Effect of strontium and gelatin on the reactivity of α-tricalcium phosphate

Acta Biomaterialia 6 (2010) 936–942

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Effect of strontium and gelatin on the reactivity of a-tricalcium phosphate E. Boanini, S. Panzavolta, K. Rubini, M. Gandolfi, A. Bigi * Department of Chemistry ‘‘G. Ciamician”, University of Bologna, 40126 Bologna, Italy

a r t i c l e

i n f o

Article history: Received 3 August 2009 Received in revised form 30 September 2009 Accepted 5 October 2009 Available online 9 October 2009 Keywords: Calcium phosphate Gelatin Strontium Hydrolysis

a b s t r a c t The hydrolysis reaction of a-tricalcium phosphate (a-TCP) is of great interest because of its widespread use in the preparation of biomaterials for hard tissue repair. The aim of this study was to investigate how this reaction is influenced by the presence of a bioactive ion, Sr2+, and of a biopolymer, gelatin, which were previously reported to affect the setting reaction of a-TCP-based cements. Hydrolysis experiments were carried out at different Sr2+ concentrations (0, 5, 10, 20 at.%) in solutions at different gelatin concentrations (0, 0.1, 0.5, 1.0 wt.%). The results indicate that Sr2+ delays the conversion of a-TCP into calciumdeficient hydroxyapatite (CDHA). The structural and morphological modifications of CDHA obtained from solutions at increasing Sr2+ concentrations indicate that during hydrolysis strontium enters the structure of CDHA, where it partially substitutes for calcium. On the contrary, a-TCP hydrolysis rate increases on increasing gelatin concentration. Gelatin promotes conversion of a-TCP into octacalcium phosphate, and strongly interacts with the nucleating and growing crystals. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

a-Tricalcium phosphate, a-Ca3(PO4)2 (a-TCP), is widely employed for the preparation of calcium phosphate bone cements (CPCs), which can be constituted of one or more calcium phosphates. Once mixed with a liquid phase, CPCs give a mouldable paste that hardens through a low-temperature reaction. The hydrolysis reaction of a-TCP occurs through dissolution and successive precipitation of a less soluble phase. The reaction yields dicalcium phosphate dihydrate (CaHPO4
2H2O; DCPD), octacalcium phosphate (Ca8H2(PO4)6
5H2O; OCP), or calcium-deficient hydroxyapatite (CDHA), depending on the working conditions [1–3]. At physiological values of pH and temperature, the product of hydrolysis is calcium-deficient hydroxyapatite (CDHA). The rate of the setting reaction of a-TCP-based CPCs can be modulated by the physical properties of the phosphate (such as particle size), powder to liquid ratio, and addition of small amounts of organic and polymeric additives, as well as inorganic salts [2,4–6]. It follows that the information acquired through the study of the hydrolysis reaction of a-TCP in the presence of additives, such as bioactive ions and molecules, can be usefully applied to the design and optimization of CPCs [2,7]. In this work we studied the effect of Sr2+ and that of gelatin on the hydrolysis reaction of a-TCP. Strontium is present in the mineral phase of bone, especially in the regions of high metabolic turnover [8]. The growing evidence of

the beneficial effect of strontium on bone justifies the increasing interest towards strontium addition into calcium phosphate bioceramics and cements [9–11]. In vitro, it increases the number of osteoblasts and reduces the number and activity of osteoclasts [12,13]; in vivo, it inhibits bone resorption and improves bone formation [14–16]. The reactivity of a-TCP was found to decrease as a function of strontium substitution for calcium [17]. However, the introduction of even small amounts of Sr2+ in the composition of a-TCP-based cements was found to promote osteoblast proliferation and differentiation and to inhibit osteoclastogenesis and osteoclast function [11]. Gelatin is a biocompatible, biodegradable polymer, with numerous applications in the biomedical field, including tissue engineering, wound dressing, drug delivery and gene therapy [18]. It is the denaturation product of collagen, and the study of its interactions with calcium phosphates during crystallization provides useful information about the growth mechanism of biominerals [19]. Moreover, gelatin addition to a-TCP-based cements was found to accelerate the setting reaction and to improve the mechanical properties of the cements [20]. In order to clarify the influence of Sr2+ ions and of gelatin on the reactivity of a-TCP, we have studied the hydrolysis reaction in solutions at increasing concentrations of Sr2+and at different gelatin concentrations.

