- Email: [email protected]
Novel biomaterials for bisphosphonate delivery
ARTICLE IN PRESS
Biomaterials 26 (2005) 2073–2080
Novel biomaterials for bisphosphonate delivery Solen Jossea, Corinne Faucheuxb, A. Soueidanb, Ga.el Grimandib, Dominique Massiotc, Bruno Alonsoc, Pascal Janviera, Samia La.ıba, Paul Piletb, Olivier Gauthierb, Guy Daculsib, # Je´rome Guicheuxb, Bruno Bujolia,*, Jean-Michel Boulerb,1 a
Faculte´ des Sciences et des Techniques, Laboratoire de Synth"ese Organique, University of Nantes, UMR CNRS 6513 and FR CNRS 2465, 2 Rue de la Houssini"ere , BP92208, 44322 Nantes Cedex 3, France b Faculte´ de Chirurgie Dentaire, Mate´riaux d’Inte´r#et Biologique, EM INSERM 99-03, BP84215, 44042 Nantes Cedex 1, France c CRMHT, UPR CNRS 4212, 1D Avenue de la Recherche Scientifique, 45071 Orle´ans Cedex 02, France Received 23 January 2004; accepted 26 May 2004 Available online 2 July 2004
Abstract One type of gem-bisphosphonate (Zoledronate) has been chemically associated onto calcium phosphate (CaP) compounds of various compositions. For that purpose, CaP powders of controlled granulometry have been suspended in aqueous Zoledronate solutions of variable concentrations. Using mainly 31P NMR spectroscopy, two different association modes have been observed, according to the nature of the CaP support and/or the initial concentration of the Zoledronate solution. b-tricalcium phosphate (bTCP) and mixtures of hydroxyapatite and b-TCP (BCPs) appear to promote Zoledronate-containing crystals formation. On the other hand, at concentrations o0.05 mol l1 CDAs (calcium deficients apatites) seem to undergo chemisorption of the drug through a surface adsorption process, due to PO3 for PO4 exchange, that is well described by Freundlich equations. At concentrations >0.05 mol l1, crystalline needles of a Zoledronate complex form onto the CDAs surface. The ability of such materials to release Zoledronate, resulting in the inhibition of osteoclastic activity, was shown using a specific in vitro bone resorption model. r 2004 Elsevier Ltd. All rights reserved. Keywords: Apatite structure; Calcium phosphate; Drug release; Osteoclast
1. Introduction ‘‘Smart’’ methods of local-delivering drugs could reduce side effects, improve efficacy of existing drugs and open the door to entire classes of new treatments. Such delivery systems are able to precisely control the timing of a drug release by adjusting the properties of the carriers. Synthetic polymers are widely used as carriers [1] since they do not cause any significant inflammation to tissues at the implantation site. In that case, the rate of drug release can be controlled by various mechanisms: diffusion out of the matrix that remains intact, simultaneous drug release and degradation of the matrix, or drug expulsion by osmotic pressure [2]. *Corresponding author. Fax: +33-251-125-402. E-mail addresses: [email protected] (B. Bujoli), [email protected] (J.-M. Bouler). 1 Also for correspondence. 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.05.019
The incorporation of biomolecules into inorganic materials, such as silica gel mainly obtained by sol–gel methods [3–10] has also been extensively studied [11]. In this context, calcium phosphate ceramics (CaPs), commonly used as implants for bone reconstruction [12–18] appear to be also good candidates as biocompatible carriers, since they can be resorbed by cells and they promote new bone formation by releasing calcium ions and phosphates. Various CaPs-based drug delivery systems have been developed, consisting either of porous ceramic sealed reservoirs filled with the drug or CaP/ drug mixtures compressed as pellets or granules [19–23]. The purpose of our work was to chemically combine CaPs with geminal bisphosphonates, usually called bisphosphonates (BPs) which are used for the treatment of post-menopausal osteoporosis [24]. Indeed, potential complementarities appear between BPs and calcium phosphate materials for bone consolidation in main osteoporosis-induced fracture sites (femur neck, distal radius, vertebral bodies). The local release of BPs could
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2074
be an interesting alternative to the oral administration, often suffering of low bioavailabilility and showing noticeable gastrointestinal disturbances [25–26]. Moreover, we aimed at determining whether this device could favor the osseous integration of metallic implants (such as hip prostheses) within osteoporotic sites. Denissen et al. [27–31] have reported the biological properties of hydroxyapatite (HA) monolithic implants incubated in 1-hydroxy-3-dimethylamino-propylidene-bisphosphonic acid (olpadronate) solutions in dental surgery, but however, no data regarding the nature of the HA–BP chemical association was reported. On the other hand, as pure hydroxyapatite dissolves too slowly, it is of little practical interest for bone substitute applications [32]. For these reasons, we have developed bioactive implants based on more soluble CaPs, such as b-TCP (b–Ca3PO4), BCPs (biphasic calcium phosphates: a mixture of HA and b-TCP) or CDAs (calcium deficient apatites, Ca10x(PO4)6x(HPO4)x(OH)2x), leading to injectable bone substitutes [33–35] showing an adapted balance between material resorption and new bone formation [36,37]. In order to maximize the expected therapeutic effect, we selected 1-hydroxy-2-(imidazol-1-yl-amino)-ethylidene-bisphosphonic acid (Zoledronate) (Fig. 1), a nitrogen-containing bisphosphonate with high antiresorptive potency, identified as the most potent inhibitor of farnesyl diphosphate synthesis and inducer of osteoclastic apoptosis [38]. In the present study, we describe the association of these CaPs with Zoledronate along with the physical and biological properties of the resulting materials.
2. Experimental 2.1. Materials and methods CDA(NH3) (Ca=P ¼ 1:56) was obtained by basic hydrolysis of 320 g of dicalcium phosphate dihydrate (DCPD), using a mixture of 440 ml of a molar solution of aqueous ammonia and 3.57 l of deionized water (4 h N
N OH
NaHO3P
PO3HNa
Fig. 1. Chemical structure of Zoledronate.
