Effect of silica gel on the cohesion, properties and biological performance of brushite cement

Acta Biomaterialia 6 (2010) 257–265

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Effect of silica gel on the cohesion, properties and biological performance of brushite cement Mohammad Hamdan Alkhraisat a,*, Carmen Rueda a, Luis Blanco Jerez b, Faleh Tamimi Mariño c, Jesus Torres d, Uwe Gbureck e, Enrique Lopez Cabarcos a a

Departamento de Química-Física II, Facultad de Farmacia, UCM, Plaza: Ramon y Cajal, sn, 28040 Madrid, Spain Departamento de Estomatología III, Facultad de Odontología, UCM, 28040 Madrid, Spain c Faculty of Dentistry, McGill University, Montreal, Que., Canada d Departamento de Ciencias de la Salud III, Facultad de Ciencias de la Salud, URJC, Alcorcon-Madrid, Spain e Department of Functional Materials in Medicine and Dentistry, University of Würzburg, 97070 Würzburg, Germany b

a r t i c l e

i n f o

Article history: Received 2 March 2009 Received in revised form 4 June 2009 Accepted 4 June 2009 Available online 10 June 2009 Keywords: Brushite Calcium phosphate cement Cohesion Bone regeneration Silica gel

a b s t r a c t The cohesion of calcium phosphate cements can be improved by the addition of substances to either the solid or liquid phase during the setting reaction. This study reports the effect of silica gel on brushite cement cohesion. The cement was prepared using a mixture of b-tricalcium phosphate (b-TCP) and monocalcium phosphate monohydrate as the solid phase, while the liquid phase comprised carboxylic acids silica gel. This cement presents a shorter final setting time (FST), better cohesion and higher amount of unreacted b-TCP than the cement prepared without silica gel. Furthermore, in vivo experiments using rabbits as an animal model showed that after 8 weeks of implantation cements modified with silica gel showed a similar new bone formation volume and more remaining graft in comparison with unmodified cements. Thus, the silica gel could be efficiently applied to reduce cement disintegration and to decrease the resorption rate of brushite cements. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Since their development by Mirtchi and co-workers, calcium phosphate cements that set by entanglement of precipitated brushite crystals have been receiving increasing interest as a bone substitute in the recent years [1]. These cements are obtained as various combinations, such as b-tricalcium phosphate (b-TCP) + monocalcium phosphate monohydrate (MCPM) and b-TCP + phosphoric acid [2,3]. The setting reaction of these cements is a continuous dissolution/precipitation mechanism at low pH values as brushite precipitates at pH <6 [2,4,5]. The relatively short setting time of brushite cements compared with hydroxyapatite forming pastes depends on both the higher solubility of the cement raw materials and the higher rate of brushite crystal growth (3.32  104 mol dicalcium phosphate dihydrate (DCPD) min1 m2) [6] compared with hydroxyapatite (2.7  107 mol Ca5(PO4)3OH min1 m2) [7]. Brushite cement is too weak to be used in load-bearing areas [8] and many studies have been dedicated to improving the mechanical properties of the set cement. Retarding the brushite crystal growth rate results in smaller sized crystals, allowing them to pack

* Corresponding author. Tel.: +34 91 3941751; fax: +34 91 3942032. E-mail address: [email protected] (M.H. Alkhraisat).

more closely together and improving the mechanical properties of brushite cements [9]. Pyrophosphate ions, sulphate ions, citrate ions, pyrophosphoric acid and carboxylic acids, such as citric, tartaric and glycolic acids, have been used to increase the cement setting time and improve its mechanical properties [10–15]. Recently, tailor-made brushite blocks with good mechanical properties have been synthesized using a 3-dimensional printing technique [16]. Interestingly, Hofmann et al. developed brushite cements as strong as 52 MPa in compression by adjusting particle size and size distribution of the b-TCP and the MCPM powder reactants and also the citric acid concentration, resulting in a decrease of cement porosity to 11% [17]. The control of cement porosity is also a useful tool to control drug release rate from the cement matrix [17,18]. Studies on drug release from brushite cements showed the efficiency of these cements as drug delivery systems and that this delivery is controlled by diffusion release kinetics and by drug interaction with cement matrix [17–20]. Furthermore, brushite cements are adequate systems to induce ionic substitution in brushite crystals, influencing the properties of the cements [21–23]. In particular, strontium and magnesium substitution have been carried out using water-soluble salts or ionically substituted reactants [21– 23]. The major advantage of brushite cements is the higher solubility of secondary protonated calcium phosphates under physiological conditions, such that brushite is dissolved at the implantation