2. Materials and methods * Corresponding author. Tel.: +39 051 2099551; fax: +39 051 2099456. E-mail address: [email protected] (A. Bigi).

a-TCP (a-Ca3(PO4)2) was obtained by solid state reaction of a mixture of CaCO3 (Carlo Erba) and CaHPO4
2H2O (Carlo Erba) in

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.10.014

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the molar ratio of 1:2 at 1300 °C for 5 h. The solid product was ground and sieved (<40 lm) before being submitted to hydrolysis reaction. Hydrolysis of a-TCP was carried out in bidistilled water at different concentrations of SrCl2
6H2O from 0% to 20% (expressed as (Sr/(Ca + Sr))  100, where Ca are the calcium atoms in the a-TCP powder). One hundred mg of a-TCP was suspended in 50 ml solution and stored at 37 or 60 °C, for different periods of time, up to 28 days. Then the products were filtered, repeatedly washed with double distilled water and dried at 37 °C. In order to increase the reaction rate, further tests were carried out after addition of 5 mg of CaHPO4
2H2O (DCPD) to the reaction vessel [21]. Some selected samples were heat-treated at 900 °C for 15 h in order to evaluate the influence of Sr2+ on the thermal stability of the hydrolysis products. Alternatively, the influence of Sr2+ on the hydrolysis of a-TCP was studied in solutions containing gelatin (type A from pig skin, 280 Bloom, Italgelatine SpA). Aqueous solutions at gelatin concentration of 0.1, 0.5 and 1 wt.% were used at 37 °C, with or without DCPD addition. pH values of a-TCP suspensions were determined by a Hamilton glass electrode connected to a Crison GLP22 pH meter. Measurements were performed in triplicate. X-ray diffraction analysis was carried out by means of a Philips PW 1050/81 powder diffractometer equipped with a graphite monochromator in the diffracted beam. CuKa radiation was used (40 mA, 40 kV). The 2h range was from 3° to 60° at a scanning speed of 0.5° min–1. The lattice parameters were determined by least-squares refinements from the well-determined positions of the most intense reflections. Silicon was used as internal standard. Morphological investigation of the samples was performed by scanning electron microscopy (SEM) using a Philips XL-20 operating at 15 kV. The samples were sputter-coated with gold before examination. Calcium and strontium contents were determined using a Perkin Elmer Analyst 400 atomic absorption spectrophotometer (k(Ca) = 422.7 nm; k(Sr) = 460.7 nm). The samples were diluted to an appropriate volume with 10 wt.% lanthanum (La(NO3)3
6H2O) in 6 M HCl, in order to suppress interferences. Thermogravimetric analysis was carried out using a Perkin Elmer TGA-7. Heating was performed in a platinum crucible in air flow (20 cm3 min–1) at a rate of 10 °C min–1 up to 900 °C. The sample weights were in the range 5–10 mg. For transmission electron microscopy (TEM) investigations, a small amount of powder was dispersed in ethanol and submitted to ultrasonication. A drop of the calcium phosphate suspension was transferred onto holey carbon foils supported on conventional copper microgrids. A Philips CM 100 transmission electron microscope operating at 80 kV was used.

Table 1 Crystalline phases identified in the powder X-ray diffraction patterns of the samples isolated from a-TCP suspensions in aqueous solutions at different Sr2+ concentrations as a function of time and of temperature. % Sr2+