at 80 C). CDA(NaOH) (Ca=P ¼ 1:57) was prepared as previously described [36–37]. The Ca/P ratio of the samples was controlled on a Philips PW 1830 diffractometer, from the X-ray diffraction powder pattern of the corresponding calcined phases, according to the literature [39]. BCPs were prepared by calcination of CDA powders at 1050 C (4 h) in air. The desired HA/b-TCP ratios were tuned by adjusting the Ca/P ratio of the starting CDA (i.e. pure b-TCP from CDA(NaOH) with Ca=P ¼ 1:5; BCP(HA25-TCP75) from CDA(NaOH) with Ca=P ¼ 1:54; BCP(HA75-TCP25) from CDA(NaOH) with Ca=P ¼ 1:63) [32,40]. The grain size of all the CaPs used in this study was adjusted to 40–80 mm. Zoledronate was a gift from Novartis Pharma Research. The determination of the phosphorus content in solution [40] was performed using a Shimadzu UV160A UV-visible spectrometer. The scanning electron microscopy (SEM) experiments were carried out on a JEOL 6400F microscope. The 31P NMR spectra, recorded in solution, were taken on a Bruker AC 200 spectrometer, with NaH2PO4 as an external standard (capillary tube insert). The solid-state 31P NMR spectra were obtained on a Bruker DSX 300 spectrometer with 85% H3PO4 as the reference. 31P{1H}CP spectra were recorded at a MAS frequency of 10 kHz, using a variable amplitude CP pulse program, a contact time of 1 ms, recycle delays of 1 or 2 s, and a pulse RF field strength of about 50 kHz. The particle size was measured using laser granulometry on a Coulter LS230 apparatus (Miami, USA). The sodium and calcium concentrations in solution were determined using a Unicam 989 atomic absorption spectrometer (Cambridge, UK). The carbon and nitrogen content of the Zoledronate-loaded CaPs was determined by chemical analyses performed by the CNRS Analysis Laboratory (Vernaison, France). 2.2. Zoledronate loadings onto the CaPs The loading of bisphosphonate was performed using (i) CDAs obtained by hydrolysis of dihydrated dicalcium phosphate with an aqueous NaOH solution (CDA(NaOH)) or aqueous ammonia (CDA(NH3)), (ii) b-TCP, and (iii) BCPs obtained by thermal decomposition of CDAs, the Ca/P ratio of which allows tuning of the composition (HA/b-TCP ratio) of the resulting BCP. Zoledronate loading onto the CaPs (40–80 mm) was performed by reacting the solid with an aqueous solution of the drug. Typically, a suspension of the desired calcium phosphate precursor (200 mg) in 1 ml of an ultrapure water (18 M O cm) solution of zoledronic acid (the disodium form) of the desired concentration (see Table 1), was placed in an assay tube that was rotated slowly and mechanically (16 rpm) for 48 h. After centrifugation, the solid was filtered and washed 4 times
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2075
Table 1 Amount of Zoledronate incorporated onto various calcium phosphatesa Entry
CaP (surface area)
% Zoledronate adsorbed from the solution (% Zoledronate incorporated onto the CaPs, by weight)
1
TCP (o3 m2/g) BCP(HA25-TCP75) (25% HA–75% TCP) (o3 m2/g) BCP(HA75–TCP25) (75% HA–25% TCP) (o3 m2/g) CDA(NaOH) (66 m2/g) CDA(NH3) (140 m2/g)
96 (7.7) 30 (2.4)
2
3
4 5
38 (3.0) 89 (7.1) 94 (7.5)
a Conditions: 200 mg CaP and 1 ml of a 0.04 mol l1 Zoledronate solution stirred for 2 days at room temperature. For each entry, experiments were run in duplicate, using two different batches of the corresponding CaP, leading to reproducible results.
with 5 ml of ultrapure water and allowed to dry at room temperature. The amount of Zoledronate incorporated onto the calcium phosphate phases was determined by difference, after measuring its residual concentration in the supernatant using two independent methods: (i) the 31P NMR spectra of the solutions showed the resonance characteristic of the Zoledronate (multiplet centered around 14.5 ppm), along with an additional peak at high field (E1.6 ppm) corresponding to phosphate species released during the reaction (Fig. 2). The residual Zoledronate concentration was measured from a calibration curve recorded with NaH2PO4 solution as a standard introduced via an internal capillary NMR tube. (ii) The phosphorus content in the solution was determined using the method described by Ames, using ascorbic acid and ammonium molybdate to determine inorganic phosphate, the phosphorus concentration being obtained from the absorbance measured at 820 nm [40]. When a preliminary ashing of the sample was performed, the total phosphorus (phosphate and phosphonate ions) content was obtained, while no mineralization led to the amount of released phosphate only. By difference, the remaining amount of Zoledronate in the supernatant was calculated (Table 1). Consistent results were observed with the two different methods, the latter being the most sensitive. Zoledronate-loaded-CDA(NaOH) (Table 1—entry 4): %C 1.12; %N 0.46. Zoledronate-loaded-CDA(NH3) (Table 1—entry 5): %C 1.42; %N 0.67. Zoledronateloaded-b-TCP (Table 1—entry 1): %C 1.65; %N 0.75.
NaH2PO4
released phosphate
zoledronate
18
16
14
12
10
8
6
4
2
0
-2
-4
Fig. 2. Typical 31P NMR spectrum recorded on isolated supernatants after reaction of CaPs with Zoledronate. The NaH2PO4 line corresponds to the external reference.
2.3. Cellular effects of Zoledronate released by CaP materials The experimental conditions for the in vitro bone resorption model were previously described [41]. To investigate the biological activity of Zoledronate-loaded CaPs, both BCP(HA25-TCP75) (Table 1—entry 2) and CDA(NaOH) (Table 1—entry 4) powders were loaded with a Zoledronate aqueous solution or its vehicle (ultrapure water). Powder samples (100 mg) were then compacted at 130 MPa for 30 s (Specac, Kent, UK) to obtain pellets 10 mm in diameter. Pellets were steam sterilized at 121 C for 20 min and placed in 6-multiwell culture plates containing two dentin slices per well. Total rabbit bone cells prepared as previously described [41], were seeded in each well in the presence of materials. As a control, total rabbit bone cell preparation established in the same conditions were cultured in the presence of vitamin D3 (VD3; 108 mol l1) and Zoledronate (Zo; 106 mol l1) or their respective vehicles. After 4 days, dentin slices were collected, sonicated and prepared for SEM studies involving a semi-automatic image analyzer (LEICA Quantimeter 500, Japan). The resorption activity of cells was expressed as percentage of dentin slice surface resorbed. Each experiment was repeated at least once with similar results. Results are expressed as the average of three SEM determinations. Comparative studies of averages were performed using one way analysis of variance followed by a post hoc test (Fisher’s projected least significant difference) with a statistical significance at po0:05:
3. Results The presence of Zoledronate in the different calcium phosphate supports was probed by chemical analyses, showing C/N ratios close to the expected value (2.5),
ARTICLE IN PRESS 2076
S. Josse et al. / Biomaterials 26 (2005) 2073–2080
except in the case of BCP, for which the amount of bisphosphonate incorporated was too low to be detected. No significant variation of the Ca/P ratio, nor significant Ca2+ release (o10 ppm) was observed during the reaction. At the same time, the concentration of sodium in the supernatant (initially present on Zoledronate) remained high, although most of the bisphosphonate was incorporated onto the CDAs or b-TCP, with a value twice as high as the released phosphate concentration. From SEM experiments, it was obvious that two types of Zoledronate associations were present, according to the nature of the starting CaP phase. For BCPs [BCP(HA25-TCP75) and BCP(HA75-TCP25) (Table 1—entries 2–3)] and b-TCP (Table 1—entry 1), crystalline needles appeared to form on the surface (Fig. 3), while no modification of the CaP surface seemed to occur in the case of the two CDA phases (Table 1— entries 4–5). 31 P CP-MAS solid state NMR spectra of the modified BCPs and b-TCP compounds showed the peaks characteristic of the calcium phosphate matrix, along with additional downfield resonances in the expected range for Zoledronate (10–20 ppm), as a massif containing at least five different lines (identical for BCPs and bTCP), that does not corresponds to the starting Zoledronate (Fig. 4a). In addition, two-dimensional 31 P–31P{1H} solid state NMR exchange experiments were performed to study spatial proximity between phosphorus centers. The exchange process is driven here by 1H spin diffusion, since off-diagonal cross peaks were only observed if proton decoupling was not applied during the mixing time [42]. When the exchange was nearly complete (Fig. 5), all peaks assigned to the bisphosphonate were correlated with each other, and no correlation was found with the phosphate signal. Therefore, the bisphosphonate unambiguously forms a single phase separated from the calcium phosphate support. The narrow lines of the 31P-MAS NMR signal
Fig. 3. SEM image of Zoledronate-modified TCP (Table 1, entry 1— magnitude: 5000).
