Hydroxyapatite bioceramic with large porosity Materials Science and

Materials Science and Engineering C 76 (2017) 985–990

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Hydroxyapatite bioceramic with large porosity M. Mbarki a,b,⁎, P. Sharrock b, M. Fiallo b, H. ElFeki a a b

Laboratory of Materials and Environmental Sciences, Faculty of Sciences of Sfax, Soukra Road km 4-B. P. no 802 − 3038, Sfax, Tunisia Université de Toulouse, SIMAD, IUT Paul Sabatier, Avenue Georges Pompidou, 81104 Castres, France

a r t i c l e

i n f o

Article history: Received 17 December 2016 Received in revised form 5 March 2017 Accepted 12 March 2017 Available online 14 March 2017 Keywords: Hydroxyapatite Gelatin Bioceramic Porosity

a b s t r a c t Calcium phosphate based biomaterials have been used as bone graft with great success in the last decade. This material is employed in orthopedic and dental applications depending on their specific properties. In this work, we made a bioceramic with a large porosity, then we measured porosity, density and compressive strength of HA bioceramic. The SEM analysis was performed to show the morphology of the structure. The mechanical properties depend on the sintering of the HA bioceramic and the amount of pores. Thus, properties can be controlled by designing bioceramics with the appropriate porosities and calcination temperatures. One advantage of using gelatin is the formation of solids of any desired shape following a short time period, as the gelatin absorbs the water and expands into a solid composite form. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Porosity is an important aspect of scaffold and biomaterials engineering [1,2]. The sizes, shapes and distribution of pores control the fate of implanted materials by biological mechanisms including colonisation, angiogenesis, vascularisation and ultimately total resorption and replacement of implants by neoformed tissues. Thus, cellular processes need enough room to prolif, multiply, evacuate toxic metabolites and regenerate normal body tissues. This requires proper selection of materials for supporting and guiding wanted cells together with adapted voids for penetration into biomaterials implanted in vivo [3]. Patents disclose methods for porosity control and describe hydrophilic and hydrophobic contents adapted for different types of pore making procedures in various materials [4]. One critical aspect is the burn out temperature of porogens inserted in other materials to create voids left by removal of the sacrificed inclusions during calcination. The rate of thermal treatment is also an important feature to master in order not to fragilize the host material [5]. For ceramics such as hydroxyapatite (HA) and other calcium phosphates (tricalcium phosphate, TCP) or glass ceramics (apatite-wollastonite glass ceramic, AWGC), the propagation of cracks spur the breakdown of hard materials and causes its fragility. Mastering pore formation allows to reinforce bioceramics by limiting crack growth by the presence of multiple nanopores, and helps prevent initial crack formation in large macropores during controlled formation of sintered bodies [6]. This is why bioceramic formation is usually a slow process with long ⁎ Corresponding author. E-mail address: marina.fi[email protected] (M. Mbarki).

http://dx.doi.org/10.1016/j.msec.2017.03.097 0928-4931/© 2017 Elsevier B.V. All rights reserved.

processing times and sometimes high rejection rates due to the presence of defects. Bioceramic implant production methods have been reviewed, and comprise many variants such as gas foaming [7,8], gas casting [9,10], polymer burn-out or sublimation of organic inclusions [11,12], freeze casting [13,14], electrophoretic deposition [15,16], 3D printing [17] etc. Water or solvent freezing can be used to form future voids following drying under reduced pressure [18,19]. On the other hand, solvent soluble inclusions or colloidal mixtures are used to form composite solids that will create pores after dissolution of molecules. One main issue is the resulting mechanical properties of the final bioceramic. For bone tissue replacement, compressive strength should be different for cortical or cancellous bone placement. Since mechanical strength varies according to implant porosity, the detailed inner structure of the bioceramic plays a critical role in the replacement of hard tissues [20]. In this report we present our results on HA bioceramic formation using gelatin expanded in water for controlled formation of pores in bloc ceramics. We show that the gelatin polymer acts as binding agent, porogen and slow retractation gel in order to form macropores in a large range of porosities. Thus, 20 wt% gelatins create a final HA ceramic with 70% porosity.

2. Materials and methods The materials used were gelatin powder (Weishardt International, Graulhet, France), hydroxyapatite powder (Prayon, Belgium) and polymethacrylic-acid as anionic polymeric dispersant (Sigma Aldrich Chemical, France).