7 days

14 days

21 days

37 °C 0 5 10 20

a-TCP a-TCP a-TCP a-TCP

a-TCP/CDHA a-TCP a-TCP a-TCP

a-TCP a-TCP a-TCP

60 °C 0 5 10 20

CDHA

CDHA

a-TCP a-TCP a-TCP

a-TCP/CDHA a-TCP a-TCP

CDHA

CDHA CDHA a-TCP/CDHA a-TCP

14 days at different Sr2+ concentrations are reported in Fig. 1. The products obtained in the presence of 5% Sr2+ still exhibits diffraction peaks characteristic of a-TCP, together with those of CDHA. No conversion is detectable at higher Sr2+ concentrations. The inhibiting effect of Sr2+ on a-TCP reactivity increases on increasing Sr2+ concentration, so that just a small amount of aTCP is converted into CDHA after 21 days in 10% Sr2+ solution at 60 °C. These results are consistent with the higher solubility of Sr-substituted hydroxyapatite [22]. The increase in solubility with strontium content in Sr-substituted hydroxyapatite was interpreted as a destabilization of the crystal structure by the larger Sr2+ ion [21], which could justify the observed inhibition of crystallization.

3. Results and discussion 3.1. Hydrolysis in Sr2+ solutions The hydrolysis of a-TCP at 37 °C is quite slow, and its speed increases on increasing temperature. The crystalline phases identified by X-ray diffraction analysis in the products of a-TCP soaking at different strontium concentrations at 37 or 60 °C are reported in Table 1. When a-TCP hydrolysis is carried out in bidistilled water at 37 °C, 21 days of soaking are needed for complete conversion into CDHA. At variance, 7 days are sufficient to observe the complete phase conversion when the reaction is carried out at 60 °C. Strontium delays the hydrolysis reaction of a-TCP. The X-ray patterns of the products obtained after soaking at 60 °C for

Fig. 1. Powder X-ray diffraction patterns of the samples obtained from a-TCP hydrolysis at 60 °C for 14 days. Sr2+ concentrations: (a) 0% (CDHA); (b) 5% (a-TCP/ CDHA) and (c) 10% (a-TCP). In (b) the most intense peaks of CDHA are indicated (j).

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DCPD is more soluble than a-TCP in a wide range of pH values and it easily hydrolizes to the more stable CDHA [23]. As a consequence, DCPD is frequently used as additive in the composition of CPCs. In particular, it was found to promote the setting reaction of a-TCP-based cements [21]. In agreement, addition of a small amount of DCPD to the hydrolysis solution accelerates the a-TCP to CDHA conversion. Table 2 reports the crystalline phases identified by X-ray diffraction analysis in the products of a-TCP soaking in solutions at different strontium concentrations at 37 or 60 °C in the presence of 5% DCPD. When the hydrolysis is carried out in the absence of strontium or in the presence of a strontium concentration of 5%, complete conversion to apatite is observed after just 24 h at 60 °C. After 7 days the reaction is complete, even in the presence of a 20% strontium concentration. Fig. 2 shows the SEM images of the starting material compared with those of the products obtained after soaking at 60 °C for different periods of time. The starting material (Fig. 2a) appears constituted of dense rounded blocks with the characteristic morphology of a-TCP. It is also possible to observe the presence of DCPD crystals, which display a plate-like morphology. After 6 h soaking in the absence of Sr2+, platelet-like crystals of CDHA appear close to the a-TCP aggregates (Fig. 2b), in agreement with the results of X-ray diffraction analysis that indicate the presence of both a-TCP and CDHA. After 7 days’ soaking, only platelet-like crystals of CDHA with dimensions of few microns are present (Fig. 2c). Strontium affects the shape of the CDHA crystals, as can be observed in Fig. 2d, which reports the SEM image of HA obtained after soaking in 20% Sr2+ solution for 7 days. Most of the crystals are rod-like and their mean width is definitely less than 1 lm. 3.2. Characterization of CDHA as a function of Sr The samples obtained through hydrolysis at 60 °C in the presence of DCPD for 7 days are completely converted into CDHA. In order to further investigate the possible modifications induced by Sr, a detailed analysis was carried out on these samples. The results of the quantitative evaluation of Sr content performed through atomic absorption spectrometry are reported in Table 3. Strontium content increases on increasing Sr/(Ca + Sr) in the soaking solution, indicating that strontium is quantitatively incorporated in the solid phase. In agreement, the lattice parameters of the apatitic phases calculated from the X-ray patterns (Table 3) increase on increasing Sr concentration, as expected for a partial substitution of calcium with the larger strontium ion inside the apatite structure. The enlargement of the cell dimensions on increasing Sr concentration is consistent with that previously observed for Sr-CaHA solid solutions [24]. In agreement with the results of SEM investigation, TEM images show that CDHA obtained in the absence of Sr2+ consists mostly of