40
35
30
25
20
15
10
5
0
-5
-10 -15 -20
31
(a)
P chemical shift /ppm
CDA
x4
40
35
30
25
20
15
10
5
0
-5
-10 -15 -20
31
(b)
P chemical shift /ppm
31
Fig. 4. (a) P CP-MAS NMR spectrum of Zoledronate-modified TCP (Table 1, entry 1). (b) 31P CP-MAS NMR spectrum of Zoledronatemodified CDA(NH3) (Table 1, entry 5).
(line width at half-height E1.5 ppm) were consistent with the presence of Zoledronate in a crystalline form, in agreement with the SEM observations, probably as a calcium complex. In contrast, the 31P CP-MAS solid state NMR spectra recorded for the modified CDA phases showed the presence of the bisphosphonate moiety as a large peak (line width at half-height E6.5 ppm) centered at 15 ppm (Fig. 4b), thus ruling out the precipitation of a bisphosphonate complex onto the surface of the CaP as a wellordered phase. Interestingly, the CaPs behaved similarly to the case of Zoledronate when exposed to aqueous solutions of the disodium salt of methylene bisphosphonic acid (NaHO3P–CH2–PO3HNa), with slightly different bisphosphonate loading: formation of a bisphosphonatebased crystalline compound on the surface of BCPs and b-TCP (for example 19% loading for BCP(HA25– TCP75)), evidenced by SEM and 31P solid state NMR, conversely to the case of CDA(NaOH) and CDA(NH3) (for example 92% loading for CDA(NH3)). It is thus very likely that the observed reactivity of the various CaPs towards Zoledronate and methylene bisphosphonate is general to the O3P–C–PO3 moiety. The evaluation of the biological activity of Zoledronate loaded materials required the development of an in vitro bone resorption model. Various osteoclastic
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2077
VD3, a well known stimulating factor of osteoclastic resorption [46]. We showed that VD3 triggers a 4-fold increase in osteoclastic resorption (Fig. 8d) thereby demonstrating that osteoclasts present in our model exhibit a valuable response to various anabolic or catabolic bone agents.
4. Discussion
Fig. 5. (31P) 31P proton-mediated two-dimensional CP-MAS NMR exchange spectrum (10 kHz) of Zoledronate-modified BCP(HA25TCP75) (Table 1, entry 2). The mixing time was 0.5 s.
models have been previously described [43]. Among these models, previous reports indicate that unfractioned rabbit bone cell model is one of the most convenient [41,44,45]. When cultured on the surface of dentin slices, vitronectin receptor positive cells exhibit typical F-actin rings indicating that bone resorption activity was taking place. This activity is evidenced by a bone resorption assay showing numerous typical resorption pits on the surface of dentin slices. Viewed together, these data indicate that in our culture conditions, the model of unfractioned rabbit bone cells supports the formation and the activity of mature resorbing osteoclasts. Therefore, to assess the biological activity of Zoledronate-loaded materials, subsequent experiments were performed using this model. Quantitative analysis of the resorptive activity of osteoclasts indicated that Zoledronate-loaded BCP(HA25-TCP75) (Table 1—entry 2; Fig. 8a) induced an almost complete inhibition of dentin slice surface resorbed. On the other hand, Zoledronate-loaded CDA(NaOH) (Table 1—entry 4; Fig. 8b) induced a 3fold decrease in the area resorbed when compared with cells cultured in the presence of Zoledronate-free CaPs. To address specifically the biological relevance of this inhibition, we next questioned whether Zoledronate in solution could affect the osteoclastic resorption. As indicated on Fig. 8c, when cells were exposed to 106 mol l1 Zoledronate for 4 days, a 3-fold decrease in dentin slice surface resorbed was evidenced, when compared to untreated cultures. As a control, we tested
In the case of TCP, the concentration of released phosphate increased in direct proportion to the amount of Zoledronate charged. High incorporation ratios (>90%) were observed for the bisphosphonate along with a constant concentration of residual Zoledronate in the supernatant (ca. 120 ppm phosphorus (i.e. B2.103 mol l1 Zoledronate)), that could mirror the solubility of the crystalline Zoledronate derivative forming on the surface of TCP. This assumption was confirmed by suspending various amounts of Zoledronate-loaded b-TCP in water (60, 120 and 200 mg in 1 ml water) for periods ranging from 8 to 72 h. Identical concentrations of released Zoledronate were invariably measured (ca. 120 ppm phosphorus). These data suggest that the chemical process involved in the preparation of the modified b-TCP samples could be the result of a partial dissolution of b-TCP occurring at the liquid– solid interface with concomitant precipitation of a crystalline Zoledronate derivative. The same end-result was achieved with BCPs, although a lower Zoledronate loading (ca. 35%) was observed, presumably because bTCP is more soluble than BCPs [32,47]. Treating CDAs with increasing quantities of Zoledronate (CZoledronate=0.01 to 0.05 mol l1) resulted in a rise of the released phosphate concentration along with a quantitative uptake of the bisphosphonate until a plateau was reached, corresponding to a ca. 10% w/w Zoledronate loading, similar for both of CDAs. The most probable situation is that chemisorption of bisphosphonate takes place on the surface of the calcium deficient hydroxyapatite phases, via polar covalent interactions with calcium atoms and displacement of PO3 species, the plateau noticed for the Zoledronate 4 uptake corresponding to the saturation of the surface. However, it is worth noting that the BET surface area of CDA(NH3) is circa twice as high as the one measured for CDA(NaOH), thereby indicating that the chemical reactivity of the two CDAs is noticeably different. Moreover, we have shown that the Freundlich equation [48] (Qe ¼ Kf Ce1=n ) was well adapted to describe the adsorption of Zoledronate onto CDAs (see Fig. 6 for CDA(NH3)—Table 2). For CZoledronate > 0:05 mol l1, we observed a rise of the amount of Zoledronate incorporated. Indeed a drastic change in the association mode took place, with the formation of a crystalline material onto the surface of CDAs, as evidenced by