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.06.010

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site more rapidly than hydroxyapatite materials. This property has been demonstrated in various in vitro [24,25] and in vivo studies [26–34]. Brushite cement is replaced by new bone and the cement-new bone composite was shown to have similar or even better mechanical properties than normal bone within 16 weeks after implantation in rabbits [26]. Osteotransductive brushite cements have been implanted in vivo in different forms, such as blocks and granules, and injected as a paste [27–32]. Different additives, such as hyaluronic acid, xanthan gum and citric acid, have been employed to improve cement injectability [13,33,34]. Xanthan gum had a negative effect on brushite cement bone regeneration, while hyaluronic acid had little effect, except for a slight decrease in initial resorption rate [34]. The rapid resorption rate of brushite cements is a result, in part, of brushite solubility under physiological conditions [27]. However, this could compromise the osteoconductive properties of brushite, as the rate of bone growth cannot fill the gap resulting from cement resorption, increasing the risk of connective tissue growth and loss of osteointegration. The addition of granular b-TCP and cement formulation with a molar b-TCP/MCPM ratio greater than 1 have been described as overcoming this problem, as b-TCP has a slower resorption rate [27,32]. The effect of b-TCP on the biological behaviour of brushite cements was studied in rabbit femoral condyles. The results showed that, except for b-TCP granules, both cement cylinders were almost completely resorbed and replaced by bone tissue after 8 weeks. At 16 weeks the bone in the cavities of both cements recovered a trabecular pattern, but only the bone trabeculae in the initial cavity of the cement with b-TCP granules became thick and mature [27]. Several processes are involved in brushite cement biodegradation, including dissolution, fragmentation [35,36,32] and in vivo macrophage phagocytosis of cement particles [28]. However, brushite transformation to a biologically more stable HA will minimize its advantages over stronger apatite cements [37]. To prevent this process either pyrophosphoric acid [12] or poorly soluble magnesium salts have been used [37]. Recently, we showed that strontium ions also prevented the precipitation of hydroxyapatite within the matrix of a secondary calcium phosphate cement [23]. Nowadays, the cohesion of brushite cements is being studied in detail to develop disintegration-resistant ones. In a previous work [15] we studied the effects of carboxylic acids and viscosity enhancing agents on brushite cement cohesion. In the present study, we report the efficacy of silica gel in preventing cement disintegration. In bioactive silica-based glasses and glass ceramics a surface silica gel layer is formed by partial network dissolution and surface polycondensation reactions that occur prior to formation of a calcium phosphate-rich layer [38]. The silica gel and hydroxyapatite layers provide adsorption sites for the cellular growth factors generated by macrophages and stem cells, promoting bone formation [39]. With the aim of incorporating silica gel, the cement liquid phase was modified by the addition of sodium silicate solution. Once gelled, a cement powder composed of b-TCP and MCPM was reacted with the gel to prepare cement cylinders. Thereafter, cements were tested for their cohesion and physico-chemical and mechanical properties. The in vivo performance of silica-modified brushite cements was analysed by implantation in bone titanium chambers placed on rabbit calvaria.

2. Materials and methods 2.1. Cement preparation All reactants with the highest purity were purchased from Sigma–Aldrich, with the exception of aerosil 200 (Degussa GmbH,

Frankfurt, Germany). b-TCP was produced by solid-state reaction of stoichiometric amounts of CaCO3 and CaHPO42H2O at 900 °C for 14 h. The brushite cement powder phase was composed of bTCP and MCPM in a molar ratio of 1.32. Pyrophosphate ions (0.54 wt.%) were used as a retardant of the setting reaction. Silica gels were prepared by dissolving 6 g aerosil 200 in 4 M NaOH at 200°C. The clear solution was adjusted with distilled water to a concentration of 60 g l1 SiO2 with a pH of 11–12. The gelation of the SiO2 was induced by the addition of citric, glycolic or tartaric acid at final concentrations of 0.5, 1 and 0.5 M, respectively. Gels were formed after 3 days for those solutions with 15.6 and 16.8 g l1 SiO2. As there was no significant difference between them, only the results of the experiments done at 16.8 g l1 SiO2 are reported. The cement setting reaction (Eq. (1)) was started by mixing the cement powder and liquid phase on a glass slab for 30 s at a powder to liquid (P/L) ratio of 2.5 g ml1.

