Scholarly article on topic 'In vitro biological activity comparison of some hydroxyapatite-based composite materials using simulated body fluid'

In vitro biological activity comparison of some hydroxyapatite-based composite materials using simulated body fluid Academic research paper on "Materials engineering"

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Academic research paper on topic "In vitro biological activity comparison of some hydroxyapatite-based composite materials using simulated body fluid"

Cent. Eur J. Chem. • 11(10) • 2013 DOI: 10.2478/s11532-013-0293-5



Central European Journal of Chemistry

In vitro biological activity comparison of some hydroxyapatite-based composite materials using simulated body fluid

Research Article

Melinda Cziko1, Erzsebet-Sara Bogya2", Reka Barabas3, Liliana Bizo2, Razvan Stefan4

department of Chemistry,

Faculty of Chemistry and Chemical Engineering,

Babes-Bolyai University, RO-400028 Cluj Napoca, Romania

2Department of Chemical Engineering, Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, RO-400028 Cluj Napoca, Romania

Department of Chemistry and Chemical Engineering of the Hungarian Line of Study, Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, RO-400028 Cluj Napoca, Romania

Received 28 March 2013; Accepted 15 May 2013

Abstract: Hydroxyapatite composites are the main biomaterials used for metal implant coatings. Their in vitro study is very important. That is why their behavior was monitored in simulated body fluid (SBF), which is a solution with ion concentrations and pH value similar to those of human blood plasma.

Silica, chitosan and gelatin-doped hydroxyapatite-based biomaterials were studied in SBF; the samples were characterized pre-, during and post-SBF immersion using infra-red, scanning and transmission electron spectroscopy and X-ray diffraction methods. The solubility of materials in SBF was determined, and the variation of Ca2+ and phosphorus concentration was also recorded during SBF experiments. The results were compared and their in vitro biological activity was determined.

Keywords: Hydroxyapatite • Bio-polymer • Silica • Composite • Simulated body fluid © Versita Sp. z o.o.

1. Introduction

In recent years, the emphasis on the progress of biomaterials has shifted from monoliths to composites. By controlling the volume fraction and distribution of the second phase in a composite, the properties of such materials can be modified to be able to meet mechanical and physiological requirements of an implant. In this respect, hydroxyapatite (HAP) based composites have widely been used in biomedical and dental applications due to HAP similarity to main mineral components of hard tissues of human body such as bone, dental enamel,

dentin and due to its biocompatibility, bioactivity and low solubility in moist media [1-5].

Hydroxyapatite (Ca10(PO4)6(OH)2) is a familiar bioactive material and has excellent properties such as bioactivity, biocompatibility and ability to induce bone tissue growth. After implantation into the body, HAP can form strong chemical bonds with natural bone and it promotes new bone growth because of its similar chemical and mineralogical composition and crystallographic structure to apatite of human living bone [6,7].

Composites of apatite crystals and natural polymers have received a great deal of attention with the view

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Table 1 SBF and human blood plasma ion concentration (10-3 mol L-1).

Na+ K+ Mg2+ Ca2+ Cl" HCO3- HPO„2- SO42-

SBF solution 142.1 5.0 1.5 2.5 124.91 27.0 0.0 0.5

Blood plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5

that the composite system can provide compositional benefits and preserve structural and biological functions of the damaged hard tissues in a manner similar, or better, to the natural system. One of the requirements of an HAP coating is its good adhesion to organic polymer substrates. In order to enhance the interaction between the inorganic apatite and the organic polymers, some researchers have introduced hydrophilic polar groups such as phosphate, carboxyl and hydroxyl groups onto hydrophobic substrates. It has been reported that organic polymers containing carboxyl groups form apatite on their surfaces in simulated body fluid (SBF) if their carboxyl groups have been previously fully combined with calcium ions [8-10].

Chitosan (CS) is a natural cationic polysaccharide that can be produced by alkaline N-deacetylation of chitin. Important properties of this material, such as biocompatibility, chemical resistance, mechanical strength, antimicrobial properties and thermal stability, have been utilized in biotechnology [11,12]. Incorporation of HAP with CS, the mineral component of a bone, could improve the bioactivity and the bone bonding ability of the CS/HAP composites [9,13].

Silicon substituted hydroxyapatites (HAP-Si) are among the most interesting bioceramics in the field of bioactive bone implants. Silicon incorporation into the apatite structure results in materials with comparable biocompatibility and mechanical properties, but with improved bioactive behavior [4,14,15].

Gelatin (GEL) is a natural biopolymer modified from the partial hydrolysis of native collagens, which are the most abundant structural proteins found in the skin, tendons, cartilage and bones in a living body [16]. Gelatin/hydroxyapatite (GEL/HAP) composites have been developed into a good candidate material for hard tissue repairs because of their similar composition to the hard tissues, good biocompatibility and high osteoconductive activity. Very recently, they have been also used as a drug delivery system for the treatment of bone infections and defects [17].