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2078
Qe = (Kf×Ce)1/n
Qe [mol.g-1]
0.2 0.15 0.1 0.05 0 0
2
6
4
8
10
Ce [mol.l-1]
Fig. 6. Isothermal Freundlich curve for Zoledronate adsorption onto CDA(NH3) at 298 K. At equilibrium: Qe: quantity of Zoledronate adsorbed per gram of CDA and Ce: remaining concentration of Zoledronate in solution, measured by a UV-visible-based method (see Materials and Methods). Logarithmic linearization lead to a r2 ¼ 0:98:
Table 2 Experimental data for Zoledronate adsorption onto CDAs at 298 K, described by isothermal Freundlich curves CaP CDA(NaOH) CDA(NH3)
Surface area (m2/g) 66 140
n
Kf 4
6.9 10 3.8 104
3.9 7.3
SEM (Fig. 7a). Concurrently, the shape of the 31P CPMAS NMR signal assigned to the incorporated Zoledronate evolved from a featureless broad line to a gradually resolved signal (Fig. 7b), consistent with the gradual formation of a crystalline Zoledronate complex onto the surface of CDAs. Repeated washings of these materials until the Zoledronate release was under the detection limit, resulted in a disappearance of the crystalline Zoledronate complex confirmed by SEM and 31P CP-MAS NMR, that showed a spectrum similar to that observed for low Zoledronate-loaded CDAs (see Fig. 4). This clearly indicates that in such conditions, the two Zoledronate association modes took place. With regard to the amount of Zoledronate incorporated onto CDA and BCP, the data obtained from the biological evaluation indicate that both materials exhibit differential patterns of Zoledronate release likely involved in the distinct cellular response. Since the inhibition of osteoclastic resorption produced by Zoledronate in solution is similar to that obtained with Zoledronate-loaded CDA, it is reasonable to conclude that Zoledronate-loaded CDA is likely to release approximately 106 mol l1 Zoledronate within 4 days. On the contrary, the bisphosphonate release with the Zoledronate-loaded BCP is controlled by the solubility constant of the crystalline Zoledronate derivative formed onto the surface of the support (amount of released Zoledronate in water: B120 ppm phosphorusE2.103 mol l1 Zoledronate). A large amount of Zoledronate is thus released, resulting in a cytotoxic effect, since this bisphosphonate is known to reduce cell
Fig. 7. (a) SEM image of Zoledronate-modified CDA(NaOH), with a bisphosphonate loading of 13% by weight (magnitude: 12 000). (b) 31P CP-MAS NMR spectrum of Zoledronate-modified CDA(NaOH), with a bisphosphonate loading of 13% by weight.
viability at concentrations in the range of 104 mol l1 [49,50].
5. Conclusion Osteoarticular disorders associated with increased bone resorption (as observed in osteoporosis, Paget’s disease and osteolytic tumors) often lead to pathological fractures, deformity or bone pain [51]. They are widely treated by the massive systemic administration of bisphosphonates, one of the most potent inhibitors of osteoclast activity [24]. In this context, our preliminary in vitro results suggest that this type of composite material could be suitable for practical application as a local drug delivery system for Zoledronate with release kinetics compatible with the inhibition of bone resorption. Further experiments regarding the association of various BPs to a series of calcium phosphates along with the release profiles of the resulting biomaterials are under intense investigation in our laboratory. This class of materials could be of interest as an alternative to oral treatments in which high doses of BPs are administrated daily or weekly [25], with more or less important gastrointestinal undesired effects. The evaluation of
ARTICLE IN PRESS
BCP
dentin slice surface resorbed (%)
(a)
(c)
* BCP zoledronate
2 1.5 1
*
0.5 0 VEH
Zo
dentin slice surface resorbed (%)
4 3 2 1 0
* CDA
(d)
2079
2.5 2 1.5 1 0.5 0
(b) dentin slice surface resorbed (%)
dentin slice surface resorbed (%)
S. Josse et al. / Biomaterials 26 (2005) 2073–2080
6 5 4 3 2 1 0
CDA zoledronate *
veh
VD3
Fig. 8. Evaluation of the biological activity of Zoledronate–loaded calcium phosphate materials. A total rabbit bone cell preparation was established on dentin slices as described in materials and methods. Cell preparation was then cultured for 4 days either in the presence of BCP or Zoledronateloaded BCP (a), CDA or Zoledronate-loaded CDA (b), 106 mol l1 Zoledronate or its vehicle (VEH) (c) or 108 mol l1 vitamin D3 or its vehicle (d). Dentin slices were next prepared for scanning electron microscopy observation linked to a semi-automatic image analyzer. Resorption activity of osteoclasts is expressed as the percentage of dentin slice surface resorbed. Results are the mean7SEM of three independent experiments. : po0:05 as compared to the respective controls.
these CaP–BP hybrids using in vivo bone resorption models are underway.
Acknowledgements This work was partially supported by the French Ministry of Research (ACI ‘‘Technologies pour la Sante´’’), the C.N.R.S. (Programme ‘‘Mate´riaux Nouveaux—Fonctionnalite´s Nouvelles’’) and the ‘‘Fondation de l’avenir pour la recherche me´dicale applique´e’’. Partial support from the Re´gion Pays de la Loire (CPER ‘‘Biomate´riaux—S3’’) is also acknowledged. We thank Novartis Pharma Research (Basel) for a generous gift of Zoledronate and J.R. Green (Novartis Pharma Research) for fruitful discussion.