Ca3 ðPO4 Þ2 þ CaðH2 PO4 Þ2  H2 O þ 7H2 O ! 4CaHPO4  2H2 O

ð1Þ

2.2. Cement characterization After mixing, the cement paste was used to fill cylindrical silicon moulds with an aspect ratio of 2:1. The cements were tested for final setting time (FST) according to the international standard ISO1566 for dental zinc phosphate cements at room temperature and humidity [40]. The cement is considered to be set when a Vicat needle of 1 mm diameter with a load of 400 g fails to make a visible circular indentation on the cement surface. The pH of the cement paste was also measured at room temperature for up to 6 h by inserting an InlabSolidÒ electrode (Mettler-Toledo, Barcelona, Spain) connected to a MP230 pH meter (Mettler-Toledo, Barcelona, Spain) into the cement paste. The pH electrode was calibrated using three standard buffer solutions. The wet diametral tensile strength of cement samples was measured by the diametral compression test and calculated from the failure load applied along the diametral plane of the specimens. The measurements were carried out using Pharma Test PTB 311 equipment after sample ageing in water for 24 h at 37 °C and a relative humidity of 100%. The solid to ageing liquid ratio was 43.75% (g ml–1). The cements were evaluated for their cohesion according to the solid weight loss method [15]. The cement sample volume was approximately 392.5 mm3 and the exposed cement surface area was 78.5 mm2. Briefly, a one face open cylinder was filled with the cement paste and left to set at room temperature and humidity for 2 min. Three specimens were prepared for each cement formulation. Afterwards, each sample was aged in 5 ml distilled water at 37 ± 1 °C for 24 h under constant stirring (70 rpm). Thereafter, the ageing medium was filtered using a Millipore membrane (Millipore Ibérica, Madrid, Spain) with a pore size of 0.10 lm that was subsequently dried and weighed. The solid weight loss is given as a percentage of the dry weight of the released particles (Pw) with respect to the total sample weight (cement dry weight + Pw). The composition of the set cement matrix was analysed by Xray diffraction using a Philips X’pert diffractometer (Cu Ka radiation, 45 kV, 40 mA). Data were collected in the interval between 2h = 3° and 60°, with a step size of 0.02° and a normalized count time of 1 s step1. The mineral composition of the cement was checked by means of structural model files of brushite (ICSD 016132) [41] and b-TCP (ICSD 06191) [42]. Fourier transform infrared (FTIR) spectra were recorded in transmission mode using a Perkin–Elmer 1720 spectrometer with a spectral resolution of 2 cm1 and pressed KBr discs. When necessary the setting reaction was stopped by immersing the cement samples in absolute ethanol for 1 min and then drying at 37 °C for 24 h.

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2.3. Biological performance The protocol of the in vivo study was approved by the ethical committee for animal experiments of the Universidad Complutense de Madrid (UCM). Experiments were conducted in accordance with the guidelines laid down by the European Communities Council Directive of November 24, 1986 (86/609/ EEC) and adequate measures were taken to minimize pain and discomfort to the animals. Herein, vertical bone regeneration was assayed over 8 weeks using the method of titanium bone conduction chamber [29]. Seven healthy 6-month-old female New Zealand rabbits of 3.8 ± 0.2 kg were used in this study. The animals were housed in the official accommodation for animal assays of the UCM at 22– 24 °C with 55–70% humidity, light cycles of 12 h and air renewal 15 times per hour. The animals were fed Panlab SL diet with water available ad libitum. 2.3.1. Surgical protocol Before surgery the rabbits were anaesthetized with an intramuscular dose of 0.75 mg/kg ketamine (Imalgene 1000Ò, Rhone Merieux, Toulouse, France) and 0.25 mg/kg xilacine (RompunÒ, Bayer, Leverkusen, Germany). The heads of the animals were shaved and disinfected with Betadine (povidone iodine) solution. The calvaria bone surface was exposed through a skin incision of approximately 6 cm in length over the linea media. A circular slit on each side of the median sagittal suture was prepared with a trephine bur (10 mm diameter) cooled with saline solution. A titanium bone conduction chamber was created by fixing a titanium cylinder (10 mm diameter, 4 mm height, 0.5 mm thick) in each slit, with slight roughening of the bony chamber floor with a round bur (Fig. 1). In each rabbit one chamber contained 250 mg granulate brushite cement (0.2–1 mm diameter) set with 1 M glycolic acid and another with 250 mg granulate brushite cement set with silica gel–1 M glycolic acid. It is worth mentioning that the granulate cement was applied dry, but upon filling the titanium chamber the granulate was wetted by blood from the operated bone. The periosteum was repositioned and sutured (DexonÒ 3/0) and the wound closed with 3/0 silk suture. TerramicinaÒ (Pfizer, Madrid, Spain) in water was given for 7 days as a post-operative antibiotic. The