It is well known that the human body environment is very destructive; therefore, each biomaterial should be tested to estimate the influence of the human body environment on its properties. In the presence of the physical and chemical conditions common in the human body, the biomaterials can lose their stability, and may

corrode or degrade. Depending on the application, for example, as a permanent prosthesis, drug delivery systems or osteoinductive material, the degradation through the body fluids can be either unwanted or it can be one of the most useful properties.

Using an environment with an ion concentration nearly equal to that of human blood plasma and a pH of 7.4 [18-20] close to human body we can simulate the composite behavior in vitro.

Since the first disclosure of the composition of simulated body fluid (SBF), several new SBF compositions have been proposed [21,22].

Table 1 contains the ion concentrations for human body plasma and the ion concentration in SBF [6].

However, none of these solutions correspond to the composition of a human blood serum. The three main differences between SBF solutions and serum are (i) the absence of proteins, whereas they are known to play an essential role in controlling apatite nucleation (nucleation inhibitors); (ii) the addition of tris(hydroxymethyl) aminomethane (TRIS) to buffer SBF solutions, and (iii) the absence of control of the carbonate content of SBF solutions, although carbonates act as pH buffer in serum (blood serum is in equilibrium with a partial pressure of carbon dioxide (CO2) close to 0.05 atm, which is a key factor in pH buffering of blood) [23-25]. Regarding these deficiencies, simulated body fluid is an important testing method to predict a potential bone-bonding behavior through the apatite layer formation on its surface. It is generally accepted that the formation of an apatite layer in body fluids facilitates attachment of osteoblasts (via proteins) on bioactive glasses (both in vitro and in vivo) and thereby enables bonding to bone [26].

The main role of SBF testing is to simulate and study the inorganic subsystem of the interaction between the biomaterial and human body plasma in a cheaper than cell culture test and in a more humane way than testing on animals.

The main goal of the present report was the in vitro study of hydroxyapatite and bio-composites (silica, gelatin and chitosan) in SBF, comparison of their behavior for various applications: to simulate an implant coating or dense ceramic implant (in form of compacted pellets) and as substrates for medicinal organic substances (in powder form).

2. Experimental procedure

2.1. Materials preparation

2.1.1. HAP synthesis

The HAP was prepared by precipitation method, described in previous studies [27], under controlled conditions. The following materials were used: calcium nitrate tetrahydrate, diammonium hydrogen phosphate, 25% ammonia solution (Merck, Germany). The reaction time was 22 h. After filtration, the resulting materials were dried for 24 hours at 105°C (ncHAP). One part of materials was heat treated at 1000°C (cHAP).

2.1.2. HAP-Si 10 synthesis

The HAP-Si 10 was prepared with precipitation method described in previous studies [28,29] under controlled conditions. The following materials were used: calcium nitrate tetrahydrate, diammonium hydrogen phosphate, 25% ammonia solution (Merck, Germany), sodium silicate (Lach:ner, Czech Republic). The reaction time was 8 h. After filtration, the resulting materials were dried for 24 hours at 105°C. The heat treatment was performed at 1000°C (cHAP-Si 10).

2.1.3. Synthesis of CS/HAP composites

The 0.1% and 0.5 wt% chitosan stock solutions were prepared by dissolving appropriate quantities of chitosan (medium viscosity) in acetic acid and distilled water and stirred about 6 h till the chitosan was dissolved.

1.5 mol L-1 calcium nitrate and 0.9 mol L-1 diammonium hydrogen phosphate solutions were added to the 0.1 or 0.5 wt% chitosan solutions, in order to obtain materials with 1.6 and 8 wt% final chitosan concentrations, respectively. The synthesis time was 22 hours. After the reaction was accomplished, the precipitate was washed with ethanol and filtered. The filtered material was dried for 24 hours at 90°C [30].

2.1.4. Synthesis of GEL/HAP composites

The preparation of GEL/HAP composites is similar to that described for CS/HAP composites: 0.1% and 0.5 wt% gelatin stock solutions were mixed with calcium nitrate (1.5 mol L-1) and diammonium hydrogen phosphate (0.9 mol L-1) solutions and stirred for 22 hours. Materials with 2 and 8 wt% final gelatin concentration were prepared. After that, the precipitate was washed with ethanol and filtered. The filtered material was dried for 24 hours at 90°C.

These materials in powder and in pellets form were used for in vitro behavior study in SBF. The pellets were prepared by pressing at 10 tons for 10 minutes.

2.2. Materials characterization

2.2.1. X-ray diffraction

X-ray diffraction measurement were carried out by a Shimadzu XRD 6000 X-ray diffractometer with CuKa radiation (A = 1.542 A) operated at 40 kV and 30 mA and data were recorded in 20°-60° 29 range.

2.2.2. Fourier Transform Infrared Spectroscopy

A Jasco FTIR-615 was used for the determination of functional groups by scanning the HAP composites in the range of 500-4000 cm -1.