References [1] Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. Polymeric systems for controlled drug release. Chem Rev 1999;99:3181–98. [2] Ranade VV. Drug delivery systems 5A. Oral drug delivery. J Clin Pharmacol 1991;31:2–16. [3] Sieminska L, Zerda TW. Diffusion of steroids from sol–gel glass. J Phys Chem 1996;100:4591–7. [4] Sieminska L, Ferguson M, Zerda TW, Couch EJ. Diffusion of steroids in porous sol–gel glass: application in slow drug delivery. Sol–gel Sci Technol 1997;8:1105–9. . [5] Bottcher H, Slowik P, Suss W. Sol–gel carrier systems for controlled drug delivery. J Sol–gel Sci Technol 1998;13:277–81. [6] Falaize S, Radin S, Ducheyne P. In vitro behavior of silica-based xerogels intended as controlled release carriers. J Am Ceram Soc 1999;82:969–76. [7] Santos EM, Radin S, Ducheyne P. Sol–gel derived carrier for the controlled release of proteins. Biomaterials 1999;20:1695–700.
[8] Ahola M, Kortesuo P, Kangasniemi I, Kiesvaara J, Yli-Urpo A. Silica xerogel carrier material for controlled release of toremifene citrate. Int J Pharm 2000;195:219–27. [9] Ahola M, S.ailynoja ES, Raitavuo MH, Salonen JI, Yli-Urpo A. In vitro release of heparin from silica xerogels. Biomaterials 2001;22:2163–70. [10] Otsuka M, Tokumitsu K, Matsuda Y. Solid dosage form preparations from oily medicines and their drug release. Effect of degree of surface-modification of silica gel on the drug release from phytonadione-loaded silica gels. J Control Release 2000;67:369–84. [11] Khan AI, Lei L, Norquist AJ, O’Hare D. Intercalation and controlled release of pharmaceutically active compounds from a layered double hydroxide. Chem Commun 2001;2342–43. [12] Passuti N, Daculsi G, Rogez JM, Martin S, Bainvel JV. Macroporous calcium phosphate ceramic performance in human spine fusion. Clin Orthop 1989;248:169–76. [13] Daculsi G, Bagot d’Arc M, Corlieu P, Gersdorff M. Macroporous biphasic calcium phosphate efficiency in mastoid cavity obliteration: experimental and clinical findings. Ann Otol Rhinol Laryngol 1992;101:669–74. [14] Piattelli A, Scarano A, Mangano C. Clinical and histologic aspects of biphasic calcium phosphate ceramic (BCP) used in connection with implant placement. Biomaterials 1996;17: 1767–70. [15] Ransford AO, Morley T, Edgar MA, Webb P, Passuti N, Chopin D, Morin C, Michel F, Garin C, Pries D. Synthetic porous ceramic compared with autograft in scoliosis surgery: a prospective, randomized study of 341 patients. J Bone Joint Surg Br 1998;80:13–8. [16] Cavagna R, Daculsi G, Bouler JM. Macroporous calcium phosphate ceramic: a prospective study of 106 cases in lumbar spinal fusion. J Long Term Eff Med Implants 1999;9:403–12. [17] Delecrin J, Takahashi S, Gouin F, Passuti N. A synthetic porous ceramic as a bone graft substitute in the surgical management of scoliosis: a prospective, randomized study. Spine 2000;25(5):563–9. [18] Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed 2002;41:3130–46. [19] Guicheux J, Gauthier O, Aguado E, Heymann D, Pilet P, Couillaud S, Faivre A, Daculsi G. Growth hormoneloaded macroporous calcium phosphate ceramic: in vitro
ARTICLE IN PRESS 2080
[20]
[21]
[22]
[23]
[24] [25] [26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35]
S. Josse et al. / Biomaterials 26 (2005) 2073–2080 biopharmaceutical characterization and preliminary in vivo study. J Biomed Mater Res 1998;40(4):560–6. Obadia L, Amador G, Daculsi G, Bouler JM. Calcium-deficient apatite: influence of granule size and consolidation mode on release and in vitro activity of vancomycin. Biomaterials 2003;24:1265–70. Slosarczyk A, Szymura-Oleksiak J, Mycek B. The kinetics of pentoxifylline release from drug-loaded hydroxyapatite implants. Biomaterials 2000;21:1215–21. Netz DJA, Sepulveda P, Pandolfelli VC, Spadaro ACC, Alencastre JB, Bentley MVLB, Marchetti JM. Potential use of gelcasting hydroxyapatite porous ceramic as an implantable drug delivery system. Int J Pharm 2001;213:117–25. Burgos AE, Belchior JC, Sinisterra RD. Controlled release of rhodium (II) carboxylates and their association complexes with cylcodextrins from hydroxyapatite matrix. Biomaterials 2002;23:2519–26. Fleich H. In: Bisphosphonates in bone disease, from the laboratory to the patients. San Francisco: Academic Press; 2000. Bisphosphonates for osteoporosis. Drug and Therapeutics Bulletin 2001;39:68–72. Doggrell SA. Zoledronate once-yearly increases bone mineral density—implications for osteoporosis. Expert Opin Pharmacother 2002;3:1007–9. . Denissen H, Van Beek E, Lowik C, Papapoulos S, Van der Hooff A. Ceramic hydroxyapatite implants for the release of bisphosphonate. Bone Min 1994;25:123–34. Denissen H, Van Beek E, Van den Bos T, De Blieck J, Klein C, Van den Hooff A. Degradable bisphosphonate-alkaline phosphatase-complexed hydroxyapatite implants in vitro. J Bone Miner Res 1997;12:290–7. Denissen H, Van Beek E, Martinetti R, Klein C, Van der Zee E, Ravaglioli A. Net-shaped hydroxyapatite implants for release of agents modulating periodontal-like tissues. J Periodont Res 1997;32:40–6. Denissen H, Martinetti R, Van Lingen A, Van den Hooff A. Normal osteoconduction and repair in and around submerged highly bisphosphonate-complexed hydroxyapatite implants in rat tibiae. J Periodont 2000;71:272–8. Denissen H, Montanari C, Martinetti R, Van Lingen A, Van den Hooff A. Alveolar bone response to submerged bisphosphonatecomplexed hydroxyapatite implants. J Periodont 2000;71:279–86. LeGeros RZ. Calcium phosphates in oral biology. In: Myers HM, editor. Monographs in oral science. Basel: Karger; 1991. Weiss P, Gauthier O, Bouler JM, Grimandi G, Daculsi G. Injectable bone substitute using a hydrophilic polymer. Bone 1999;25(Suppl. 2):67S–70S. Daculsi G, Weiss P, Bouler JM, Gauthier O, Millot F, Aguado E. Biphasic calcium phosphate/hydrosoluble polymer composites: a new concept for bone and dental substitution biomaterials. Bone 1999;25(Suppl. 2):59S–61S. Gauthier O, Boix D, Grimandi G, Aguado E, Bouler JM, Weiss P, Daculsi G. A new injectable calcium phosphate biomaterial for immediate bone filling of extraction sockets: a preliminary study in dogs. J Periodontol 1999;70:375–83.