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animals were sacrified 8 weeks after the intervention with an overdose of sodium pentobarbital IV (DolethalÒ, Vétoquinol, Lure, France). Bone samples were collected and fixed in 10% formaldehyde buffered at pH 7.0. 2.3.2. Histology and histomorphometry Sample dehydration, embedding and sectioning were performed according to a previous protocol [43,44]. Samples stained with toluidine blue were examined under optical microscopy for histological evaluation. Micrographs (magnification 6) of the biopsy slices were captured with a digital camera and analysed using histomorphometry software IAS 2000 (Delta Sistemi, Rome, Italy). Six randomly selected slices were analysed for each biopsy. The area inside the cylinder was included for histomorphometric evaluation. Total sample volume, augmented bone volume (BV), and remaining graft (RG) volume were determined for each sample.

BVð%Þ ¼ ½ðnewly formed bone volumeÞ=ðtotal sample volumeÞ  100

RGð%Þ ¼ ½ðremaining graft volumeÞ=ðtotal sample volumeÞ  100 The Mann–Whitney test at P < 0.05 (SPSS 7.0, Chicago, IL, USA) was applied for statistical analysis of the histomorphometric measurements. 3. Results 3.1. Cement characterization Herein we have investigated the effect of silica gel on the properties of brushite cements set with glycolic, tartaric and citric acid. Fig. 2 shows that the FST of the cements prepared with citric, glycolic and tartaric acid (17, 7 and 3 min, respectively) was considerably reduced by the addition of silica gel to the cement liquid phase (7, 5 and 1 min, respectively). Clinically, such an acceleration in setting rate may be suitable for galenic formulations, enabling the fast setting of brushite cements upon application, thus reducing the surgery time and the risk of cement disintegration.

Fig. 1. Titanium bone chamber model used in this study to evaluate bone regeneration with the cement granulate.

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Fig. 2. Solid weight loss and FST of brushite cements set with: (i) 0.5 M citric acid; (ii) 0.5 M citric acid and silica gel (16.8 g l1); (iii) 1 M glycolic acid; (iv) 1 M glycolic acid and silica gel (16.8 g l1); (v) 0.5 M tartaric acid; (vi) 0.5 M tartaric acid and silica gel (16.8 g l1). The cement weight loss error was set at the 95% confidence interval on the adjusted values.

The carboxylic acid silica gels had higher pH values than their corresponding carboxylic acid solutions (Table 1). Changes in cement paste pH were followed during the setting reaction. Fig. 3 presents the results for the cements set with 0.5 M tartaric acid and 0.5 M tartaric acid silica gel. In both cases the pH increased as function of time. The cement prepared with silica gel reached equilibrium faster and at a more acidic pH than the cement set with tartaric acid. Interestingly, the pH changes were marked by the appearance of an inflection at 40 min after mixing the cement with silica gel. This inflection appeared later (80 min) in the case of cement set with tartaric acid. XRD and FTIR analyses were carried out to investigate structural changes within the cement matrix. FTIR spectra and XRD patterns were recorded at 21 and 35 min after mixing with silica gel and at 75 and 80 min after mixing with tartaric acid. The XRD patterns show an increase in the intensity of brushite peaks and a decrease in the b-TCP peaks above the inflection point (Fig. 4), while no differences were observed in the FTIR spectra. These results indicate that there is no solid phase change at the inflection point. The reduction in the FST of silica gel cements was reflected in their mechanical properties, as depicted in Fig. 5. The diametral tensile strength (DTS) of the brushite cement set with 0.5 M citric acid was significantly reduced, from 4 to 2.3 MPa, by the presence of silica gel. However, this reduction was negligible for brushite cements set with 0.5 M tartaric acid. Furthermore, the incorporation of silica gel significantly improved brushite cement cohesion (Fig. 2). Cement samples were incubated in water after 2 min of mixing the solid and liquid phases. The presence of silica gel reduced the solid weight loss by 59%, 28% and 79% for cements set with citric, glycolic and tartaric acid, respectively. Statistical analysis of the results shown in Figs. 2 and 5 was performed using two-way ANOVA as we have two independent nominal variables (acid type and presence of silica gel). The results showed that cement cohesion was significantly affected by the