2.2.3. Scanning electron microscopy

The morphology of the ceramic powders and pellets pre- and post-SBF incubation were studied by a FEI Quanta 3D FEG scanning electron microscope (SEM). The powders were pre-coated with a thin layer of gold and investigated at high vacuum mode using EDT (Everhart Thornley 171 Detector) and the pellets were examined in extended low vacuum mode (ESEM) with water vapor.

2.2.4. Transmission electron microscopy

The microstucture of the samples before and after SBF soaking was studied with a Hitachi H-7650 transmission electron microscope apparatus.

2.3. SBF preparation

The used simulated body fluid exhibits ion concentrations nearly equal to those of human blood plasma. The SBF was prepared by dissolving reagent grade NaCl (Reactivul, Romania), NaHCO3 (Merck, Germany), KCl (Reactivul, Romania), K2HPO4^3H2O (Lach:ner, Czech Republic), MgCl2^6H2O (Lach:ner, Czech Republic), CaCl2 (Nordic, Romania) and Na2SO4 (Reactivul, Romania) in distilled water with final ion concentrations presented in Table 2 [18]. The solution was buffered to obtain pH 7.25 - 7.40 with hydrochloric acid (Reactivul, Romania) and TRIS [(CH2OH)3CNH2] (Merck, Germany) at 37°C. The pH was monitored with Electrode SenTix 41-3 pH electrode.

2.4. Materials soaking in simulated body fluid

Two parallel measurement sets were established: I. on compacted pellets

40 mg of each material was pressed into pellets and kept in SBF (20 mL) for 28 days. The following parameters were monitored: Ca2+/phosphorus concentration and weight variation. The temperature was maintained at 37°C.

Table 2. Prepared materials and their symbolization.

Materials Symbol

Calcined HAP cHAP

Non Calcined HAP ncHAP

Calcined HAP containing 10 wt% of SiO2 cHAP-Si 10

Non-calcined HAP containing 2 wt% of gelatin 2% GEL/HAP

Non-calcined HAP containing 8 wt% of gelatin 8% GEL/HAP

Non-calcined HAP containing 1.6 wt% of chitosan 1.6% CS/HAP

Non-calcined HAP containing 8 wt% of chitosan 8% CS/HAP

The samples were characterized. II. on powder

Hydroxyapatite-based material powders (40 mg) were introduced in 20 mL SBF for 28 days. Ca2+/phosphorus concentration and weight variation was studied. After this period the samples were characterized by TEM, SEM, IR and XRD.

The Ca2+ concentration was determined with 0.02 M ethylenediaminetetraacetic acid (Merck, Germany) titration using ERIO T (Reactivul, Romania) as indicator.

The phosphor concentration variation was measured by UV-VIS spectrophotometer (Jasco- 550) using: 2% (NH4)2MoO4 (Reactivul, Romania), 0.1 M L(+) ascorbic acid (Riedel-de Haen, China) and 1 N H2SO4 (Reactivul, Romania).

The weight variation was measured with a four digital Kern ALJ 220-NM balance.

3. Results and discussion

3.1. Materials characterization

The purpose of this report was to simulate the in vivo behavior of HAP and their chitosan and gelatin composites by studying in vitro in SBF in different forms: powder and pellets. The differentiation was applied in order to monitor their behavior with respect to their application: in form of pellets to simulate an implant coating or dense ceramic implant and in powder form in case of substrate for medicinal organic substances (e.g. ibuprofen, ampicillin, etc.). Their morphological, structural characteristics pre and post-SBF soaking were compared, and their in vivo biological activity -new apatite layer formation - was foreseen from their SBF behavior pattern.

3.1.1. X-ray diffraction

Pre-SBF soaking the characteristic HAP peaks are visible on the spectrums at: 25.87°; 32.19°; 31.77°; 39.87°; 46.7° and 49.46° (PDF 09-0169) [31]. It can be seen

that hydroxyapatite and their composites have similar XRD patterns. The diffraction peaks can be assigned to mono-phase crystalline hydroxyapatite, since no peaks from other calcium phosphate phases are detected. It indicates that the involvement of chitosan and gelatin does not change the crystallographic structure of HAP in the composite. Slight broadening of the CS/HAP nano-biocomposite peaks compared to pure hydroxyapatite can signify decreased size and crystallinity of the hydroxyapatite in the presence of biopolymers matrix [32,33].

Post-SBF immersion in the case of pure HAP resulted in no additional peak, which means that the HAP structure did not change during the treatment period. Although the peaks are sharper and more pronounced for samples soaked in SBF, which suggests a better crystallized hydroxyapatite structure, probably due to the dissolution/recrystallization processes [34].

On the other hand, in the case of GEL and CS composite materials after 28 days SBF soaking, two new peaks can be identified at: 45.2° and 45.8° (Fig.1 D). The peak at 45.8° is due to Na ions substituted HAP (Na-HAP) and at 45.2° is due to K-HAP resulting from K ions incorporation in the crystal structure from the SBF solution. The XRD study also reveals that Na and K ions from the SBF solution are incorporated in the newly formed apatite layer as an impurity in HAP matrix [32].