[36] Gauthier O, Bouler JM, Weiss P, Bosco J, Daculsi G, Aguado E. Kinetic study of bone ingrowth and ceramic resorption associated with the implantation of different injectable calcium-phosphate bone substitutes. J Biomed Mater Res 1999;47:28–35. [37] Bourgeois B, Laboux O, Gauthier O, Obadia L, Aguado E, Daculsi G, Bouler JM. Calcium-deficient apatite: a first in vivo study concerning bone ingrowth. J Biomed Mater Res 2003;65A(3):402–8. [38] Wilder L, Jaeggi KA, Glatt M, Muller . K, Bachmann R, Bisping M, Born A, Cortesi R, Guiglia G, Jeker H, Klein R, Ramseier U, Schmid J, Schreiber G, Seltenmeyer Y, Green JR. Highly potent geminal bisphosphonates. From pamidronate (Aredia) to zoledronic acid (Zometa). J Med Chem 2002;45:3721–38. [39] Ishikawa K, Ducheyne P, Radin S. Determination of the Ca/P ratio in calcium deficient hydroxyapatite using X-ray diffraction analysis. J Mater Sci Mater Med 1993;4:165–8. [40] Ames BN. Assay of inorganic phosphate, total phosphate and phosphatases. In: Colowick SP, Kaplan NO, editors. Methods in enzymology, Vol. 8. Orlando: Academic Press; 1966. p. 115–8. [41] Guicheux J, Heymann D, Rousselle AV, Gouin F, Pilet P, Yamada S, Daculsi G. Growth hormone stimulatory effects on osteoclastic resorption are partly mediated by insulin-like growth factor I: an in vitro study. Bone 1998;22:25–31. [42] Schmidt-Rohr KW, Spiess H. In: Harcourt Brace and Company, editors. Multidimensional solid-state NMR and polymers. New York: Academic Press, 1994. [43] Heymann D, Guicheux J, Gouin F, Passuti N, Daculsi G. Cytokines, growth factors and osteoclasts. Cytokine 1998;10(3):155–68. [44] David JP, Neff L, Chen Y, Rincon M, Horne WC, Baron R. A new method to isolate large numbers of rabbit osteoclasts and osteoclast-like cells: application to the characterization of serum response element binding proteins during osteoclast differentiation. J Bone Miner Res 1998;13(11):1730–8. [45] Tezuka K, Sato T, Kamioka H, Nijweide PJ, Tanaka K, Matsuo T, Ohta M, Kurihara N, Hakeda Y, Kumegawa M. Identification of osteopontin in isolated rabbit osteoclasts. Biochem Biophys Res Commun 1992;186(2):911–7. [46] Roodman GD. Advances in bone biology: the osteoclast. Endocr Rev 1996;17(4):308–32. [47] Bouler JM, LeGeros RZ, Daculsi G. Biphasic calcium phosphates: influence of three synthesis parameters on the HA/betaTCP ratio. J Biomed Mater Res 2000;51:680–4. [48] Freundlich H. In: Hatfield HS, editor. Colloid and capillary chemistry. London: Methuen, 1926. [49] Li EC, Davis LE. Zoledronic acid: a new parenteral bisphosphonate. Clin Therapeut 2003;25:2669–708. [50] Reinholz GG, Getz B, Pederson L, Sanders ES, Subramaniam M, Ingle JN, Spelsberg TC. Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts. Cancer Res 2000;60:6001–7. [51] Consensus Development Conference, ‘Diagnosis, prophylaxis and treatment of osteoporosis’, conference report. Am J Med 1993;94(6):646–50.
Biomaterials 26 (2005) 2073–2080
Novel biomaterials for bisphosphonate delivery Solen Jossea, Corinne Faucheuxb, A. Soueidanb, Ga.el Grimandib, Dominique Massiotc, Bruno Alonsoc, Pascal Janviera, Samia La.ıba, Paul Piletb, Olivier Gauthierb, Guy Daculsib, # Je´rome Guicheuxb, Bruno Bujolia,*, Jean-Michel Boulerb,1 a
Faculte´ des Sciences et des Techniques, Laboratoire de Synth"ese Organique, University of Nantes, UMR CNRS 6513 and FR CNRS 2465, 2 Rue de la Houssini"ere , BP92208, 44322 Nantes Cedex 3, France b Faculte´ de Chirurgie Dentaire, Mate´riaux d’Inte´r#et Biologique, EM INSERM 99-03, BP84215, 44042 Nantes Cedex 1, France c CRMHT, UPR CNRS 4212, 1D Avenue de la Recherche Scientifique, 45071 Orle´ans Cedex 02, France Received 23 January 2004; accepted 26 May 2004 Available online 2 July 2004
Abstract One type of gem-bisphosphonate (Zoledronate) has been chemically associated onto calcium phosphate (CaP) compounds of various compositions. For that purpose, CaP powders of controlled granulometry have been suspended in aqueous Zoledronate solutions of variable concentrations. Using mainly 31P NMR spectroscopy, two different association modes have been observed, according to the nature of the CaP support and/or the initial concentration of the Zoledronate solution. b-tricalcium phosphate (bTCP) and mixtures of hydroxyapatite and b-TCP (BCPs) appear to promote Zoledronate-containing crystals formation. On the other hand, at concentrations o0.05 mol l1 CDAs (calcium deficients apatites) seem to undergo chemisorption of the drug through a surface adsorption process, due to PO3 for PO4 exchange, that is well described by Freundlich equations. At concentrations >0.05 mol l1, crystalline needles of a Zoledronate complex form onto the CDAs surface. The ability of such materials to release Zoledronate, resulting in the inhibition of osteoclastic activity, was shown using a specific in vitro bone resorption model. r 2004 Elsevier Ltd. All rights reserved. Keywords: Apatite structure; Calcium phosphate; Drug release; Osteoclast
1. Introduction ‘‘Smart’’ methods of local-delivering drugs could reduce side effects, improve efficacy of existing drugs and open the door to entire classes of new treatments. Such delivery systems are able to precisely control the timing of a drug release by adjusting the properties of the carriers. Synthetic polymers are widely used as carriers [1] since they do not cause any significant inflammation to tissues at the implantation site. In that case, the rate of drug release can be controlled by various mechanisms: diffusion out of the matrix that remains intact, simultaneous drug release and degradation of the matrix, or drug expulsion by osmotic pressure [2]. *Corresponding author. Fax: +33-251-125-402. E-mail addresses: [email protected] (B. Bujoli), [email protected] (J.-M. Bouler). 1 Also for correspondence. 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.05.019
