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Investigation of Growth and Decomposition of Ca-Deficient Hydroxyapatite

Koji NISHIO, Masato TAMAI, Mitsuhiro NAKAMURA and Toshiyuki ISSHIKI Nano-Structural Science Laboratory, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Contents

• Introduction of hydroxyapatite (HAp)
• Growth of calcium-deficient hydroxyapatite (Ca-def HAp)
• T. Isshiki et al., In: Proc. ICEM15, 2002, Durban, 1, 1047
• High-temperature behavior of Ca-def HAp
• M. Tamai et al., J. Mater. Sci. in Med. 14 (2003) in print
• M. Tamai et al., In: Proc. ICEM15, 2002, Durban, 1, 1057

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Hydroxyapatite

Hydroxyapatite (Ca10(PO4)6(OH)2: HAp)

• Crystal structure: Similar to a bone

Hexagonal (a = 0.943 nm, c = 0.688 nm) ⇓ widely noticed and expected

• As an alternative material for bone
• Application to a biosensor making

good use of biocompatibility

[00¯ 1] a b [100] b c

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Synthesis of Hydroxyapatite

Dry process

⇒ Stoichiometric HAp (s-HAp) 6CaHPO4 + 4CaCO3 → s-HAp + 2H2O + 4CO2

Wet process

⇒ Non-stoichiometric HAp

• Sol-gel method
• Hydrolysis method
• A. Nakahira et al., J. Am. Ceram. Soc. 82 (1999) 2029-2032

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Hydrolysis Method

α-tricalcium phosphate (α-Ca3(PO4)2: α-TCP) ⇓ Hydrolysis in a mixture of water and alcohol Calcium-deficient HAp

(Ca10−Z(HPO4)Z(PO4)6−Z(OH)2−Z ⋅ nH2O (Z = 0-1): Ca-def HAp)

Advantage of this method:

• Easy to synthesis within several hours

under mild conditions

• Easy to control of crystal morphology

plate-, blade-, whisker-like shape, etc.

⇓ ⇓ ⇓ ⇓ ⇓

before hydrolysis before hydrolysis before hydrolysis before hydrolysis before hydrolysis after after after after after α-TCP particle α-TCP particle α-TCP particle α-TCP particle α-TCP particle Ca-def HAp Ca-def HAp Ca-def HAp Ca-def HAp Ca-def HAp whiskers whiskers whiskers whiskers whiskers

1 µm 1 µm 1 µm 1 µm 1 µm 500 nm 500 nm 500 nm 500 nm 500 nm Nano-Structural Science Laboratory, Kyoto Institute of Technology

Ca-Deficient Hydroxyapatite

Ca-def HAp → s-HAp + β-TCP Important reaction to develop new biomaterials Whisker-like shape is favorable as a source to produce porous biomaterials

⇒ In-situ high-temperature SEM

• bservation of sintering process
• f Ca-def HAp whiskers

770 °C 770 °C 770 °C 770 °C 770 °C 900 °C 900 °C 900 °C 900 °C 900 °C 980 °C 980 °C 980 °C 980 °C 980 °C 1040 °C 1040 °C 1040 °C 1040 °C 1040 °C 1 µm 1 µm 1 µm 1 µm 1 µm Nano-Structural Science Laboratory, Kyoto Institute of Technology

Growth of Ca-def HAp

• Introduction of HAp
• Growth of Ca-def HAp

HRTEM observation of ♦ initial stage of hydrolysis ♦ growth of HAp whiskers

• High-temperature behavior
• f Ca-def HAp

α-TCP Hydrolysis ↓ Ca-def HAp Annealing ↓ s-HAp + β-TCP

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Experimental Procedure

Specimen preparation

Specimen I Specimen II α-TCP powder α-TCP powder for mass production Thin α-TCP for HRTEM

• bservation

Dropping on a TEM grid Thinning Compressing and sintering Hydrolysis Hydrolysis

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Hydrolysis

Hydrolysis in a water solvent suspending 1-octanol Mixture α-TCP : water : 1-octanol 0.01 mol : 60 ml : 100 ml Initial pH 11 (adjusted with NH4OH) Temperature 70 °C Time 0 - 48 hours

Instruments

TEM JEOL, JEM-2010/SP JEOL, JEM-2000EX XRD Rigaku, RINT 2000

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Initial stage of hydrolysis

Specimen I

(Thinned α-TCP) before hydrolysis before hydrolysis before hydrolysis before hydrolysis before hydrolysis α-TCP α-TCP α-TCP α-TCP α-TCP 300 nm 300 nm 300 nm 300 nm 300 nm

hydrolyzed for 2 h hydrolyzed for 2 h hydrolyzed for 2 h hydrolyzed for 2 h hydrolyzed for 2 h amorphous layer amorphous layer amorphous layer amorphous layer amorphous layer