In Fig. 1 A. the XRD spectra of 1.6% CS/HAP before and after 7 and 28 days of immersion are represented. It can be observed, that the introduction of K and Na ions appears only on the spectra after 28 days postimmersion. The same measurements were done for all the composite samples and the similar results were obtained: K+ and Na+ substituted hydroxyapatite were formed after 28 days of soaking.

Feki et al. determined that the sodium is localized mainly in Ca(2) cationic site. Furthermore, carbonate ions occupy phosphate sites leading to a B-type carbonate apatite. These simultaneous substitutions affect the OH- position in the channel, as well as the metal-oxygen interatomic distances [35].

In the case of cHAP-Si 10 three new peaks appeared post-SBF immersion at 21.63°, 35.8° and 46.29° (see Fig. 1E), which corresponds to the crystal planes (101), (200) and (113) of the crystalline cristobalite, respectively [36]. These peaks do not appear pre-SBF treatment because the silica is either incorporated in the hydroxyapatite structure or is in an amorphous form.

3.1.2. Fourier Transform Infrared Spectroscopy

Figs. 2A-2C present the FT-IR spectra for HAP based composites before and after SBF study.

A. 1.6% CS/HAP

Before SBF After SBF

-Before SBF

-After SBF

30 40 50

2 thêta (degree)

В. ncHAP

2 thêta(degree)


2 thêta (degree)


2 til eta (degree)

E. cHAP-Si 10

Figure 1. XRD pattern of the HAP / hydroxyapatite-composite powders pre-, during and post-SBF immersion. XRD spectrum of HAP-based materials after (blue line) and before soaking in SBF solution (black line).

The most pronounced peaks for hydroxyapatite materials are due to the PO43" group vibrations: 963, 1036, 1095 cm-1 - ul, u2, u3 and at 608, 572 cm-1 stretching (u4) and the bending modes (u5). The peaks at 3500-3200 and 631 cm -1 are due to vibrational bands of the OH- group.

For silica substituted materials the band at 798 cm-1 can be related to the Si-O-Ca vibration band [37].

The main characteristic bands for chitosan are at 1632 cm-1 (amide I) and 1557 cm-1 (NH2 bending) and

1382 cm-1 (amide III), at 1199 cm-1 (anti-symmetric stretching of the C-O-C bridge), 1082 and 1042 cm-1 (skeletal vibrations involving C-O stretching) and at 2890-2980 cm-1 due to the aliphatic C-H stretching. The broad band centered at 3210 cm-1 is assigned to the NH stretching, which overlaps the OH stretching band occurring in the same region [38,39].

The IR absorption spectrum for GEL/HAP exhibits several characteristic peaks, and they are amide absorption at 1631 cm-1, -CH2- bending at 1454 cm-1,

Ь 1.0.

105 с

3500 3000


-After 1 day

-After 2 days

-After 3 days

-After 7 days

—- After 28 days

3500 3000

- Before SBF

-After 1 day

-After 2 days

-After 3 days

-After 7 days

-After 28 days

Wavenumber (cm-1)


Wavenumber (cm-1)

B - 8% CS/HAP

с га С

(Я 0.6-


- nc HAP 2 % GEL/HAP cHAP-Si 10

Wavenumber (cm-1)

C - ncHAP, 2% GEL/HAP and cHAP-Si 10IR spectra after 28 days SBF immersion

Figure 2 FT-IR spectrum of HAP-based materials pre and post-SBF immersion.

and PO4 band at 1286.8 cm-1. The shift of N-H bending indicates the presence of hydrogen bonding with GEL/ HAP [40].

The IR spectra show that the intensity of HAP bands such as OH (3500 and 3180 cm-1) and phosphate (560 and 1030 cm-1) and carbonate groups (u3 bands around 1449 cm-1) in HAP spectrum increased postimmersion at 7 and 28 days of SBF-soaking compared to pre-immersion supporting the formation of an apatite biolayer [41 ]. In the case of ncHAP and cHAP materials, the carbonate group vibration does not appear pre-SBF treatment, meaning that they are carbonate free before SBF immersion. In addition, the adsorbed TRIS molecule can be detected by the amide I, II and III type

vibration in the range of 1280-1740 cm-1, which the peaks are interfering with the peaks characteristic to chitosan and gelatin.

The shoulder signals around 1493 cm-1 and 1296 cm-1 and the doublet peaks around 863 cm-1 are characteristic of carbonated ions substituted into the phosphate site in an apatite structure, the so called B-type apatite [39].

3.1.3. Scanning electron microscopy

The SEM images for powder and pellets are presented in Figs. 3-5.