plate-like thin crystals (Fig. 3a). The morphology changes in the presence of Sr: the thickness of the crystals increases and the shape becomes rod-like, as can be observed in Fig. 3b for CDHA obtained in the presence of 20% Sr2+. Thermal stability was tested by heat treatment at 900 °C for 15 h of the hydrolysis products. The X-ray diffraction patterns of the heat-treated samples show the presence of numerous peaks characteristic of b-tricalcium phosphate (b-TCP). The sample obtained in the absence of Sr shows also a peak of low relative intensity at about 31.8° of 2h (Fig. 4a), in agreement with the presence of a small quantity of CDHA as a second phase. This peak is barely appreciable in the sample obtained from 5% Sr solution (Fig. 4b), and no longer detectable at higher concentrations (Fig. 4c), indicating that Sr promotes the thermal conversion of the apatitic phase into b-TCP. The lattice parameters of the b-TCP samples containing Sr are larger than those of the sample prepared in the absence of Sr (Table 4), in agreement with a partial substitution of Sr for Ca into the crystal structure of b-TCP [25]. 3.3. Hydrolysis in gelatin solutions The presence of gelatin promotes a-TCP hydrolysis. The crystalline phases identified by X-ray diffraction analysis in the products of a-TCP soaking in gelatin solutions at 37 °C are reported in Table 5. At the lowest gelatin concentration (0.1%), 14 days are necessary for complete phase conversion into CDHA in the absence of Sr2+. However, the hydrolysis of a-TCP occurs faster than in the absence of gelatin (Table 1), and the reaction rate increases on increasing gelatin concentration. At gelatin concentration of 0.5%, a-TCP converts into an OCP/CDHA mixture in 3 days, and OCP is obtained as a pure phase after just 3 days in 1% gelatin solution. At longer soaking times CDHA appears as a second phase. The time course of the a-TCP hydrolysis in 1% gelatin solution is illustrated in Fig. 5a–c, which reports the powder X-ray diffraction patterns of the products obtained after 1, 3 and 14 days, respectively. Under SEM investigation, OCP displays the characteristic blade crystals clustered in spherulites (Fig. 6). Gelatin reduces the inhibitory effect of Sr2+: even if no conversion at all can be appreciated in the presence of strontium in solutions containing 0.1% gelatin, 0.5% gelatin is sufficient to yield a partial conversion into OCP in solution containing 5% Sr after 7 days. After the same period and for the same Sr concentration, a-TCP is no longer detectable in the X-ray patterns of the product obtained in 1% gelatin solution, which shows the presence of OCP only. At longer soaking times CDHA appears as a second phase. After 28 days of soaking only the sample obtained in the presence of 20% Sr still contains a relevant amount of a-TCP, together with OCP as a second phase. The conversion of a-TCP into OCP during the reaction of hydrolysis can be ascribed to the presence of gelatin. Fig. 7 reports the

Table 2 Crystalline phases identified in the powder X-ray diffraction patterns of the samples isolated from a-TCP suspensions in aqueous solutions containing DCDP at different Sr2+ concentrations as a function of time and temperature. % Sr2+

2 days

3 days

37 °C + DCPD 0 5 10 20

a-TCP/CDHA a-TCP a-TCP a-TCP

CDHA

CDHA

CDHA

CDHA

a-TCP a-TCP a-TCP

a-TCP a-TCP a-TCP

a-TCP/CDHA a-TCP a-TCP

a-TCP/CDHA a-TCP a-TCP

% Sr2+

3h

6h

1 day

3 days

7 days

60 °C + DCPD 0 5 10 20

a-TCP/CDHA a-TCP a-TCP a-TCP

a-TCP/CDHA a-TCP/CDHA a-TCP a-TCP

CDHA CDHA a-TCP/CDHA a-TCP/CDHA

CDHA CDHA CDHA a-TCP/CDHA

CDHA CDHA CDHA CDHA

7 days

10 days

14 days

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Fig. 2. SEM images of (a) the starting mixture (a-TCP + DCPD); the samples obtained after soaking in bidistilled water at 60 °C for (b) 6 h (a-TCP/CDHA) and (c) 7 days (CDHA); (d) the sample obtained after soaking in 20% Sr solution at 60 °C for 7 days (CDHA).