↓ ↓ ↓ ↓ ↓

α-TCP α-TCP α-TCP α-TCP α-TCP 20 nm 20 nm 20 nm 20 nm 20 nm

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Dendritic structure on the amorphous layer

hydrolyzed hydrolyzed hydrolyzed hydrolyzed hydrolyzed for 2 h for 2 h for 2 h for 2 h for 2 h amorphous layer amorphous layer amorphous layer amorphous layer amorphous layer 30 nm 30 nm 30 nm 30 nm 30 nm 4 h 4 h 4 h 4 h 4 h 100 nm 100 nm 100 nm 100 nm 100 nm Nuclei

• not on the α-TCP
• but on the surface of

the amorphous layer ⇒ Dendrites composed of

• twigs with a few nm

in width

• trunks with several

tens nm in width ⇒ Embryo of Ca-def HAp

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Growth of Ca-def HAp whiskers

20 25 30 35 40 Intensity [a.u.] Diffraction angle 2θ [degree] 0 h 1 h 2 h 3 h HAp 20 40 60 80 100 60 120 180 Conversion rate [%] Hydrolyzed time [min]

Specimen II

(α-TCP powder)

before hydrolysis before hydrolysis before hydrolysis before hydrolysis before hydrolysis

hydrolyzed for 1 h hydrolyzed for 1 h hydrolyzed for 1 h hydrolyzed for 1 h hydrolyzed for 1 h 2 h 2 h 2 h 2 h 2 h 3 h 3 h 3 h 3 h 3 h α-TCP α-TCP α-TCP α-TCP α-TCP α-TCP α-TCP α-TCP α-TCP α-TCP 500 nm 500 nm 500 nm 500 nm 500 nm

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Summary (Growth of Ca-def HAp) ⇒ ⇒ ⇒ ⇒ ⇒ ⇒ ⇒ ⇒ ⇒ ⇒ ⇓ ⇓ ⇓ ⇓ ⇓ ⇐ ⇐ ⇐ ⇐ ⇐ ⇐ ⇐ ⇐ ⇐ ⇐

α-TCP

Amorphous layer on the surface of α-TCP Nucleation of HAp on the amorphous layer Dendritic structures

• n the nuclei

Needle-like HAp crystals

Ca-def HAp whiskers

Nano-Structural Science Laboratory, Kyoto Institute of Technology

• H. T. Behavior of Ca-def HAp
• Introduction of HAp
• Growth of Ca-def HAp
• High-temperature behavior
• f Ca-def HAp

HRTEM observation of the Ca-def HAp whiskers annealed at various conditions

α-TCP Hydrolysis ↓ Ca-def HAp Annealing ↓ s-HAp + β-TCP

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Experimental Procedure

Specimen

Source: Whisker-like shape, Ca-def HAp synthesized by hydrolysis method (Ca/P molar ratio = 1.58) Heat Annealed at 200-1100 °C in air treatment: (heating rate: 5 °C/min, keep time: 2-6 hours)

Instruments

TEM JEOL, JEM-2010/SP JEOL, JEM-2010F∗ (EDS) Noran, Vantage JEOL, JED-2300T∗ XRD Rigaku, RINT 2000 FT-IR JEOL, IR-WINSPEC100

∗Acknowledgements: Dr. E. Okunishi, Application & Research Center, JEOL Ltd.

Nano-Structural Science Laboratory, Kyoto Institute of Technology

TEM images of samples before & after annealing

before annealing before annealing before annealing before annealing before annealing annealed at 600 °C annealed at 600 °C annealed at 600 °C annealed at 600 °C annealed at 600 °C 700 °C 700 °C 700 °C 700 °C 700 °C 800 °C 800 °C 800 °C 800 °C 800 °C 900 °C 900 °C 900 °C 900 °C 900 °C 500 nm 500 nm 500 nm 500 nm 500 nm Ca-def HAp whiskers

Length (c axis): 2 ∼ 5 µm Width: ∼ 0.1 µm Ca/P molar ratio: 1.58 Heating rate: 5 °C/min Keep time: 2 hours

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Analysis of XRD patterns

25 30 35 40 45 Intensity [a.u.] Diffraction angle 2θ [degree]

900 °C 900 °C 900 °C 900 °C 900 °C 800 °C 800 °C 800 °C 800 °C 800 °C 600 °C 600 °C 600 °C 600 °C 600 °C before annealing before annealing before annealing before annealing before annealing

1100 °C 1000 °C 900 °C 800 °C 600 °C 400 °C 200 °C before annealing

β-TCP HAp 500 nm 500 nm 500 nm 500 nm 500 nm

→ → → →

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Ca-def HAp whisker annealed at 800 °C

50 nm 50 nm 50 nm 50 nm 50 nm

HAp HAp HAp HAp HAp HAp HAp HAp HAp HAp HAp HAp HAp HAp HAp

← ← ← ← ← → → → → → → → → → →

[010] [010] [010] [010] [010] 100 100 100 100 100 002 002 002 002 002

3 nm 3 nm 3 nm 3 nm 3 nm

(100) (100) (100) (100) (100)

Planar defect / Layered precipitate

• traversing in the whisker
• parallel to (100) plane

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Planar defect and layered phase