It can be seen from Fig. 3 that the surface morphology of the powders immersed in SBF is changed. Pre-SBF

immersion agglomerated particles can be seen and ncHAP shows almost spherical granulation. The SEM micrograph of the calcined hydroxyapatite powder shows that the single particles stick together because of the sintering process at 1000°C. After SBF soaking for 28 days, the surface of the powder became smoother, the melted particles of the heat-treated HAP and HAP-Si 10 were no longer visible and a uniformly distributed new layer appeared. This is consistent with the findings for the other materials also (Fig. 4). This can be due to the formation of a new apatite layer, a hypothesis supported also by the following results presented in the weight variation section.

In the case of the pressed pellets, no obvious changes on the surface were evident after SBF immersion. The surface of HAP-based materials was found to be plain and dense (Fig. 5). Normally, the formation of a dense apatite layer is observed on porous bioactive materials from SBF solution, which in our case is not evidenced.

In the case of powder materials, the surface morphology changes and the formation of a new layer can be observed with scanning electron microscopy (Figs. 4, 5). The XRD measurements support that in the case of composite materials, the new layer is hydroxyapatite containing K+ and Na+ ions.

3.1.4. Transmission electron microscopy

Materials in powder form were studied with transmission electron microscopy. By comparing the pre and postimmersion images the particle size and morphology, a variation can be observed.

The TEM images of powder samples are presented in Figs. 6 and 7. They show that the nano-crystals of cHAP-Si 10 sample are sphere like before SBF treatment [42], and in the case of calcined and ncHAP, they are rod-like particles both for pre- and post-SBF immersion. The sizes of the particles are uniform which provides a regular surface texture. The average grain sizes of the presented samples in Fig. 6 are distributed in the range of 40-60 nm length and 10-25 nm width for the rod-like particles. After 28-day SBF treatment, the single particles are better defined, an outcome that can be caused by the dissolution/recrystallization of the hydroxyapatite. Fig. 7 presents the chitosan and gelatin hydroxyapatite composites transmission electron microscopy micrographs after 28 days of SBF soaking. The particles are rod-like in the range of 20-30 nm length and 5-20 nm width, smaller than the particles of pure and silica-doped hydroxyapatite.

As TEM images show, hydroxyapatite and their composites have nano-sized particles after 28 days of SBF immersion.

3.2. Weight and Ca/P ion concentration variation during simulated body fluid study soaking experiments for pellets and powder material

When a material is incubated in SBF solution, the formation of apatite layer on the surface of pellet/ powder goes through a sequence of chemical reactions such as spontaneous precipitation, nucleation and growth of calcium phosphate. It has been suggested that surface chemistry plays an important role in this process and even the functional groups of materials have a large effect on the bone-bonding property [32].

3.2.1. Weight variation

In order to demonstrate the formation of a new apatite layer the weight variation of the materials both in powder and pellet form was monitored.

The mass variation is different for the powders and pellets, although, they behave according to the same pattern. For all the materials both in powder and pellet form in the first 3-5 days, a mass loss (2-3 mass%) can be observed with further increase/decrease in the soaking time, suggesting a continuous precipitation of the bone-like apatite. However, the mass of the HAP powders decreases with an increase in soaking time in the early stage, indicating the dissolution of the HAP powders [43]. It can also be seen from Fig. 8 that the weight variation is oscillating, with slight differences, but with the same pattern for all the materials both in powder and pellet forms. This phenomenon can be explained by the dissolution and re-precipitation of the newly formed hydroxyapatite layer.

The formation of the new apatite layer was described by Lu and Leng with the following equations [44]:

Ca2++HPO2" ^ CaHPO4 (1)

4Ca2++HPO2" + 2PO4" ^ Ca4(HPO2")(PO4")2 (2)

5Ca2+ + 3PO4- + OH" ^ Ca5 (PO4 )3 (OH) (3)

The dissolution reaction of apatite can be described by Eqs. 4 and 5 as follows [45]:

Ca5 (PO4 )3 (OH) ^ 5Ca2+ + 3PO34" + OH" (4)

СаЛРО,) (OH) + 7H+<->

о (5)

^ 5Ca + 3H2PO4 + H2O

Pre-SBF immersion Post-SBF immersion

Figure 3. SEM micrographs of HAP-based materials in powder form before and after SBF soaking.

In the 5-7 pH range, the phosphate site protonates to form EP-OH and within the pH range 8-10, the calcium site deprotonates to form ECa-OH.

The highest mass variation was recorded for cHAP-Si 10, which showed a high mass loss in the first 3 days, with the final weight variation above 4%. The chitosan and gelatin-hydroxyapatite composite are more stable, the weight loss is more reduced and the final mass variation is above 3.5%. This phenomenon supports

that the introduction of chitosan and gelatin increases the in vitro stability of the hydroxyapatite composites. The average mass increase after 28 days occurs for all materials between 3-5%.

3.2.2. Variation of ions Ca'+and phosphor concentration

Fig. 9 shows the changes of the Ca and phosphorous ion concentration variation with time during SBF soaking separately for powders and pellets. As it can be seen,

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Figure 4. SEM images of HAP composite materials in powder form post-SBF immersion.