Table 3 Lattice parameters of CDHA obtained from a-TCP hydrolysis at 60 °C in the presence of DCPD as a function of Sr2+ concentration in solution and in the solid phase. % Sr2+

a = b (Å)

c (Å)

V (Å3)

Sr content (at.%)

0 5 10 20

9.434(6) 9.447(3) 9.462(2) 9.483(2)

6.869(6) 6.892(3) 6.905(2) 6.921(1)

529(1) 532(1) 535(1) 539(1)

0 3 9 13

pH variations of the suspensions in bidistilled water and in 0.5% gelatin as a function of time. The initial pH of a-TCP suspension in aqueous solution is slightly higher than 9.0. At variance, the initial pH of the a-TCP suspensions in gelatin is about 6.0. The slightly acidic conditions promote the conversion of a-TCP into OCP, whereas at relatively high values of pH the hydrolysis is quite slow and yields CDHA, with no appreciable presence of OCP as intermediate phase. DCPD addition slightly reduces the pH values of the aqueous suspension, whereas it does not induce significant varia-

tions in the pH values of gelatin suspensions (Fig. 7). Nonetheless, DCPD provokes a modest acceleration of the hydrolysis reaction also when the experiment is performed in the presence of gelatin, as can be observed in Table 6, which confirms the catalytic role of DCPD on the hydrolysis reaction [21]. The relative content of gelatin in some selected samples was evaluated by thermogravimetric (TG) and differential thermogravimetric (TG-DTG) analysis. The TG/DTG plots of the samples hydrolyzed in the absence of gelatin show just a small weight loss between 37 and 200 °C, due to absorbed water. In the plots of the products obtained at increasing gelatin concentration, two weight losses appear in this range of temperature, and a further weight loss is present between 250 and 700 °C (Fig. 8). The two weight losses between 37 and 200 °C can be ascribed to the presence of OCP. As a matter of fact, the TG-DTG plot of OCP in this range of temperature displays two thermal processes: the first one, which is reversible, takes place at temperatures lower than 80 °C and has been ascribed to the reversible removal of one water molecule from the OCP structure; the second one, at about

Fig. 3. TEM images of the CDHA samples obtained through hydrolysis at 60 °C in the presence of DCPD for 7 days at (a) 0 and (b) 20% Sr concentration.

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Fig. 4. Powder X-ray diffraction patterns of the CDHA samples after heat treatment at 900 °C: (a) 0, (b) 5% and (c) 10% Sr. The peak at 31.8° of 2h of CDHA is indicated with (j).

Table 4 Lattice parameters of b-TCP obtained after heat treatment at 900 °C of CDHA as a function of Sr2+ concentration. % Sr2+

a = b (Å)

c (Å)

V (Å3)

0 5 10 20

10.433(4) 10.471(3) 10.485(2) 10.525(4)

37.328(5) 37.433(1) 37.494(1) 37.706(2)

3518(1) 3554(1) 3569(1) 3617(1)

130–150 °C, corresponds to the removal of two water molecules per OCP molecule [26]. The derivative peak at about 130–150 °C is clearly appreciable in the plot of the sample obtained from 1%

Fig. 5. Powder X-ray diffraction patterns of the products obtained from hydrolysis in 1% gelatin solution after (a) 1 day (a-TCP/OCP); (b) 3 days (OCP) and (c) 14 days (OCP/CDHA). The most intense peaks of CDHA (j) and of a-TCP (.) are indicated.

