Preparation and characterisation of calcium phosphate cement made

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In this study, the compressive strength and bioactivity of strong polymeric calcium phosphate cement (PCPC), made by mixing a calcium phosphate powder (a ...
Preparation and characterisation of calcium phosphate cement made by poly(acrylic/ itaconic) acid S. Hesaraki*1, D. Sharifi2, R. Nemati1 and N. Nezafati1 In this study, the compressive strength and bioactivity of strong polymeric calcium phosphate cement (PCPC), made by mixing a calcium phosphate powder (a mixture of tetracalcium phosphate and dicalcium phosphate dihydrate) and an aqueous solution of poly(acrylic/itaconic) acid, were investigated. The characteristics of the cement such as phase composition, setting reaction products and microstructure were analysed and compared to those of a control sample made by the same solid phase and water as a liquid. The hard tissue healing capability of PCPC was tested in a rabbit model by radiographical observations of the healing process as well as the cement condition. The results showed that the compressive strength of the set PCPC was y35 MPa before soaking in a simulated body fluid (SBF), which was much higher than that of the control specimen. However, it sharply decreased when the cement was immersed in the SBF. Xray diffraction analysis revealed that tricalcium phosphate was formed in the set PCPC and only a small amount of hydroxyapatite was produced after seven days soaking. In contrast, hydroxyapatite was almost the only phase of the control specimen after the soaking period. Radiography tests showed a cement (PCPC) with an irregular macrostructure after three months implantation, with a decreased radiopacity, and without any periosteal or intercortical callus formation. Keywords: Calcium phosphate, Bone cement, Polycarboxilic acids, Biocomposites

Introduction Calcium phosphate cements (CPC) are osteoconductive materials that can be transformed into bone-like poorly crystalline apatite in vitro/in vivo.1,2 They are attractive materials for dental and medical applications, because they are replaced by the newly formed bone during their resorption period.3,4 Traditional CPCs are generally composed of a solid phase, e.g. a mixture of dicalcium phosphate dihydrate (DCPD) and tetracalcium phosphate (TTCP) and a liquid phase such as distilled water or a solution of Na2HPO4.5,6 Unfortunately, these materials are mechanically weak and their applications are limited to non-load bearing sites.7 To make strong CPCs, an approach is the use of a reactive polymeric solution as the liquid phase of these cements. Matsuya et al.8 used poly(methylvinylether/maleic) acid, PMVE/ MA, for preparing a polymeric calcium phosphate cement (PCPC). The compressive strength reported by the authors were 23–48 MPa regarding the particle size of the cement solid phase. In Tenhuisen and Brown’s work,9 a hardened mass could be achieved using a 1 2

Materials and Energy Research Center, PO Box 31787/316, Tehran, Iran Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

*Corresponding author, email [email protected]

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solution of polyacrylic acid as the liquid phase of PCPC. Miyazaki et al.10 prepared a rapid settable PCPC using poly(acrylic/itaconic) acid solution so that its compressive strength was about 80–87 MPa even after one month storing in distilled water. In the present study, PCPC was made by mixing TTCP–DCPD powder and poly(acrylic/itaconic) acid liquid. The compressive strength, setting reaction products, cement morphology and the hard tissue healing capability of the cement were evaluated.

Experimental procedure At first, TTCP was synthesised through a solid state reaction of calcium carbonate and dicalcium phosphate anhydrate (both from Merck, Germany) as described elsewhere.11 A homogenous powder consisting of 1 mole of TTCP (with an average particle size of 12 mm) and 1 mole of DCPD (with an average particle size of 6 mm) was used as the solid phase of the cement and a solution of poly(acrylic/itaconic) acid (Fuji, Japan) was used as its liquid phase. The cement paste was made by mixing the powder (P) and the liquid (L) at a suitable P/L ratio. The same solid phase was mixed with distilled water to prepare control samples so that its characteristics were compared with the PCPC. ß 2009 Institute of Materials, Minerals and Mining Published by Maney on behalf of the Institute Received 16 January 2008; accepted 17 March 2008 DOI 10.1179/174367508X306514

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2 Fourier transform infrared spectra of hardened PCPC powder a before and b after soaking sample in SBF

1 X-ray diffraction patterns of a PCPC and b control cement after different periods of setting/soaking: T – tetracalcium phosphate, t – a tricalcium phosphate, B – brushite (dicalcium phosphate dihydrate), H – hydroxyapatite 1 solid phase of cement; 2 set cement after 24 h of setting (37uC); 3 set cement after one day of soaking; 4 set cement after seven days of soaking

To make a workable cement, pastes made with different P/L ratios (0?5–4 g mL21) were tested in terms of consistency. In brief, 1 g of the cement paste was pressed between two glassy plates at a constant load of 20 N (an approximate force applied to cement during filling a bone defect). The cement was classed as workable, if no fragmentation or cracking phenomena were observed. The results of the consistency test showed that appropriate and practical P/L ratios were 1 and 3 g mL21 for PCPC and control samples respectively. Thus, in the following study, the specimens made at these P/L ratios were used. The test of compressive strength (CS) was made on the cylindrical specimens (12 mm in height and 6 mm in diameter) of all cements after being incubated for 24 h (37uC and 100% humidity) and immersed in the

