203

5-2-1 Frequency dependence of dielectric measurements of pure calcium phosphate Samples……………………………………………………………

209

5-2-2 Temperature dependence of dielectric measurements of pure calcium phosphate Samples……………………………………………………………

213

5-2-3 Frequency dependence of dielectric measurements of biphasic calcium phosphate (BCP) doped with various metals………………………………….

215

5-2-4 Temperature dependence of dielectric measurements of biphasic calcium phosphate (BCP) substituted with various metals……………………………..

219

Chapter V Conclusions…………………………………………………………

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References……………………………………………………………………..

225

List of reactions………………………………………………………………..

241

Arabic Summary………………………………………………………………

vii

Acknowledgement

ACKNOLAGMENT

ACKNOWLEDGMENT

Thanks and indebtedness is directed first and always to ALLAH for all his graves, without the power he gave me, the accomplishment of his work would have been certainly impossible. The author feels a deep sense of gratitude to all the people associated in various ways during the progress and completion of this work. My sincere gratefulness to Prof. Dr. W. I. Abdelfattah, Prof. of Ceramics and Bioceramics, Biomaterials Division , Inorganic Chemical Industries & Mineral Resources Research Division ,

National Research Centre,

for

suggesting the essential points of this subject. She is also deeply thanked for his supervision, advice, constructive discussion and giving me uninterrupted freedom during the course of the work. I would like to express my deep gratitude, appreciation and sincerest thanks to Prof.. Dr. F.M. Reicha, Prof of Experimental Solid State Physics, Physics department, Faculty of Science, Mansoura University for his supervision, useful discussions, advice and great help during the course of the work. Finally, I would like to extend my deep gratitude and appreciation to my mother and father who gift me the life, my large family and small family (my wife and my kids) for their help, understanding and continuous encouragement, truly dedicate this work.

viii

Abstract

Abstract

Abstract The first aim of the present work is to prepare various types of calcium phosphates in nanometric scale through curing with microwave and confirm there structures and purity percentage by using several characterization methods. The second major aim is to incorporate transitional and rare earth metal ions in the calcium phosphate crystal structure to improve its bioactivity behavior in vitro. Three phases of calcium phosphates being hydroxyapatite, beta tricalcium phosphate and biphasic calcium phosphate were prepared from calcium deficient hydroxyapatite by chemical precipitation method, microwave curing, and calcination. Additionally, transitional metal ions (tantalum oxide Ta 2 O 5 and B

B

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niobium oxide Nb 2 O 5 ) and rare earth metal ions (neodymium oxide Nd 2 O 3 ) B

B

B

B

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B

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were incorporated to study their effect on the ceramic parameters and crystalline structure by XRD, FT-IR, TGA, Density measurements as well as TEM. The biomimetic surface deposition was followed for calcined samples immersed in corrected-simulated body fluid (c-SBF) to assess biolayer formation. Dielectric and A.C. conductivity of the calcined phases were measured to discuss relation to structure. Structural analyses (XRD and FT-IR) emphasized that the transformation of calcium deficient hydroxyapatite (CDHA) to beta tricalcium phosphate (βTCP), biphasic calcium phosphate (BCP) and hydroxyapatite (HA) depends on the degree of calcium deficiency present in the parent CDHA. The most pure βTCP sample was obtained by doping with 3% ammonium sulfate and calcination at 1100 o C for 3 h. While samples prepared under microwave curing exhibited a P

P

minor amount of hydroxyapatite phase impurities due to its heat effect on the initial Ca/P molar ratio. In situ preparation of BCP compounds with different xi