Fig. 3. Cement paste pH for brushite cements set with 0.5 M tartaric acid and 0.5 M tartaric acid and silica gel (16.8 g l1).

Fig. 4. X-ray diffraction patterns of CPCs set with 0.5 M tartaric acid and silica gel (16.8 g l1). These patterns were recorded at different times. The most prominent peaks of brushite (*) and b-TCP (+) are marked.

type of carboxylic acid used and the presence of silica gel. However, the effects of these two factors on cement cohesion were not independent, as their interaction was significant (Table 2). Similar conclusions can be derived from statistical analysis of the DTS values (Table 3). The regression coefficients (Table 4) indicate that the factors citric acid, absence of silica gel and glycolic acid had a positive effect on the cement solid weight loss. The strongest effect was for the factor citric acid in the absence of silica gel. This factor also had a positive effect on the cement DTS. Rietveld analysis of X-ray diffraction patterns shows that the cements are composed predominantly of brushite and unreacted

Table 1 The effect of silica gel on the phase composition of brushite cements. Cement liquid phase

Solution pH

Cement composition (%) DCPD

b-TCP

0.5 M citric acid 0.5 M citric acid in silica gel (16.8 g l1 SiO2) 0.5 M Tartaric acid 0.5 M tartaric acid in silica gel (16.8 g l1 SiO2) 1 M glycolic acid 1 M glycolic acid in silica gel (16.8 g l1 SiO2)

1.74 ± 0.01 4.39 ± 0.02 1.76 ± 0.01 4.52 ± 0.02 1.84 ± 0.01 4.71 ± 0.02

87 74 87 72

13 26 13 28

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the cement matrix, of complexes between calcium ions and the oxygen atoms of the gel hydroxy groups. 3.2. Biological performance Animal recovery after surgery was uneventful and no animal loss was registered. The animals weighted 4.8 ± 0.2 kg at the moment of death.

Fig. 5. DTS of brushite cements set with: 0.5 M citric acid; 0.5 M citric acid with silica gel (16.8 g l1); 0.5 M tartaric acid; 0.5 M tartaric acid with silica gel (16.8 g l1). The error bars correspond to the 95% confidence interval on the adjusted values.

b-TCP. However, setting the cements with carboxylic acid silica gel increased the residual fraction of unreacted b-TCP, with a consequent decrease in the brushite component within the cement matrix (Table 1). To further investigate the relationship between cement composition and cohesion we obtained FTIR spectra of the cements prepared with silica gel. Fig. 6 shows the FTIR spectra of cements set with tartaric acid silica gel as well as those prepared with glycolic acid silica gel. The vibration bands observed at 1589 and 1385 cm1 are attributed to the asymmetric and symmetric stretching vibrations of the carboxylic group, respectively. The presence of peaks at 970 and 668 cm1 (insets in Fig. 6A and B) is attributed to Si–O stretch vibration and Si–O–Si vibration, respectively [45]. These bands could indicate the formation, within