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2.1. Preparation of green bodies The ceramic preparation process involves three main steps: preparation hydroxylapatite suspension, drying and sintering. Three different mixtures of HA-gelatin were prepared by mixing 5, 10 and 20% of gelatin (percentages with respect to solid HA) with appropriate amounts of hydroxyapatite, and corresponding distilled water contents. The obtained paste was cast and placed in molds that can be opened (10 × 10 × 2.5 cm or 20 × 20 × 2.5 cm). After opening the molds the wet HA samples were unmolded gently and dried at room temperature until they hardened. They were then placed in a circulating air oven at 70 °C for final drying (the total time for drying was between 48 h and 72 h). Then, the samples were calcined following with the following temperature program: 7 h from 25 °C to 700 °C. They were sintered 1 h at different temperatures (900, 1000 and 1200° C) with a heating rate of 100 °C/h. The obtained bioceramic is shown in Fig. 1. 2.2. Characterization of obtained materials 2.2.1. X-ray diffraction The final product was characterized using powder X-ray diffraction (XRD) on a Bruker D2 diffractometer using copper radiation (radiation K α = 1.5418 Å) voltage and current 40 kV and 40 mA to identify and to prove the purity of the present phase. The scanning speed of the goniometer was 0.05° per second and scan ranged from 10° to 65°. 2.2.2. Infrared spectroscopy The FTIR spectrum of HA-gelatin powder was recorded with a Nicolet Impact 410 spectrometer in the DRIFT mode with no sample preparation. 2.2.3. Thermal analysis Thermal analysis was investigated with a Q600 from TA-DSC Instruments under 100 ml/min air flow, and the sample sizes varied between 10 and 20 mg. 2.2.4. Density and porosity The density and porosity were measured according to the standard ASTM C20 (Standard Test Methods for apparent porosity, water absorption, apparent specific gravity, and bulk density of burned refractory brick) by determining the mass of the dry sample and of the same sample immersed in distilled water. The trapped air was removed under vacuum until air bubbles stopped. 2.2.5. Scanning electron microscopy (SEM) The morphology and microstructure of porous ceramics were examined using scanning electron microscopy (SEM) using an ESEM XL30

Fig. 2. X-ray diffraction (XRD) obtained from HA before (a) and after (b) sintering at a temperature of 1000 °C.

from Philips. Some samples were embedded in epoxy resin and polished before examination under the microscope.

2.2.6. Mechanical testing The mechanical properties of macroporous hydroxyapatite ceramics were determined using compression tests. Specimens were polished to make flat and parallel surfaces to long axes. Mechanical tests were carried out on ZWICK-ROLL Z020 fitted with a 1 KN load cell. The loads were applied until the scaffolds cracked. The load of displacement curves were registered and compared together to weed out specimens failing prior rupture.

3. Results 3.1. X-ray diffraction The XRD pattern Fig. 2 corresponding to the HA powder before sintering (a) is similar to that of standard ceramic HA. The XRD pattern indicates that all the diffraction lines correspond to an apatite phase and correspond to the Joint Committee for Powder Diffraction Data Standards (JCPDS) reference (File Card No 9-432) [21]. The profiles are characteristic of a unique pure apatite phase. Comparing of the XRD patterns can be seen that the peaks in the diffractogram (b), after sintering, are sharper than that of (a) which shows that heat treatment increased the crystallinity of the product.

Fig. 1. Bioceramic with (10 × 10 × 2.5 cm) dimension.


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Table 1 The porosity of the ceramic 5%, 10% and 20% of gelatin as a function of calcination temperatures (n = 5). Temperature (°C)

900

1000

1200

Porosity(%) with 5% G Porosity(%) with 10% G Porosity(%) with 20% G

62.1 68.5 73.5

50.3 66.6 70.5

43.58 65.5 68.5

Table 2 The density of the ceramic 5%, 10% and 20% of gelatin as a function of calcination temperatures (n = 5). Temperature (°C)

900

1000

1200

Density (g/cm3) with 5% G Density (g/cm3) with 10% G Density (g/cm3) with 20% G

1.196 0.995 0.838

1.570 1.055 0.933

1.783 1.176 0.995

Fig. 3. IR spectrum of HA and gelatin (a), HA/gelatin (b) after sintering, and original HA powder alone (c).