Abstract

initial Ca/P ratio revealed different ratios of HA to β-TCP phases, where the HA phase increases as the initial Ca/P ratio increases. Morphological analysis revealed the ability of microwave radiation to reduce and homogenize the particle size of synthesized calcium deficient hydroxyapatite powders compared to non irradiated samples. The particle size calculated from transmission electron microscope (TEM) for undoped calcium phosphate samples prepared by microwave curing exhibited nanoparticles. While, the doped samples exhibited an increase in particle length due to the effect of ions incorporation such as carbonate, sulfate, niobate, and tantalate in the phosphate site and neodymium ions in calcium site. The calculation of lattice parameter from Rietveld refinement beside IR spectra indicated that Nd 3+ would be incorporated as a lower valence to P

P

compensate the charge balance between neodymium and calcium in both HA and β-TCP structure. While, both tantalum and niobium ions were incorporated as niobate and tantalate group in the phosphate site of HA lattice due to the difference in ionic radii and valances between both tantalum and niobium ions and calcium ions. In-vitro test revealed that the presence of resorbable calcium sulfate and β-TCP phases accelerated the nucleation process of amorphous calcium phosphate (ACP) inside the created lacunae and delayed the crystallization of carbonated CDHA. Also, the presence of niobium and tantalum ions in the HA structure accelerated the deposition and crystallization of apatite layer through the formation of calcium tantalate and calcium niobate. Neodymium ions incorporation improved the bioactivity of the prepared calcium phosphate samples through formation of Nd-OH group which is considered as a site of apatite nucleation.

xii

Abstract

A detailed study on A.C. conductivity and dielectric properties of pure and doped samples at various temperatures and frequencies ranged from 304 to 385 K and from 10 Hz to 100 kHz, respectively to act as additional tool for structure validation. The hydroxyapatite sample exhibited an increase in conductivity and dielectric constant with respect to β-TCP and biphasic samples due to its hydroscopic and hygroscopic ability and mobility of its structural hydroxyl group. The highly resistive oxides formed after calcination of doped samples segregated on the grain boundaries of HA phase resulted in a decrease in conductivity. It was possible to obtain three pure phosphatic phases being βTCP, HA, and BCP. The nano range was achieved which mimic the natural phases. Incorporation of ions enhanced their bioactivity and created surface layer deposition that would satisfy requirement as a bone substitution.

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Chapter I Introduction and Aim of the Work

Chapter I Introduction and Aim of the Work I-1- Introduction:

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As health care is improving, our life expectancy is increasing but as we get older we lose bone density due to osteoporosis. At present the treatment for severe cases of osteoporosis and hip fractures is the total hip replacement. It uses bio-inert materials to replace damaged bone. These materials cause further loss in bone density and eventually the replacement need to be replaced. Also bone resorption in the maxilla or mandible in elderly persons need to be grafted bone to allow avoid mal function in the field of maxillofacial surgeries. Additionally, the reaction of areas of cysts leaving cavities need to be grafted too. Current treatments typically rely on donor tissues obtained either from the patient himself or from another banks. The former raises the issue of supply, whereas the latter poses the risk of rejection and disease transfer. This has prompted orthopedic surgeons and scientists to look for viable alternatives [1]. As an alternative to the above mentioned three types of bone grafts, synthetic substances are gaining much interest for use as bone graft materials [2]. Calcium phosphates based materials are preferred as bone grafts in hard tissue engineering because of their superior biocompatibility and bioactivity. However, this group of bioceramics exhibits poor mechanical performance, which restricts their uses in load bearing applications. The recent trend in bioceramic research is mainly concentrated on bioactive and bioresorbable ceramics, i.e. hydroxyapatite (HA), bioactive glasses, tricalcium phosphates (β-TCP and α-TCP) and biphasic calcium phosphates (BCP) as they exhibit superior biological properties over other materials [3]. Hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ; HA) has been extensively B

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studied and clinically applied for its bioactive properties in medicine. HA forms

1

Chapter I

Introduction and Aim of the Work

the mineral ingredient of bones, tooth and calcified tissues in vertebrate. Manmade HA is served for human implant coatings possessing beneficial biocompatibility and osteoconductivity resulting in bonding to a human hard tissue. It is known as a substrate for effective adhesion of proteins, peptides, lipids, bacteria, and strains [4]. On the other hand, beta tricalcium phosphates (Ca 3 (PO 4 ) 2 : β-TCP) are B