gelatin solution, whereas it is just a shoulder in the plots of the products obtained from solutions at smaller gelatin concentrations. The presence of OCP in these samples is so small that it was not appreciable in the relative X-ray patterns. The weight loss between 250 and 700 °C is due to gelatin decomposition and combustion. The thermogravimetric plot of gelatin usually displays three thermal processes. The first process occurs between 25 °C and about 250 °C, and it is due to loss of water; the second one between

Table 5 Crystalline phases identified in the powder X-ray diffraction patterns of the samples isolated from a-TCP suspensions in gelatin solutions at different Sr2+ concentrations as a function of time. Gelatin content was evaluated from TGA analysis of the samples at 28 days. Sr2+ %

1 day

3 days

7 days

14 days

28 days

Gelatin content (wt.%)

0.1% gel 0 5 10 20

a-TCP a-TCP a-TCP a-TCP

a-TCP a-TCP a-TCP a-TCP

a-TCP a-TCP a-TCP a-TCP

CDHA a-TCP a-TCP a-TCP

CDHA a-TCP a-TCP a-TCP

5±1 nd nd nd

0.5% gel 0 5 10 20

a-TCP a-TCP a-TCP a-TCP

OCP/CDHA a-TCP a-TCP a-TCP

OCP/CDHA

a-TCP /OCP a-TCP a-TCP

CDHA/OCP a-TCP /OCP a-TCP a-TCP

CDHA a-TCP /OCP a-TCP a-TCP

7±1 5±1 nd nd

1% gel 0 5 10 20

a-TCP /OCP a-TCP a-TCP a-TCP

OCP a-TCP a-TCP a-TCP

OCP OCP a-TCP a-TCP

OCP/CDHA OCP/CDHA a-TCP /OCP a-TCP

OCP/CDHA OCP/CDHA OCP/CDHA a-TCP /OCP

8±1 8±1 9±1 6±1

nd: not detectable.

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Table 6 Crystalline phases identified in the powder X-ray diffraction patterns of the samples isolated from a-TCP suspensions in gelatin solutions containing DCDP at different Sr2+ concentrations as a function of time. Sr2+ %

Fig. 6. SEM image of the product of a-TCP hydrolysis in 1% gelatin solution after 3 days.

Fig. 7. pH variation as a function of time of a-TCP suspension in bidistilled water ( ), in bidistilled water containing DCPD ( ) and in 0.5% gelatin solution ( ). The values measured on a-TCP suspension in 0.5% gelatin solution containing DCPD are not reported since they do not vary appreciably with respect to those recorded in the absence of DCPD ( ).

250 °C and 450 °C involves gelatin decomposition, and the third one between 450 °C and 700 °C corresponds to the combustion of the residual organic component [21]. In the TG-DTG plots of the hydrolysis products the combustion peak appears just as a shoulder of the decomposition peak. A similar behaviour was previously observed for biologically calcified samples, such as bone and calcified tendon, and for gelatin-containing calcium phosphate bone cements [27,21], and it indicates a close relationship between gelatin and the inorganic phase. The weight losses between 450 and 700 °C evaluated for some selected samples are reported in Table 5. The data indicate that gelatin content increases on increasing gelatin concentration in solution from 5 ± 1 to 8 ± 1 wt.%. Gelatin content of the samples is not appreciably affected by Sr presence during hydrolysis, whereas it seems to be related to the extent of

3 days

7 days

21 days

28 days

0.1% gel + DCPD a–TCP 0 a-TCP 5 a-TCP 10 a-TCP 20

1 day

a-TCP /OCP a-TCP a-TCP a-TCP

CDHA a-TCP a-TCP a-TCP

CDHA OCP/CDHA a-TCP a-TCP

CDHA CDHA a-TCP a-TCP

0.5% gel + DCPD a–TCP/OCP 0 a-TCP 5 a-TCP 10 a-TCP 20

OCP/CDHA a-TCP /OCP a-TCP a-TCP

OCP/CDHA a-TCP /OCP a-TCP a-TCP

CDHA OCP/CDHA a-TCP a-TCP

CDHA OCP/CDHA OCP/CDHA a-TCP

1% gel + DCPD 0 a-TCP/OCP a-TCP 5 a-TCP 10 a-TCP 20

OCP OCP a-TCP/OCP a-TCP

OCP OCP a-TCP/OCP a-TCP

OCP/CDHA OCP/CDHA OCP/CDHA a-TCP

OCP/CDHA OCP/CDHA OCP/CDHA a-TCP/OCP

Fig. 8. TG-DTG plots of the samples obtained after 28 days at increasing gelatin concentration in the absence of strontium: (a) 0.1%; (b) 0.5% and (c) 1.0%.