3.2. Infrared spectroscopy Infrared spectra of the HA powder (C) and HA/gelatin (80%/10%) after sintering Fig. 3b, shows vibration band characteristic of hydroxyapatite [22]. Besides the main bands identified in the spectrum of HA powder and HA/gelatin corresponding to the functional groups of (PO4), hydroxyl (OH) and water, note the presence of a broad peak centered at 3330 cm−1 characteristic of the (OH) group related to absorbed water (H2O) [23]. In addition, the small bands appearing at 961 cm−1 and 1018 cm−1 correspond to HPO4 and PO4. Fig. 3a, before sintering, presents all the bands due to HA and several bands related to gelatin: amide bands at 1634 cm− 1 (C_O) and (N\\H deformation) at 1550 cm− 1 [24,25]. The peaks attributed to gelatin do not appear in the infrared spectra of HA/gelatin after sintering, which indicate that no traces of gelatin remain in our ceramics. The thermal treatment removes gelatin from the ceramic.

3.3. Thermal analysis Thermogravimetric analysis Fig. 4 revealed HA/gelatin mixture contained 1.72% by weight of water, which could be removed by heating to 150 °C, an endothermic peak appear at low temperature corresponding to the evaporation of (H2O) [26]. The sample also contained 8.15% by weight of gelatin. The mass loss is explained by thermal decomposition of gelatin from 300 °C to 500 °C [27]. Two exothermic peaks appeared at 400 °C and 500 °C, respectively, corresponding to the decomposition of

gelatin and combustion of carbon residues. These results are similar to those reports by Martínez-Vázquez et al. [21]. 3.4. Density and porosity The variation of the density and porosity of macroporous ceramics prepared according to the amount of porogen is listed in Tables 1 and 2. These results show that the increase of gelatin amounts during the preparation of green bodies increase the percentage of porosity after sintering. For example from 5% to 20% gelatin although the porosity pass from 50.3% to 70.5% respectively at T = 1000 °C. Note that when the temperature increases from 900 °C to 1200 °C, the porosity decreases from 62.1% to 43.58% for 5% of gelatin and 73.5% to 68.5% for 20% of gelatin. Thus, that two parameters, gelatin's percentage and temperature, affect the porosity so that the increasing of the amount of gelatin due to increasing of porosity. On other hand, the density of the ceramic is greater at T = 1200 °C than at T = 900 °C. The relative density at this temperature is 56.42% to 5% of gelatin; it is small compared with values reported in the literature and is about 98% [28–31]. This difference can be explained by the fact that no compression was performed in ceramic preparation during preparation. While the density is 1.783 g/cm3, it is a value similar to the density of cortical bone, which is 1.80 g/cm3. The density decreases in proportion with the amount of gelatin, therefore the porosity increases, which shows in Figs. 5 and 6. 3.5. Microstructure: scanning electron microscope (SEM) The pore size in our HA bioceramics are distributed in the range of de 5 μm. From the SEM images, we see that the pores are

Fig. 4. TG-DSC of HA/gelatin (80%/10%).

Fig. 5. The density and porosity of the ceramic as a function of % gelatin at T = 1200 °C.

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Fig. 6. The porosity as function of time at T = 1200 °C.

interconnected. The SEM micrographs given in Fig. 7 display the microstructure of macroporous bioceramics produced with 62.8% porosity. Large pores exist in the bioceramics, this phenomenon occurs because the gelatin particles have an important size in order to produce a large pore (size of the gelatin swollen in water).

3.6. Mechanical testing Compression fracture is applied to the ceramic until they cracked. The test is performed on the average of five samples for each amount of gelatin. In this section, we present the value of compressive strength of ceramic at three different temperature (T = 900 °C, 1000 °C and 1200 °C) and three amounts of gelatin (5%, 10% and 20%). The variation of the compressive strength is shown in Fig. 8 as it can be seen that the increasing of temperature increase the resistance of the sample rupture. Each ceramic with different amount of gelatin has average compression strength of 0.6 MPa, 1.12 MPa and 7.59 MPa, respectively, at T = 900 °C, 1000 °C and 1200 °C with 5% of gelatin. In addition, it can be seen that the addition of gelatin decrease compression strength of the ceramic. This fact occurs because, the variation of amount of gelatin increase the percentage of porosity causing the creation of porous structure. Although high porosity and large pore size promote interaction between the biomaterial and tissue [32].

Fig. 8. The compression strength of the ceramic with 5%, 10% and 20% of gelatin as a function of temperature.