The incorporation of biomolecules into inorganic materials, such as silica gel mainly obtained by sol–gel methods [3–10] has also been extensively studied [11]. In this context, calcium phosphate ceramics (CaPs), commonly used as implants for bone reconstruction [12–18] appear to be also good candidates as biocompatible carriers, since they can be resorbed by cells and they promote new bone formation by releasing calcium ions and phosphates. Various CaPs-based drug delivery systems have been developed, consisting either of porous ceramic sealed reservoirs filled with the drug or CaP/ drug mixtures compressed as pellets or granules [19–23]. The purpose of our work was to chemically combine CaPs with geminal bisphosphonates, usually called bisphosphonates (BPs) which are used for the treatment of post-menopausal osteoporosis [24]. Indeed, potential complementarities appear between BPs and calcium phosphate materials for bone consolidation in main osteoporosis-induced fracture sites (femur neck, distal radius, vertebral bodies). The local release of BPs could
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2074
be an interesting alternative to the oral administration, often suffering of low bioavailabilility and showing noticeable gastrointestinal disturbances [25–26]. Moreover, we aimed at determining whether this device could favor the osseous integration of metallic implants (such as hip prostheses) within osteoporotic sites. Denissen et al. [27–31] have reported the biological properties of hydroxyapatite (HA) monolithic implants incubated in 1-hydroxy-3-dimethylamino-propylidene-bisphosphonic acid (olpadronate) solutions in dental surgery, but however, no data regarding the nature of the HA–BP chemical association was reported. On the other hand, as pure hydroxyapatite dissolves too slowly, it is of little practical interest for bone substitute applications [32]. For these reasons, we have developed bioactive implants based on more soluble CaPs, such as b-TCP (b–Ca3PO4), BCPs (biphasic calcium phosphates: a mixture of HA and b-TCP) or CDAs (calcium deficient apatites, Ca10x(PO4)6x(HPO4)x(OH)2x), leading to injectable bone substitutes [33–35] showing an adapted balance between material resorption and new bone formation [36,37]. In order to maximize the expected therapeutic effect, we selected 1-hydroxy-2-(imidazol-1-yl-amino)-ethylidene-bisphosphonic acid (Zoledronate) (Fig. 1), a nitrogen-containing bisphosphonate with high antiresorptive potency, identified as the most potent inhibitor of farnesyl diphosphate synthesis and inducer of osteoclastic apoptosis [38]. In the present study, we describe the association of these CaPs with Zoledronate along with the physical and biological properties of the resulting materials.
2. Experimental 2.1. Materials and methods CDA(NH3) (Ca=P ¼ 1:56) was obtained by basic hydrolysis of 320 g of dicalcium phosphate dihydrate (DCPD), using a mixture of 440 ml of a molar solution of aqueous ammonia and 3.57 l of deionized water (4 h N
N OH
NaHO3P
PO3HNa
Fig. 1. Chemical structure of Zoledronate.
at 80 C). CDA(NaOH) (Ca=P ¼ 1:57) was prepared as previously described [36–37]. The Ca/P ratio of the samples was controlled on a Philips PW 1830 diffractometer, from the X-ray diffraction powder pattern of the corresponding calcined phases, according to the literature [39]. BCPs were prepared by calcination of CDA powders at 1050 C (4 h) in air. The desired HA/b-TCP ratios were tuned by adjusting the Ca/P ratio of the starting CDA (i.e. pure b-TCP from CDA(NaOH) with Ca=P ¼ 1:5; BCP(HA25-TCP75) from CDA(NaOH) with Ca=P ¼ 1:54; BCP(HA75-TCP25) from CDA(NaOH) with Ca=P ¼ 1:63) [32,40]. The grain size of all the CaPs used in this study was adjusted to 40–80 mm. Zoledronate was a gift from Novartis Pharma Research. The determination of the phosphorus content in solution [40] was performed using a Shimadzu UV160A UV-visible spectrometer. The scanning electron microscopy (SEM) experiments were carried out on a JEOL 6400F microscope. The 31P NMR spectra, recorded in solution, were taken on a Bruker AC 200 spectrometer, with NaH2PO4 as an external standard (capillary tube insert). The solid-state 31P NMR spectra were obtained on a Bruker DSX 300 spectrometer with 85% H3PO4 as the reference. 31P{1H}CP spectra were recorded at a MAS frequency of 10 kHz, using a variable amplitude CP pulse program, a contact time of 1 ms, recycle delays of 1 or 2 s, and a pulse RF field strength of about 50 kHz. The particle size was measured using laser granulometry on a Coulter LS230 apparatus (Miami, USA). The sodium and calcium concentrations in solution were determined using a Unicam 989 atomic absorption spectrometer (Cambridge, UK). The carbon and nitrogen content of the Zoledronate-loaded CaPs was determined by chemical analyses performed by the CNRS Analysis Laboratory (Vernaison, France). 2.2. Zoledronate loadings onto the CaPs The loading of bisphosphonate was performed using (i) CDAs obtained by hydrolysis of dihydrated dicalcium phosphate with an aqueous NaOH solution (CDA(NaOH)) or aqueous ammonia (CDA(NH3)), (ii) b-TCP, and (iii) BCPs obtained by thermal decomposition of CDAs, the Ca/P ratio of which allows tuning of the composition (HA/b-TCP ratio) of the resulting BCP. Zoledronate loading onto the CaPs (40–80 mm) was performed by reacting the solid with an aqueous solution of the drug. Typically, a suspension of the desired calcium phosphate precursor (200 mg) in 1 ml of an ultrapure water (18 M O cm) solution of zoledronic acid (the disodium form) of the desired concentration (see Table 1), was placed in an assay tube that was rotated slowly and mechanically (16 rpm) for 48 h. After centrifugation, the solid was filtered and washed 4 times
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2075
Table 1 Amount of Zoledronate incorporated onto various calcium phosphatesa Entry
CaP (surface area)
% Zoledronate adsorbed from the solution (% Zoledronate incorporated onto the CaPs, by weight)
1
TCP (o3 m2/g) BCP(HA25-TCP75) (25% HA–75% TCP) (o3 m2/g) BCP(HA75–TCP25) (75% HA–25% TCP) (o3 m2/g) CDA(NaOH) (66 m2/g) CDA(NH3) (140 m2/g)
96 (7.7) 30 (2.4)
2
3
4 5
38 (3.0) 89 (7.1) 94 (7.5)
a Conditions: 200 mg CaP and 1 ml of a 0.04 mol l1 Zoledronate solution stirred for 2 days at room temperature. For each entry, experiments were run in duplicate, using two different batches of the corresponding CaP, leading to reproducible results.