Pre-SBF immersion


Post-SBF immersion

s té v * >

v-. 'V - ;r.i «ft* '«w®- ; - :. ■ •■

"¿-'v ï-'îlS




1.6% CS/HAP

Figure 5 SEM micrographs of HAP-based composites pellets before and after SBF soaking.

Figure 6. TEM images of the HAP-based materials in powder form before and after SBF soaking.



Figure 7 TEM images of the gelatin and chitosan-HAP composites post-SBF immersion.

a- powder materials

b pellets

Figure 8. Weight variation of powder and pellets HAP materials in function of time during SBF soaking.

their behavior is influenced by heat treatment, structural modification by silica, bio-polymer addition and sample preparation (powder or compacted pellets).

The chitosan and gelatin composite powder materials Ca and P ion concentration variation kinetics follows a similar pattern, which consists of an oscillation of the concentrations, an abrupt decrease, followed by a rapid or a more prolonged increase, and finally mostly a decrease of the Ca ions concentration and an increase in the phosphorus concentration. For calcined-, ncHAP and gelatin composites in powder form the Ca ion concentration in SBF reaches a maximum value after

3 days of immersion and then decreases with further increase in the soaking times until it becomes almost constant (Fig. 9A). This can be explained by the fact that the dissolution of the amorphous phases of the HAP powders is dominant during the early stage of soaking, as described in Eqs. 4-5, and then the precipitates begin to form and increase gradually as the soaking time increases. This kind of variation is more pronounced for the non-calcined powder materials, which have a more amorphous structure. For calcined materials, the dissolution is less likely due to their high crystallinity, and the fact that the precipitates can form almost

a. powder

b. pellets

10 15 20


10 15 20

t (days)


0.030 0.025

10 15 20

t (days)

10 15 20

t (days)

0.1000.095 0.0900.085

P 0.080

" 0.075 0.070 0.065

0.020 _

0.01 ] <Q 0.01 ) "S ra

1.6% CS/HAP

0.020]+ S IF (Z

0.0153 0


0.025 0.020"*+ s JT^

0.01 / 0

10 15 20

t (days)

0.100 0.095

10 15 20

t (days)

10 15 20

t (days)

10 15 20

t (days)


Figure 9 Calcium and phosphorous ion concentration variation of powder and pellets HAP materials in function of time during SBF soaking.

a. powder

b. pellets

3 0.11-

r .1 w

0.10- k

0.050 0.045 0.040

0.035 [

0.03 ] 0.02 l 0.020 0.015



0 5 10 15 20 25

t (days)

0 5 10 15 20 25

t (days)

là 0.08 (o

0 5 10 15 20 25 30

t (days)

0.035 0.030 0.025 00 ]+ 0.0) 0.010 0.005

0 5 10 15 20 25

t (days)




Î & 0J

10 15 20 25

t (days)

10 15 20 25

t (days)

cHAP-Si 10

ContinUedFigure 9 Calcium and phosphorous ion concentration variation of powder and pellets HAP materials in function of time during SBF soaking.

immediately after immersion. Kim et al. claimed that the process of bone-like apatite formation on sintered hydroxyapatite in SBF was the formation of both a Ca-rich amorphous calcium phosphate (ACP) and a Ca-poor ACP, which eventually crystallized into bone-like apatite [46]. According to Kim's theory, the heat treated powders exhibit negative surface charge due to the surface hydroxyl and phosphate groups; therefore, they will interact with the positive calcium ions to form the Ca-rich ACP when they are soaked in SBF, and thus gain positive surface charge. Then, the Ca-rich ACP formed

on the sintered powders interacts with the negative phosphate ions in the SBF to form Ca-poor ACP, which eventually crystallizes into bone-like apatite [43].

However, the non-calcined HAP and their chitosan and gelatin composites powders used in this study have a different mechanism of bone-like apatite formation during SBF soaking. This is because the HAP powders have a much lower crystallinity and a relatively weak negative surface potential. When they are immersed in SBF, the dissolution of the amorphous phases of the HAP powders dominates rather than the formation of

Figure 10. Calcium and phosphorous final ion concentration consumption in the case of powder and compacted HAP materials after 28 days SBF soaking.

the bone-like apatite, which is in agreement with results presented by Sun et al. [43].

In the case of compacted pellet materials, on one hand, Ca ion concentration in SBF decreases in the first stage, exception being the case of cHAP-Si 10. The Ca ions and P ions are consumed, which means that the nucleation and growth of apatite outclasses the dissolution process. After 5-7 days of immersion, the apatite dissolution rate increases as the Ca ions concentration in SBF increases and finally approaches the equilibrium stage (Fig. 9B). In addition, during late stage, the P ion concentration shows a decreasing tendency due to precipitation of P ions on the surface. The general characteristic for both, powders and pellets is an oscillation in Ca and P ion concentration, which is more pronounced for ncHAP and their gelatin and chitosan composites. This phenomenon is well correlated with the weight oscillation presented in Fig. 8.