3.2.1. Histology No signs of inflammation or infection were observed in histological slices of the grafted titanium bone chambers. After 8 weeks of granulate implantation new bone formation was observed to occur from the external surface of the parietal bone alongside the cylinder walls towards the centre of the chamber (Fig. 7). Similar behaviour was observed in other studies of bone regeneration using the titanium chamber model [29,46]. We also observed the presence of more remaining graft in the chamber grafted with cement set by silica gel (Fig. 7). The remaining graft was surrounded by new bone and evenly distributed within the titanium chamber (Fig. 7). The newly regenerated bone reached a height of >3 mm, occupying most of the chamber volume. As illustrated in (Fig. 8A), at higher magnification osteoblasts can be seen embedded in unmineralized bone matrix (osteoid) at the granulate–bone interface. This matrix was later mineralized to woven bone. It can be seen that unmineralized matrix is closer to the cement granules than woven bone, indicating that new bone formation occurred after granulate resorption (Fig. 8A). This newly formed bone had a lamellar orientation and surrounded osteoblast cells, inducing their transformation to osteocytes (Fig. 8A). Signs of graft resorption, such as surface pitting and phagocytosis by macrophages, were also observed (Fig. 8B). Fig. 8B also shows that Hawship’s lacunaw were formed by osteoclast cells, which is clear evidence of bone remodelling.

Table 2 Two-way ANOVA analysis of cement solid weight loss (%) results. Source

Degrees of freedom

Sum of squares

Mean square

F value

P < 0.05

Acid type Silica gel Acid type  silica gel

2 1 2

8.48 2.51 1.63

4.24 2.51 0.82

2559.9 1514.1 492.2

Yes Yes Yes

Table 3 Two-way ANOVA analysis of DTS (MPa) results. Source

Degrees of freedom

Sum of squares

Mean square

F value

P < 0.05

Acid type Silica gel Acid type  silica gel

1 1 1

2.28 4.17 1.52

2.28 4.17 1.52

10.9 19.9 7.3

Yes Yes Yes

Table 4 Regression coefficients (B) obtained from the statistical analysis of the results for cement solid weight loss and diametral tensile strength. Parameter

Intercept Citric acid Glycolic acid Absence of silica gel Citric acid  absence of silica gel Glycolic acid  absence of silica gel SE, standard error. a Not significant at the 5% level.

Cement solid weight loss (%)

Diametral tensile strength (MPa)

B

SE

B

SE

0.086 1.043 0.699 0.340 1.257 0.039a

0.023 0.033 0.033 0.033 0.047 0.047

2.192 0.123a

0.153 0.216

0.033a 1.527

0.216 0.305

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Fig. 6. (A) FTIR spectrum of brushite cement set with 0.5 M tartaric acid and silica gel. The inset depicts the stretching vibration band of Si–O at 970 cm1 [45]. (B) FTIR spectrum of brushite cement set with 1 M glycolic acid and silica gel. The inset shows the vibration band of Si–O–Si at 668 cm1 [45].

Fig. 7. Bone formation promoted by granulate brushite cement set with 1 M glycolic acid (right) and silica gel modified brushite cement (left). Bone formation starts from the external surface of the parietal bone alongside the lateral wall of the titanium cylinder and continues towards the centre of the chamber. Cement granules are evenly distributed and surrounded by new bone formation.

Fig. 8. (A) Bone formation around brushite cement granules surrounded by unmineralized bone matrix maturing to woven bone. This indicates that bone formation followed granulate resorption. Osteoblasts were embedded within the osteoid. (B) Cement particles engulfed by macrophages (right) and Hawship’s lacuna formation (left), which indicates bone remodelling by osteoclasts.

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Fig. 9. (A) Remaining graft (RG) after 8 weeks cement implantation in vivo. The remaining graft is significantly higher for brushite cements set with glycolic acid silica gel (P < 0.05). (B) Augmented bone volume (BV) after 8 weeks implantation provoked by granulate brushite cement. There are no significant differences between brushite cement set by glycolic acid with silica gel and cement set by glycolic acid alone (P > 0.05).