600 °C 600 °C 600 °C 600 °C 600 °C 700 °C 700 °C 700 °C 700 °C 700 °C 800 °C 800 °C 800 °C 800 °C 800 °C [021] [021] [021] [021] [021] [010] [010] [010] [010] [010] [010] [010] [010] [010] [010]

20 nm 20 nm 20 nm 20 nm 20 nm

5 nm 5 nm 5 nm 5 nm 5 nm ↑ ↑ ↑ ↑ ↑

planar defect planar defect planar defect planar defect planar defect (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (001) (001) (001) (001) (001) 1.43 nm 1.43 nm 1.43 nm 1.43 nm 1.43 nm

↓ ↓ ↓ ↓ ↓ ↑ ↑ ↑ ↑ ↑

Nano-Structural Science Laboratory, Kyoto Institute of Technology

TEM-EDS microanalysis

Counts [a.u.] Energy [keV] 1 2 3 4 5 C O P Ca Ca

A (layered phase)

atomic molar ratio

area Ca/P

Ca P [atom. %]

A 1.78

64 36

B 1.40

59 42

s-HAp

1.67 62.5 37.5 [010] [010] [010] [010] [010] 2 nm 2 nm 2 nm 2 nm 2 nm

⇐ ⇐ ⇐ ⇐ ⇐ ⇒ ⇒ ⇒ ⇒ ⇒

A A A A A B B B B B

⇒ Ca-rich composition

Counts [a.u.] Energy [keV] 1 2 3 4 5 C O P Ca Ca

B (Ca-def HAp)

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Analysis of IR absorption spectra

Wave number [cm−1] Transmittance [a.u.] 3800 3300 2800 900 °C 800 °C 600 °C 400 °C 3570 cm−1

← 3538 cm−1

Wave number [cm−1] Transmittance [a.u.] 1400 900 400 900 °C 800 °C 600 °C 400 °C

↑

744 cm−1

IR absorption spectrum of the sample annealed at 800 °C ⇓

• IR absorption peak at

3570 cm−1 shifted to 3538 cm−1

• IR absorption peak ap-

peared at 744 cm−1 ⇓

Ca-rich HAp

• G. Bonel et al., Ann. NY
• Acad. Sci. 523 (1988) 115

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Ca-rich metastable phase

The unidentified phase appeared in the annealed Ca-def HAp TEM ⇓ 600 °C: planar defect 700-800 °C: layered phase 900 °C: not observed ⇓ Metastable phase ⇓ ⇓ TEM-EDS, IR EDS: higher Ca/P molar ratio than of s-HAp IR: shift & appearance

• f absorption peaks

⇓ Ca-rich phase ⇓ Ca-rich metastable phase

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Relation between the Ca-def HAp and the phase

[010]HAp [010]HAp [010]HAp [010]HAp [010]HAp [011]HAp [011]HAp [011]HAp [011]HAp [011]HAp [001]HAp [001]HAp [001]HAp [001]HAp [001]HAp

(100) (100) (100) (100) (100) → → → → → ← ← ← ← ← 1.43 nm 1.43 nm 1.43 nm 1.43 nm 1.43 nm

2 nm 2 nm 2 nm 2 nm 2 nm

100 100 100 100 100 002 002 002 002 002 100 100 100 100 100 01¯ 1 01¯ 1 01¯ 1 01¯ 1 01¯ 1 100 100 100 100 100 010 010 010 010 010

Ca-def HAp metastable phase [010] [011] [001]

0.943 nm 0.688 nm 2.86 nm

Metastable phase a = 2.86, b = 0.943, c = 0.688 [nm] (Orthorhombic) (100)HAp || (100)MSP [010]HAp || [010]MSP

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Interface between HAp and β-TCP

850 °C 850 °C 850 °C 850 °C 850 °C 200 nm 200 nm 200 nm 200 nm 200 nm ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ β-TCP β-TCP β-TCP β-TCP β-TCP HAp HAp HAp HAp HAp β-TCP β-TCP β-TCP β-TCP β-TCP 2 nm 2 nm 2 nm 2 nm 2 nm HAp [010] HAp [010] HAp [010] HAp [010] HAp [010] β-TCP [81¯ 2] β-TCP [81¯ 2] β-TCP [81¯ 2] β-TCP [81¯ 2] β-TCP [81¯ 2]

Nano-Structural Science Laboratory, Kyoto Institute of Technology

Summary (H. T. behavior of Ca-def HAp)

• Thermal decomposition of the Ca-def HAp whiskers pre-

pared by the hydrolysis method begins at about 800 °C and finishes at about 900 °C.

• Planar defects, often observed in the specimen annealed at

600-800 °C, are considered as a metastable phase.

• The results of EDS microanalysis and IR analysis suggest

the metastable phase is a Ca-rich phase.

• Lattice constants of the phase are analyzed into a = 2.86,

b = 0.943, c = 0.688 nm with orthorhombic crystal system.

Nano-Structural Science Laboratory, Kyoto Institute of Technology