Table 1 Typical composition (SBF) solution

Composition

of

simulated

body

fluid

Weight (g) per litre distilled water

Sodium chloride (NaCl) 8.2187 Potassium chloride (KCl) 0.2260 0.3860 Calcium chloride (CaCl2.2H2O) 0.3337 Dipotassium hydrogen phosphate (K2HPO4.3H2O) Sodium sulphate decahydrate (Na2SO4.10H2O) 0.1697 Magnesium chloride hexahydrate (MgCl2.6H2O)0.3366 0.3508 Sodium bicarbonate (NaHCO3)

simulated body fluid (SBF) for one, three and seven days. The typical composition of the SBF is shown in Table 1. A universal testing machine (Zwick/Roell-HCR 25/400) with a crosshead speed of 1 mm min21 was employed for the examination of specimens. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses were performed on the set and soaked specimens using a diffractometer (Philips PW3710 with Cu Ka radiation) and a spectrometer (Bruker Vector 33) respectively. For these purposes, the soaked specimens were washed with distilled water and then, dried at room temperature. All specimens were ground to powder, weighted and then, analysed. The microstructure of the gold coated fractured surfaces of the specimens was observed through a scanning electron microscope (Stereoscan S 360, Cambridge Ltd). In vivo studies were performed on 18 adult New Zealand male rabbits of 3?0¡0?250 kg weight in accordance with the University of Tehran law on animal experimentation. After securing the supine position of each rabbit, the craniolateral surface of the right radial bone was exposed and a piece of full thickness bone with a length of 1 cm was removed from the medial section of the radial bone using an electrical bone vibrator. The produced gap was then filled with the PCPC paste. Radiographs were taken after creation of the gap and one, two and three months of the surgery. Antibiotics (penicillin G procaine 40 000 IU kg21 IM every 12 h), dexamethasone (0?6 mg/kg bw), vitamin B complex (0?2 mg/kg bw) and analgesic such as Tramadol hydrochloride (5 mg/kg bw IM every 12 h) were administered for three days after the operation.

Results and discussion In the present work, the characteristics of two different types of workable calcium phosphate cements were compared. The workability of a cement paste determines its ability to shape and mould in the defect sites without any undesirable changes (such as cracking or fragmentation) in its quality. It depends on the powder to liquid ratio of the cement and the composition of the reagents. In control sample, the cement paste showed sufficient

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a

b

c

d

3 Scanning electron micrographs of specimens: a 24 h incubated PCPC, b 24 h incubated control, c seven days soaked PCPC and d seven days soaked control

consistency when the solid phase was mixed with the liquid phase at a P/L ratio of 3 g mL21. This value was 1 g mL21 for PCPC. Lower P/L ratios led to weak cements whereas higher ratios yielded cements with poor workability, because of the increased viscosity of the pastes. In addition, the pilot tests revealed that the setting time of the PCPC increased with decreasing P/L ratio. The setting time of a workable PCPC was y2 min, which was considerably lower than that of the control one (16 min). Figure 1a shows the XRD patterns of PCPC after different setting/soaking times. The patterns of both the original cement powder (the solid phase 1) and the control specimen (Fig. 1b) are also shown for comparison. All peaks in the XRD pattern of the original cement powder belonged to TTCP and DCPD and no further phase was found (note that main peaks have been merely marked in Fig. 1). The predominant phase detected in both set and soaked PCPC was a tricalcium phosphate (a-TCP), which was accompanied with brushite (DCPD) and a small amount of TTCP. The composition of PCPC was slightly influenced by the soaking process so that a small quantity of poorly crystalline hydroxyapatite was found in the cement soaked. However, for the control specimen, the pattern of the set cement was similar to that of the solid phase

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and poorly crystalline apatite was the most predominant phase after soaking for seven days. In this cement, the setting reaction (apatite formation) is a hydraulic process that can be controlled by the solubility of the reactants at the early stages of the reaction (the dissolution mechanism) and by the diffusion mechanism at the later stages (during soaking). However, for calcium phosphate cement with a polymeric liquid phase, the setting reaction is completely different.

4 Compressive strengths of PCPC and control cement specimens after different soaking times

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5 Radiographs of a defected radial bone, b gap immediately after filling with PCPC, filled gaps c two months after implantation and d three months after implantation

Some researchers8 have proposed the following acid– base reaction as the setting mechanism of bone cements with tetracalcium phosphate powder and polycarboxilic acid liquid Ca4 (PO4 )2 Oz2RCOOH~ R

COO

Ca

COO

RzCa3 (PO4 )2 zH2 O

(1)

In this study, the appearance of the TCP peaks, in the XRD pattern of the set PCPC, approved the given reaction (equation (1)). It was expected that hydroxyapatite was

precipitated in PCPC with a little delay (after at least seven days of soaking), but it was almost inhibited owing to the presence of acrylate substances surrounding the calcium phosphate particles. Thus, a small amount of HA was detected after seven days, which probably was formed through the following reaction Ca4 (PO4 )2 OzCaHPO4 :2H2 O~ (2)