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known for their biodegradable characteristics and they are essential components of natural bone [5]. Highly purified β-TCP could be absorbed and replaced by newly formed bone in clinical use. Highly purified β-TCP appears to be advantageous in comparison to HA for surgeries involving bone tumors consequent to the nature of remodelling and superior osteoconductivity due to the high degradability of TCP which may be favorable in osteogenesis because dissolution of TCP can provide Ca and P rich environment needed for bone formation [6]. This property is a significant advantage of TCP compound to other biomedical materials that are not resorbable. These factors give β-TCP an edge over other biomedical materials when it comes to resorbability and replacement of the implanted TCP in vivo by the new bone tissue. Recently, βTCP had been used in tissue engineering field with bone marrow stromal cells (BMSCs) for repairing the critical-sized segmental mandibular defects in canines [7]. In fact, tricalcium phosphate ceramic has a stoichiometry similar to amorphous bone precursors, whereas hydroxyapatite has a stoichiometry similar to bone mineral [8]. Biphasic calcium phosphate (BCP) ceramics consisting of hydroxyapatite (HA) and tricalcium phosphate (TCP) has been used as a bone graft material during the last few decades. Currently, BCP bioceramics are recommended for use as an alternative or additive to autogeneous bone for orthopedic and dental application due to its superior bioreactivity which can be controlled by

2

Chapter I


manipulating the composition (HA/β-TCP ratio) and/or the crystallinity of the BCP [9]. Recently, biphasic calcium phosphate with composition of 80% HA/ 20% β-TCP accompanied with poly-D,L-lactide-co-glycolide showed high level of osseous regeneration [10]. Also, a bilayered calcium phosphate consisting of 20 µm hydroxyapatite layer and 30 µm biphasic calcium phosphate (60%HA40%β-TCP) coated Ti6Al4V alloy might enhance bone apposition in the early stages because of the superior bioactivity of the BCP layer while the more stable HA layer might sustain bone bonding over long periods [11]. Hydroxyapatite can be synthesized by a variety of methods such as chemical precipitation method [12], sol-gel [13], mechanochemical methods [14], microemulsion route [15,16], electrospinning technique [17], and ultrasound treatment [18]. Among these methods, hydroxyapatite prepared with microwave irradiation has an advantage of very short time, small particle size, narrow particle size distribution and high purity [19]. Also, the method of chemical precipitation from aqueous solutions provides a versatile and economic route [20]. Nanomaterials are considered as a new class of material as it possesses superior properties over its microscale counterpart. It defined as those materials with very small components and/or structural features (such as particles, fibers, and/or grains) with at least one dimension in the range of 1–100 nm [21]. Nanocrystalline HA promotes osteoblast cells adhesion, differentiation, and proliferation, osteointegration and deposition of calcium containing minerals on its surface better than microcrystalline one; thus enhancing the formation of new bone tissue within a short period [22]. Therefore, in order to obtain the three types of calcium phosphate ceramics in nanosacle and in economic route, both microwave and chemical precipitation methods has been chosen for the preparation of β-TCP, HA and

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Chapter I


BCP phases. A number of synthesis methods have been used to produce β-TCP powders. Both mechano-chemical method [5] and wet chemical precipitation methods [3] are the most common methods for β-TCP preparation. The disadvantage of chemical precipitation method for β-TCP preparation is the formation of phosphatic impurities after calcination of β-TCP above 700 o C. The P

P

most common impurities exist with β-TCP after calcination is hydroxyapatite (HA; Ca 10 (PO 4 ) 6 (OH) 2 ) and pyrophosphate (CPP; Ca 2 P 2 O 7 ) depending on the B

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value of Ca/P ratio (>1.5 or