the hydrolysis reaction. The plots of the samples, which still consist just of a-TCP, do not show any weight loss due to gelatin, indicating that a-TCP crystals do not appreciably adsorb gelatin even after 28 days’ soaking in solution. On the other hand, the TG/DTG plots of the samples, which are partially or completely converted, do exhibit significant weight losses due to gelatin. Moreover, at the same gelatin concentration the weight losses of the samples which are just partially converted into OCP are smaller than those obtained for the samples which are completely converted into OCP and/or CDHA. On this basis, it can be suggested that gelatin is adsorbed on the inorganic crystals (OCP and/or CDHA) during the hydrolysis reaction. As a matter of fact, gelatin might provide its charged groups as nucleation sites for the precipitating crystals, accelerating their nucleation and growth. This hypothesis would lead to a close interaction between gelatin and the inorganic phase in the composite crystals, in agreement with the shape of the gelatin decomposition peak in the TG/DTG plots of the partially and completely converted samples.

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4. Conclusions Strontium and gelatin display significant and opposite influence on the hydrolysis reaction of a-TCP. Sr2+ inhibits a-TCP conversion into CDHA, although it quantitatively replaces calcium in the apatite structure, and promotes CDHA thermal conversion into b-TCP. At variance, gelatin promotes the hydrolysis reaction of a-TCP, which in this case occurs through OCP as an intermediate phase. The effect of gelatin is not just due to the slight acidic pH of its solutions, which promotes a-TCP conversion into OCP, but most likely to a close structural relationship with the growing inorganic crystals on which gelatin is quantitatively adsorbed during hydrolysis. Acknowledgements This research was carried out with the financial support of MIUR, and of the Fondazione Monte dei Paschi di Siena (Progetto ‘‘Traslazione Clinica Ingegneria Tissutale Muscolo-Scheletrica”). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 6 and 7, 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.2009.10.014. References [1] Durucan C, Brown PW. a-Tricalcium phosphate hydrolysis to hydroxyapatite at and near physiological temperature. J Mater Sci Mater Med 2000;11: 365–71. [2] Durucan C, Brown PW. Reactivity of alpha-tricalcium phosphate. J Mater Sci 2002;37:963–9. [3] Bigi A, Boanini E, Botter R, Panzavolta S, Rubini K. a-Tricalcium phosphate hydrolysis to octacalcium phosphate: effect of sodium polyacrylate. Biomaterials 2002;23:1849–54. [4] Camiré CL, Gbureck U, Hirsiger W, Bohner M. Correlating crystallinity and reactivity in an alpha-tricalcium phosphate. Biomaterials 2005;26(16):2787–94. [5] Bohner M, Malsy AK, Camiré CL, Gbureck U. Combining particle size distribution and isothermal calorimetry data to determine the reaction kinetics of a-tricalcium phosphate–water mixtures. Acta Biomater 2006;2:343–8. [6] Brunner TJ, Grass RN, Bohner M, Stark WJ. Effect of particle size, crystal phase and crystallinity on the reactivity of tricalcium phosphate cements for bone reconstruction. J Mater Chem 2007;17:4072–8. [7] Sogo Y, Ito A, Kamo M, Sakurai T, Onuma K, Ichinose N, et al. Hydrolysis and cytocompatibility of zinc-containing a-tricalcium phosphate powder. Mater Sci Eng C 2004;24:709–15.