Ca5 (PO4 )3 (OH)z2H2 O

Figure 2 shows FTIR spectra of PCPC sample after setting

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(a) and seven days of soaking (b). The bands around 1415 and 1560 cm21 are assigned to the symmetric and asymmetric stretches of the carboxyl group of calcium acrylate matrix.9 These bands largely appeared in PCPC before the soaking process but decreased after soaking the sample. This indicates that the polyacrylate matrix is a water soluble salt. Figure 3 shows the scanning electron micrographs (SEM) of the control specimen and the PCPC after 24 h setting and seven days soaking. It was observed for PCPC (Fig. 3a) that unreacted particles of reactants were embedded in a uniform matrix of polyacrylate salt. The micrograph of the control specimen shows a lumpy morphology of reactant particles connected to each other (Fig. 3b). For the soaked PCPC, microstructural degradation could be seen owing to dissolution of the cement constituents (Fig. 3c) and no embedded reactants were observed. Some granular crystals (y1 mm size) were also found over the particle surfaces. It is suggested that these crystals are precipitated hydroxyapatite.12 A completely different morphology was observed in the soaked control specimens, i.e. as entangled, nanosized and needle-like apatite crystals (Fig. 3d). In Fig. 4, the CS values of both PCPC and control specimens are compared. Before soaking, although the P/L ratio of the control specimen was higher than that of the PCPC, the CS of the PCPC was nearly 11 times higher than that of the control specimen. However, the results were totally different after soaking the specimens in SBF so that unlike to the control specimen, the CS of PCPC was gradually decreased. At the end of the 7th day, the CS of the control specimens was three times higher than that of the PCPC. In other words, the PCPC had an adequate CS after setting, but it was gradually weakened when it was immersed in a liquid medium such as SBF. The mechanical behaviour of calcium phosphate cements can be explained by their microstructural features. For the control specimen, entanglement of the nanosized apatite crystals was responsible for the evaluated compressive strength whereas in PCPC, dissolution of the calcium polyacrylate phase (which binds the remained calcium phosphate particles to each other) out of the matrix is suggested to weaken the material. In vivo tests revealed that no operative or postoperative complications occurred. All of the rabbits well tolerated the operation and survived at the end of

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experimental periods. No wound opening or infections were observed as well. Radiographs of the defected bone and the filled gap, three months after the implantation, are shown in Fig. 5. It can be seen that no sign of infection is shown on the edges of all defects. The stabilisation of the defected bones and their maintenance in the correct position was also observed in the tested rabbits. The radiopacity and regularity of the PCPC bulk was gradually decreased (just as the compressive strength decreased) and also, no external and intercortical callus was observed even after three months of implantation.

Conclusions Although the PCPC prepared by mixing TTCP/DCPD powder and poly(acrylic/itaconic) acid solution had a good initial compressive strength, this property was not maintained in the liquid medium (SBF) and it gradually became weaker. In addition, the PCPC did not show an appropriate hard tissue healing capability during the evaluating period and thus, it cannot be considered as a suitable substitute in the bone/dental defect treatment.

Acknowledgement The authors wish to acknowledge Ms N. Nosoudi and Mr M. Zardkouhi for their help.

References 1. M. Takechi, Y. Miyamoto, K. Ishikawa, T. Toh, T. Yuasa, M. Nagayama and K. Suzuki: Biomaterials, 1998, 19, 2057–2063. 2. S. Hesaraki, F. Moztarzadeh and M. Solati-Hashjin: J. Biomed. Mater. Res. B, 2006, 79B, 203–209. 3. L. C. Chow: J. Ceram. Soc. Jpn, 1991, 99, 954–964. 4. R. P. Del Real, J. G. C. Wolke, M. Vallet-Regi and J. A. Jansen: Biomaterials, 2002, 23, 3673–3680. 5. S. Hesarki, F. Moztarzadeh and D. Sharifi: J. Biomed. Mater. Res. A, 2007, 83A, 80–87. 6. S. Takagi, L. C. Chow and K. Ishikawa: Biomaterials, 1998, 19, 1593–1599. 7. E. Fernandez, S. Sarda, M. Hamcerencu, M. D. Vlad, M. Gel, S. Valls, R. Torres and J. Lopez: Biomaterials, 2005, 26, 2289–2296. 8. Y. Matsuya, S. Matsuya, J. M. Antonucci, S. Takagi, L. C. Chow and A. Akamine: Biomaterials, 1999, 20, 691–697. 9. K. S. Tenhuisen and P. W. Brown: J. Dent. Res., 1994, 73, 598–606. 10. K. Miyazaki, T. Horibe, J. M. Antonucci, S. Takagi and L. C. Chow: Dent. Mater., 1993, 9, 41–45. 11. S. Hesaraki and D. Sharifi: Bio-med. Mater. Eng., 2007, 17, 29–38. 12. R. Xin, Y. Leng, J. Chen and Q. Zhang: Biomaterials, 2005, 26, 6477–6486.