3.2.2. Histomorphometry The hismorphometric analysis showed significantly higher remaining graft (17 ± 3%) of the cements set with glycolic acid silica gel, indicating slower resorption than of the brushite cement set with glycolic acid (3 ± 1%) (Fig. 9A). The brushite cement set with silica gel (47 ± 3%) were as efficient as the brushite cement set with glycolic acid (43 ± 6%) in promoting bone regeneration, as indicated by the absence of statistically significant differences (Fig. 9B). 4. Discussion Silicon is an essential trace element for metabolic processes associated with the development of bone and connective tissues [47]. Its presence in hydroxyapatite implants resulted in greater bone apposition at the implant surface [48]. The bioactivity and osteointegration of bioglasses stem from the formation of a silica gel layer on their surfaces upon contact with body fluids and the precipitation of calcium and phosphate ions (within the gel) as poorly crystalline apatite [49]. Herein the simultaneous use of carboxylic acids and silica gel has resulted in shorter cement FST values than those for carboxylic acids alone (Fig. 2). These acids inhibit the growth of brushite crystals through the binding of calcium ions by carboxylic groups exposed on the brushite surface [50]. The FTIR spectra of carboxylic acid silica gels show the presence of vibration bands at 970 and 668 cm1, corresponding to the Si–O stretch vibration and Si–O– Si vibration, respectively (Fig. 6) [45], indicating the possible formation of calcium complexes with OH groups of the silica gel. This complex could serve as a nucleus for brushite crystal precipitation, accelerating setting of the cement. This coincides with previous studies showing a catalytic effect of Si–OH groups on apatite nucleation [51,52]. The pH increase within the cement paste set with tartaric acid silica gel was faster and achieved a more acidic pH at equilibrium (Fig. 3). This is surprising, since the pH value of the cement liquid phases were less acidic for the silica-modified samples (Table 1). Basically, the pH changes in brushite cements is a result of: (i) dissolution of highly soluble MCPM, which lowers the cement pH; (ii) precipitation of DCPD crystals, lowering the cement pH; (iii) dissolution of b-TCP, which consumes H+ and increases the pH [53]. Rietveld analysis showed more residual b-TCP particles and lesser DCPD within tartaric acid silica gel set cement (Table 1) [54]. Consequently, a lesser amount of b-TCP was available for the setting reaction and to increase cement pH. We attribute this to the higher pH value of the tartaric acid silica gel, as basic b-TCP is more soluble at lower pH, and to the faster setting reaction. Moreover,

tartaric acid could be complexed to b-TCP via calcium–carboxylate interaction [15]. The addition of silica gel was highly efficient in preventing cement disintegration (Fig. 2), a determinant factor for calcium phosphate cement (CPC) applications in highly blood perfused regions, such as vertebroplasty, as small volumes of intravascular CPCs resulted in right ventricle occlusion and massive pulmonary embolism [55]. It has been reported that cement microparticles might also act as a scaffold for platelet aggregation, and the coagulation cascade is enhanced by free calcium ions [55]. The cohesion of calcium phosphate cements is a transient property, so that cements with shorter FST are expected to have better cohesion [15,56]. Consequently, a decrease in cement FST could explain the enhanced cohesion of brushite cements set with carboxylic acid silica gel. The decrease in FST also affected the mechanical properties of the cements. The decrease in DTS was significant for citric acid cements, however, it was negligible for cements set with tartaric acid (Fig. 5). Thus, the addition of silica gel to brushite cements could facilitate their clinical application, particularly in highly blood perfused regions, by decreasing potential haematological risks and reducing surgery time. The in vivo experiments were aimed at evaluating the effect of silica gel on the osteoconductive properties of brushite cements. Bone conduction chambers are a useful instrument with which to evaluate vertical bone regeneration under variable conditions in both rats, goats and rabbits [57,58]. As the bone chamber is unlikely to be completely filled with bone, the effects of growth factors, the processing of bone and biomaterials within these chambers can be evaluated, as differing final amounts of bone are formed [58]. This model was successfully used in a previous study to survey bone regeneration promoted by brushite cements [29]. Recently, a review has been published on the physicochemical and biological properties of calcium phosphate-based bone substitute materials [59]. The set brushite cements evaluated in this study were shown to be biocompatible, as no signs of inflammation or infection were detected. These cements were osteotransductive, as their resorption by multinuclear/mononuclear phagocytic cells and surface pitting [28,29] were accompanied by new bone formation (Figs. 7 and 8). Additionally, the presence of Hawship’s lacunae indicated bone remodelling by osteoclasts (Fig. 8B). The addition of silica gel did not significantly influence the bone regeneration achieved by brushite cements (Fig. 9B). However, more remaining graft was present after 8 weeks implantation (Fig. 9A). This is probably due to the higher TCP content of brushite cements set with glycolic acid silica gel, as the resorption rate of bTCP is slower than that of DCPD. This property is associated with the higher solubility of brushite (pKsp = 6.59, calculated solubility