4. Discussion Several chemical procedures exist today, manufacture hydroxyapatite bioceramics with large porosity simulating nature bone, which could be used for biomedical field (orthopedic use). Michael T. Malanga et al. [4] invented a new fabrication technique to product a high porosity ceramic materials. The process comprises the use of mixture which contains two or more porogens, one has a hydrophobic character, the other porogen has a hydrophilic character. In addition, Peón et al. [34] prepared a porous block of hydroxyapatite weighing the Hap, naphthalene as porogenic agent and polyvinyl alcohol. The mixture pressed and sintered at different temperatures in order to remove the naphthalene. The advantage of our preparation methods is the using of gelatin instead of naphthalene, according to his high capacity of absorption of free water to give a solid instead of a paste. So we started with a paste that we can mold and then quickly became solid (5 to 10 min) to be demolded and calcined. Reference to Michael T. Malanga et al. [4], the mixture may also contain organic compounds to facilitate the shaping of mixture like, binder, lubricants and dispersants, as described in International to the Principales of Ceramics Processing [35]. What is needed is a process that will increase the overall yield by reducing the number of bodies which crack without increasing the time needed to create a bioceramic with large porosity. For this purpose, this study investigated the fabrication of porous HA with addition of gelatin powders. The gelatin was used because it acts as a binder for cohesiveness of the green body, it is a composite material can cause voids due to burn out of

Fig. 7. SEM sample's with 10% of gelatin.


organic components, which allows easily obtain a high porosity (60% or more after calcination) and high porosity in pores sizes. Pore size should correlate with normal bone porosity with an approximate diameter of 100–200 μm. Hulbert et al. examined such correlation in vivo in 46% porosity calcium aluminate pellets [41]. They found that the nature of the penetrated tissue is directly related to pores' sizes. With small pores (10–44 and 44–75 μm), the pellets were penetrated by fibrous tissue only. With an increase of pores size to 75–100 μm, an ingrowth of unmineralised osteoid tissue started to appear. Gauthier et al. similarly showed that a pore size of 500 μm better supported bone formation compared to 300 μm pore size [42]. The main advantage to use gelatin powders was also the simple preparation of green bodies allows us to manufacture any form of ceramics and any dimension. The use of gelatin powders can be achieved with high and large ceramics stability. What is needed is a process that will increase the overall yield by reducing the number of bodies which crack without increasing the time needed to create a bioceramic with large porosity [4]. In the other hand, during processing of ceramic mixture to form ceramic bodies, proper control of the during process can result in significant reduction in cracking and resulting increase in productivity of the process by selection of desirable temperature and rate of calcination. That is why, the bioceramic were produced by using a two-step heat treatment to a void cracking during the burning-out of the gelatin. The samples were calcined following the temperature program: 7 h from 25 °C to 700 °C with a heating rate of 100 °C/h. Therefore, heating rate was performed at 100 °C/h to prevent the deformation of porous ceramics and micro crack formation. Besides, TGA experiments indicated the decomposition of gelatin from 300 °C to 500 °C, this result is in accord with results proved by Ward AG and Cem Bulent Ustundag [27,5]. In this case, the gelatin was completely burnt which allows the creation of macropores. This type of pores plays an important role in the biological fixation and mechanical bonding of scaffolds to hard living tissues. Furthermore, this type of homogeneous pore structure is an important factor for environmental applications. Bioceramic such as hydroxyapatite (HA) and tricalcium phosphate (TCP), have gained much recognition in biomaterials development due to their density, porosity, mechanical and biomimetric properties similar to nature bone structure [36–38]. By the way, a bone implant requires biocompatibility mechanical strength and porosity to promote interaction between the biomaterial and tissue without undesirable reaction [33]. In addition, the porosity affects mechanical performance of ceramic material. The mechanical strength of the material decreases by the increasing pore content this result is similar to the result found by Razali et al. [20]. On the other hand, N. Ozgur et al. [39] and J.E. Barralet et al. [40] reported that the addition of porogen increase the amount of pore, therefore the decrease of the mechanical properties this is in accord with our results. In this topic F.J. Martinez et al. reported that the compressive strength decrease from 3.8 MPa to 2.7 MPa improved by variation of porosity that is also in accord with our results, where three amounts of gelatin 5%, 10% and 20% were given 50.3%, 68.5% and 70,5% of porosity respectively. The porosity obtained 70% is suitable for human osteoblast cell.

5. Conclusion HA-gelatin macroporous bioceramics are fabricated with simple and speed preparation. The mechanical strength of the sample is decrease by addition of gelatin powders. Besides, the fact of reducing the compressive strength due to the high percentage of formation of pore during the heat treatment of samples by reducing the resistance therefore increases its porosity. However, the high percentage of porosity obtained (70%) is suitable for human osteoblast cell. Thus, in our preparation gelatin powders able to create a large porosity for cell proliferation. Hence, in this work the results proved that the ceramics are a suitable material for tissue engineering application [21].

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