Fig. 10 summarizes the final amount of calcium and phosphorus ions precipitated from the SBF for powder and compacted materials. In the case of hydroxyapatite composite materials, a much higher P ion consumption can be observed, compared to cHAP-Si 10, non-calcined and cHAP. This can be explained by the formation of a larger quantity of Na and K ion substituted hydroxyapatite, which is supported by the XRD results. This suggests that gelatin and chitosan addition increases the ion exchange properties of the hydroxyapatite. P ion consumption is higher for the chitosan composites in the case of the pellets, and for the gelatin ones in the case of powder materials. For the heat-treated hydroxyapatite also the P ion consumption

is higher, and the amount Ca2+ introduction in the newly formed bone-like apatite is small, which suggests a Ca-poor ACP formation tendency. On the other hand, for ncHAP and cHAP-Si 10 the Ca2+ consumption is high and the P ion is low, suggesting the precipitation of Ca-rich ACP in the first stage. After 28 days of SBF immersion, all the materials formed a well-crystallized bone-like apatite structure, as supported by the XRD results.

Finally, comparing the powder materials to the compacted ones, it can be said that the powder materials are more soluble; in the first stage the dissolution of the material is the predominant controlling step, which can be due to the higher specific surface that increases the chemical activity of the materials. All the materials promote the formation of bone-like apatite on their surface; however the mechanism differs in function of the material phase composition and their form: powder or green compacts.

4. Conclusions

Pure and silica doped hydroxyapatite and their gelatin and chitosan composites biological activity were tested in vitro by their behavior study in simulated body fluid. The influence of heat treatment of pure hydroxyapatite, the effect of silica doping and bio-polymers addition, the immersion time and the form of the material (powder and compacted) was monitored and discussed. The materials were characterized pre-, during and post-SBF soaking by different methods.

The results show that after 28 days of SBF soaking:

• the materials have a better crystallized hydroxyapatite structure confirmed by XRD results, and in the case of gelatin, chitosan composites and silica doped hydroxyapatite a new phase appeared: K+ and Na+ substituted HAP and respectively cristobalite for cHAP-Si;

• a new apatite bio-layer formation can be observed on SEM images and it is supported by FT-IR spectra's also;

• TEM micrographs show that hydroxyapatite and their composites are nano-sized;

• for all the materials both in powder and pellet form in the first 3-5 days a mass loss (2-3 mass%) was observed with further increase/decrease in the soaking time, suggesting a continuous precipitation of the bonelike apatite;

• the weight loss is more reduced for the chitosan and gelatin-hydroxyapatite composites, so the introduction of chitosan and gelatin increases the in vitro stability of the hydroxyapatite composites;

• the final Ca and P ion consumption reveals that gelatine and chitosan composites form Na and K ion substituted hydroxyapatite, cHAP has a Ca-poor ACP formation tendency and in the case of ncHAP and cHAP-Si 10 Ca-rich ACP formation is most likely.

Finally, comparing the powder materials to the compacted ones, it can be said that the powder materials are more soluble; in the first stage the dissolution of the material is the predominant controlling step. All the materials promote the formation of bone-like apatite on their surface; however the mechanism differs with function of the material phase composition and their form: powder or green compacts.

The SBF soaking results revealed that all the studied materials are biologically active. Although in order to achieve the highest efficiency for a specific application, it is essential that the strong relation between synthesis parameters (silica doping, bio-polymer addition, sintering) and materials characteristics be taken into consideration.


The authors wishes to thank for the financial support provided project POSDRU 89/1.5/S/60189, and for the SEM measurements for the pellets supported from the grant agreement no. TAMOP 4.2.1./B-09/KMR-2010-0003.


[1] L.D. Silvio, M. Dalby, W. Bonfield, J. Mater. Sci: Mater. Med. 9, 12 (1998)

[2] A.M.Ambrosio, J.S. Sahota, Y. Khan, C.T. Laurencin, J. Biomed. Mater. Res. 58, 3 (2001)

[3] A. Afshar, M. Ghorbani, N. Ehsani, M. R. Saeri, C.C. Sorrell, Intern. J. Eng. Trans. B: Appl. 16, 4 (2003

[4] M. Vallet-Regi, D. Arcos, J. Mater. Chem. 15, 15 (2005)

[5] P. Ducheyne, Q. Qui, Biomaterials 20, 23 (1999)

[6] X. Fan, J. Chen, J-P. Zou, Q. Wan, Z-C. Zhou, J-M. Ruan, Trans. Nonferrous Met. Soc. China 19, 2 (2009)

[7] X. Zhao, L. Yang, Y. Zuo, J. Xiong, Chin. J. Chem. Eng. 17, 4 (2009)

[8] X. Pang, I. Zhitomirsky:, Mater. Charact. 58, 4 (2007)

[9] Q. Hu, B. Li, M. Wang, J. Shen, Biomaterials 25, 5 (2004)