[8] Blake GM, Zivanovic MA, McEwan AJ, Ackery DM. Sr-89 therapy: strontium kinetics in disseminated carcinoma of the prostate. Eur J Nucl Med 1986;12:447–54. [9] Leroux L, Lacout JL. Preparation of calcium strontium hydroxyapatites by a new route involving calcium phosphate cements. J Mater Res 2001;16:171–8. [10] Landi E, Tampieri A, Celotti G, Sprio S, Sandri M, Logroscino G. Sr-substituted hydroxyapatites for osteoporotic bone replacement. Acta Biomater 2007;3: 961–9. [11] Panzavolta S, Torricelli P, Sturba L, Bracci B, Giardino R, Bigi A. Setting properties and in vitro bioactivity of strontium enriched gelatin-calcium phosphate bone cements. J Biomed Mater Res A 2008;84:965–72. [12] Canalis E, Hott M, Deloffre P, Tsouderos Y, Marie PJ. The divalent strontium salt S12911 enhances bone cell replication and bone formation in vitro. Bone 1996;18:517–23. [13] Chang W, Tu C, Chen T, Komuwes L, Oda Y, Pratt S, et al. Expression and signal transduction of calcium-sensing receptors in cartilage and bone. Endocrinology 1999;40:5883–93. [14] Ammann P, Shen V, Robin B, Mauras Y, Bonjour JP, Rizzoli R. Strontium ranelate improves bone resistance by increasing bone mass and improving architecture in intact female rats. J Bone Miner Res 2004;19:2012–20. [15] Grynpas MD, Hamilton E, Cheung R, Tsouderos Y, Deloffre P, Hott M, et al. Strontium increases vertebral bone volume in rats at a low dose that does not induce detectable mineralization defect. Bone 1996;18:253–9. [16] Marie PJ, Hott M, Modrowski D, De Pollak C, Guillemain J, Deloffre P, et al. An uncoupling agent containing strontium prevents bone loss by depressing bone resorption and maintaining bone formation in estrogen-deficient rats. Bone Miner Res 1993;8:607–15. [17] Jegou Saint-Jean S, Camiré CL, Nevsten P, Hansen S, Ginebra MP. Study of the reactivity and in vitro bioactivity of Sr-substituted a-TCP cements. J Mater Sci Mater Med 2005;16:993–1001. [18] Mano JF, Silva GA, Azevedo HS, Malafaya PB, Sousa RA, Silva SS, et al. Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. J R Soc Interface 2007;4:999–1030. [19] Kniep R, Simon P. Hidden hierarchy of microfibrils within 3D-periodic fluorapatite-gelatine nanocomposites: development of complexity and form in a biomimetic system. Angew Chem Int Ed 2008;47:1405–9. [20] Bigi A, Cantelli I, Panzavolta S, Rubini K. a-Tricalcium phosphate-gelatin composite cements. J Appl Biomater Biomech 2004;2:81–7. [21] Bigi A, Panzavolta S, Rubini K. Setting mechanism of a biomimetic bone cement. Chem Mater 2004;16:3740–5. [22] Pan H, Li Z, Lam WM, Wong JC, Darvell BW, Luk KDK, et al. Solubility of strontium-substituted apatite by solid titration. Acta Biomater 2009;5: 1678–85. [23] Fernandez E, Gil FJ, Ginebra MP, Driessens FCM, Planell JA, Best SM. Calcium phosphate bone cements for clinical applications. Part II: precipitate formation during setting reactions. J Mater Sci: Mater Med 1999;10:177–83. [24] Bigi A, Boanini E, Capuccini C, Gazzano M. Strontium-substituted hydroxyapatite nanocrystals. Inorg Chim Acta 2007;360:1009–16. [25] Bigi A, Foresti E, Gandolfi M, Gazzano M, Roveri N. Isomorphous substitutions in b-tricalcium phosphate: the different effects of zinc and strontium. J Inorg Biochem 1997;66:259–65. [26] Bigi A, Cojazzi G, Gazzano M, Ripamonti A, Roveri N. Thermal conversion of octacalcium phosphate into hydroxyapatite. J Inorg Biochem 1990;40:293–9. [27] Bigi A, Cojazzi G, Panzavolta S, Ripamonti A, Roveri N, Romanello M, et al. Chemical and structural characterization of the mineral phase from cortical and trabecular bone. J Inorg Biochem 1997;68:45–51.