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87 mg l1) compared with b-TCP (pKsp = 28.9, calculated solubility 0.20 mg l1) [60]. Another question is what happens to the added silica? The solubility of amorphous silica (solubility at 25 °C = 2  103 mol l1 = 120 mg l1) [61] could provoke the breaking of Si–O–Si bonds and the formation of Si(OH)4, which polycondensates in the silica gel layer [38]. This layer was reported to induce the precipitation of calcium and phosphate ions into poorly crystalline hydroxyapatite [38,49]. 5. Conclusions A new approach to enhance the cohesion of brushite cements is reported, using silica gel. The combination of carboxylic acids with silica gel was highly efficient in improving cement cohesion, significantly decreasing particle release from the cement surface. Cement modification by silica gel did not affect its efficacy in promoting bone regeneration, however, more remaining graft was available after 8 weeks implantation. Acknowledgements This work was supported by the Ministry of Science and Technology (MAT2006-13646-C03-01) and the BSCH-UCM program for research groups (GR58/08). The authors also thank the CAI service (UCM) for X-ray diffraction measurements. Appendix. . Figures with essential colour discrimination. Certain figures in this article, particularly Figures 1, 3, 4, 7, 8, and 9, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio.2009.06.010. References [1] Mirtchi AA, Lemaitre J, Terao N. Calcium phosphate cements: study of the betatricalcium phosphate–monocalcium phosphate system. Biomaterials 1989;10:475–80. [2] Lemaitre J, Mirtchi A, Mortier A. Calcium phosphate cements for medical use: state of the art and perspectives of development. Silicates Industrials 1987;52:141–6. [3] Bohner M, Lemaitre J, Ring T. Hydraulic properties of tricalcium phosphatephosphoric acid–water mixtures. In: Duran P, Fernandez JF, editors. Third euro-ceramics. Castellon de la Plana, Spain: Faenza Editrice Iberica; 1993. [4] Bohner M. Propriétés physico chimiques et ostéogéniques d’un biociment hydraulique à base de phosphates de calcium. Ph.D. Thesis No. 1171, Swiss Federal Institute of Technology Lausanne (EPFL): Lausanne; 1993. [5] Driessens FCM, de Maeyer EAP, Fernández E, Boltong MG, Berger G, Verbeeck RMH, et al. Amorphous calcium phosphate cements and their transformation into calcium deficient hydroxyapatite. In: Bioceramics, vol. 9. In: Kokubo T, Nakamura T, Miyaji F, editors. Proceedings of the ninth international symposium on ceramics in Medicine. Pergamon, Otsu; 1996, p. 231–4. [6] Hassan KAR. The microelectrophoretic behaviour of sparingly soluble salts. MS Thesis. NY: SUNY, Buffalo; 1984 [7] Zawacki SJ, Koutsoukos PB, Salimi NH, Nancollas GH. The growth of calcium phosphates. In: Davis JA, Hayes KF, editors. Geochemical processes at mineral surfaces, vol. 323. Am Chem Soc Symp Series; 1996. p. 650–62. [8] Grover LM, Gbureck U, Wright AJ, Barralet JE. Cement formulations in the calcium phosphate H2O–H3PO4–H4P2O7 system. J Am Ceram Soc 2005;88:3096–103. [9] Grover LM, Gbureck U, Young AM, Wright AJ, Barralet JE. Temperature dependent setting kinetics and mechanical properties of b-TCPpyrophosphoric acid bone cement. J Mater Chem 2005;15:4955–62. [10] Bohner M, Merkle HP, Landuyt PV, Trophardy G, Lemaitre J. Effect of several additives and their admixtures on the physico-chemical properties of a calcium phosphate cement. J Mater Sci Mater Med 2000;11:111–6. [11] Bohner M, Lemaitre J, Ring TA. Effects of sulfate, pyrophosphate, and citrate ions on the physicochemical properties of cements made of b-tricalcium phosphate– phosphoric acid–water mixtures. J Am Ceram Soc 1996;79:1427–34. [12] Grover LM, Gbureck U, Wright AJ, Tremayne M, Barralet JK. Biologically mediated resorption of brushite cement in vitro. Biomaterials 2006;27:2178–85.

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