[10] A.A. Haroun, V. Migonney:, Int. J. Biol. Macromol. 46,3 (2010)

[11] W.W. Thein-Han, R.D.K. Misra, Acta Biomater. 5, 4 (2009)

[12] K. Zhang, M. Zhao, L. Cai, Z-k. Wang, Y-f. Sun,

Q-l. Hu, Chin. J. Polym. Sci. 28, 4 (2010)

[13] O.C. Wilson Jr., J.R. Hull, Mater. Sci. Eng., C 28, 3 (2008)

[14] A.E. Porter, N. Patel, J.N. Skepper, S.M. Best, W. Bonfield, Biomaterials 24, 25 (2003)

[15] N. Patel et al., J. Mater. Sci. Mater. Med. 13, 12 (2002)

[16] J.H. Ko et al., Macromol. Res. 18, 2 (2010)

[17] S. Teng, L. Chen, Y. Guo, J. Shi, J. Inorg. Biochem. 101, 4 (2007)

[18] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, J. Biomed. Mater. Res. 24, 6 (1990)

[19] H. Maeda, T. Kasuga, M. Nogami, J. Eur. Ceram. Soc. 24, 7 (2004)

[20] S.B. Cho, F. Miyaji, T. Kokubo, K. Nakanishi, N. Soga, T. Nakamura, J. Mater. Sci. Mater. Med. 9, 5 (1998)

[21] T. Kokubo, H. Takadama, Biomaterials 27, 15 (2006)

[22] T. Kokubo, S. Ito, M. Shigematsu, S. Sanka, T. Yamamuro, J. Mater. Sci. 22, 11 (1987)

[23] J.A. Juhasz, S.M. Best, A.D. Auffret, W. Bonfield, J. Mater. Sci. Mater. Med. 19, 4 (2008)

[24] J. Garnett, P. Dieppe, Biochem. J. 266, 3 (1990)

[25] S.L. Teitelbaum, Science 289, 5484 (2000)

[26] M. Bohner, J. Lemaitre, Biomaterials 30, 12 (2009)

[27] M. Jarcho, C.H. Bolen, M.B. Thomas, J. Bobick, J.F. Kay, R.H. Doremus, J. Mater. Sci. 11, 11 (1976)

[28] Al-C. Dancu, R. Barabas, E.S. Bogya, Cent. Eur. J. Chem. 9, 4 (2011)

[29] E.S. Bogya, R. Barabas, V.R. Dejeu, Al. Csavdari, I. Baldea, Chem. Pap. 63, 5 (2009)

[30] R. Barabas, M. Cziko, I. Dekany, L. Bizo, E.S. Bogya, Chem. Pap. (in press)

[31] A. Slosarczyk, Z. Paszkiewicz, C. Paluszkiewicz, J. Mol. Struct. 744 (2005)

[32] P.N. Chavan, M.M. Bahir, R.U. Mene, M.P. Mahabole, R.S. Khairnar, Mater. Sci. Eng., B 168, 1 (2010)

[33] L. Wang, C. Li, Carbohydr. Polym. 68, 4 (2007)

[34] L. Kong, Y. Gao, G. Lu, Y. Gong, N. Zhao, X. Zhang, Eur. Polym. J. 42, 12 (2006)

[35] H.E. Feki, J.M. Savariault, A.B. Salah, M. Jemal, Solid State Sci. 2, 5 (2000)

[36] L-T. Zhang, W-F. Xie, Y-D. Wu, H. Xing, A-W. Li, W. Zheng, Y-S. Zhang, Chinese Phys. Lett. 20, 8 (2003)

[37] M.S. Sadjadi, H.R. Ebrahimi, M. Meskinfam, K. Zare, Mater. Chem. Phys. 130, 1 (2011)

[38] N. Davidenko, R.G. Carrodeguas, C. Peniche, Y. Solis, R.E. Cameron, Acta Biomater. 6, 2 (2010)

[39] P. Jongwattanapisan et al., Mater. Sci. Eng., C 31, 2 (2011)

[40] T-J.M. Luo, C-C. Ko, C-K. Chiu, J. Llyod, U. Huh, J. Sol-Gel Sci. Technol. 53, 2 (2010)

[41] K.R. Mohamed, Z.M. El-Rashidy, A.A. Salama, Ceram. Int. 37, 8 (2011)

[42] S. Gao, R. Sun, Z. Wei, H. Zhao, H. Li, F. Hu, J. Fluorine Chem. 130, 6 (2009)

[43] R. Sun, M. Li, Y. Lu, A. Wang, Mater. Char. 56, 3 (2006)

[44] X. Lu, Y. Leng, Biomaterials 26, 10 (2005)

[45] J. Oliva, J. Cama, J.L. Cortina, C. Ayora, J. De Pablo, J. Hazard. Mater. 213 (2012)

[46] H.M. Kim, T. Himeno, T. Kokubo, T. Nakamura, Biomaterials 26, 21 (2005)