with 5 ml of ultrapure water and allowed to dry at room temperature. The amount of Zoledronate incorporated onto the calcium phosphate phases was determined by difference, after measuring its residual concentration in the supernatant using two independent methods: (i) the 31P NMR spectra of the solutions showed the resonance characteristic of the Zoledronate (multiplet centered around 14.5 ppm), along with an additional peak at high field (E1.6 ppm) corresponding to phosphate species released during the reaction (Fig. 2). The residual Zoledronate concentration was measured from a calibration curve recorded with NaH2PO4 solution as a standard introduced via an internal capillary NMR tube. (ii) The phosphorus content in the solution was determined using the method described by Ames, using ascorbic acid and ammonium molybdate to determine inorganic phosphate, the phosphorus concentration being obtained from the absorbance measured at 820 nm [40]. When a preliminary ashing of the sample was performed, the total phosphorus (phosphate and phosphonate ions) content was obtained, while no mineralization led to the amount of released phosphate only. By difference, the remaining amount of Zoledronate in the supernatant was calculated (Table 1). Consistent results were observed with the two different methods, the latter being the most sensitive. Zoledronate-loaded-CDA(NaOH) (Table 1—entry 4): %C 1.12; %N 0.46. Zoledronate-loaded-CDA(NH3) (Table 1—entry 5): %C 1.42; %N 0.67. Zoledronateloaded-b-TCP (Table 1—entry 1): %C 1.65; %N 0.75.
NaH2PO4
released phosphate
zoledronate
18
16
14
12
10
8
6
4
2
0
-2
-4
Fig. 2. Typical 31P NMR spectrum recorded on isolated supernatants after reaction of CaPs with Zoledronate. The NaH2PO4 line corresponds to the external reference.
2.3. Cellular effects of Zoledronate released by CaP materials The experimental conditions for the in vitro bone resorption model were previously described [41]. To investigate the biological activity of Zoledronate-loaded CaPs, both BCP(HA25-TCP75) (Table 1—entry 2) and CDA(NaOH) (Table 1—entry 4) powders were loaded with a Zoledronate aqueous solution or its vehicle (ultrapure water). Powder samples (100 mg) were then compacted at 130 MPa for 30 s (Specac, Kent, UK) to obtain pellets 10 mm in diameter. Pellets were steam sterilized at 121 C for 20 min and placed in 6-multiwell culture plates containing two dentin slices per well. Total rabbit bone cells prepared as previously described [41], were seeded in each well in the presence of materials. As a control, total rabbit bone cell preparation established in the same conditions were cultured in the presence of vitamin D3 (VD3; 108 mol l1) and Zoledronate (Zo; 106 mol l1) or their respective vehicles. After 4 days, dentin slices were collected, sonicated and prepared for SEM studies involving a semi-automatic image analyzer (LEICA Quantimeter 500, Japan). The resorption activity of cells was expressed as percentage of dentin slice surface resorbed. Each experiment was repeated at least once with similar results. Results are expressed as the average of three SEM determinations. Comparative studies of averages were performed using one way analysis of variance followed by a post hoc test (Fisher’s projected least significant difference) with a statistical significance at po0:05:
3. Results The presence of Zoledronate in the different calcium phosphate supports was probed by chemical analyses, showing C/N ratios close to the expected value (2.5),
ARTICLE IN PRESS 2076
S. Josse et al. / Biomaterials 26 (2005) 2073–2080
except in the case of BCP, for which the amount of bisphosphonate incorporated was too low to be detected. No significant variation of the Ca/P ratio, nor significant Ca2+ release (o10 ppm) was observed during the reaction. At the same time, the concentration of sodium in the supernatant (initially present on Zoledronate) remained high, although most of the bisphosphonate was incorporated onto the CDAs or b-TCP, with a value twice as high as the released phosphate concentration. From SEM experiments, it was obvious that two types of Zoledronate associations were present, according to the nature of the starting CaP phase. For BCPs [BCP(HA25-TCP75) and BCP(HA75-TCP25) (Table 1—entries 2–3)] and b-TCP (Table 1—entry 1), crystalline needles appeared to form on the surface (Fig. 3), while no modification of the CaP surface seemed to occur in the case of the two CDA phases (Table 1— entries 4–5). 31 P CP-MAS solid state NMR spectra of the modified BCPs and b-TCP compounds showed the peaks characteristic of the calcium phosphate matrix, along with additional downfield resonances in the expected range for Zoledronate (10–20 ppm), as a massif containing at least five different lines (identical for BCPs and bTCP), that does not corresponds to the starting Zoledronate (Fig. 4a). In addition, two-dimensional 31 P–31P{1H} solid state NMR exchange experiments were performed to study spatial proximity between phosphorus centers. The exchange process is driven here by 1H spin diffusion, since off-diagonal cross peaks were only observed if proton decoupling was not applied during the mixing time [42]. When the exchange was nearly complete (Fig. 5), all peaks assigned to the bisphosphonate were correlated with each other, and no correlation was found with the phosphate signal. Therefore, the bisphosphonate unambiguously forms a single phase separated from the calcium phosphate support. The narrow lines of the 31P-MAS NMR signal
Fig. 3. SEM image of Zoledronate-modified TCP (Table 1, entry 1— magnitude: 5000).
40
35
30
25
20
15
10
5
0
-5
-10 -15 -20
31
(a)
P chemical shift /ppm
CDA
x4
40
35
30
25
20
15
10
5
0
-5
-10 -15 -20
31
(b)
P chemical shift /ppm
31
Fig. 4. (a) P CP-MAS NMR spectrum of Zoledronate-modified TCP (Table 1, entry 1). (b) 31P CP-MAS NMR spectrum of Zoledronatemodified CDA(NH3) (Table 1, entry 5).
(line width at half-height E1.5 ppm) were consistent with the presence of Zoledronate in a crystalline form, in agreement with the SEM observations, probably as a calcium complex. In contrast, the 31P CP-MAS solid state NMR spectra recorded for the modified CDA phases showed the presence of the bisphosphonate moiety as a large peak (line width at half-height E6.5 ppm) centered at 15 ppm (Fig. 4b), thus ruling out the precipitation of a bisphosphonate complex onto the surface of the CaP as a wellordered phase. Interestingly, the CaPs behaved similarly to the case of Zoledronate when exposed to aqueous solutions of the disodium salt of methylene bisphosphonic acid (NaHO3P–CH2–PO3HNa), with slightly different bisphosphonate loading: formation of a bisphosphonatebased crystalline compound on the surface of BCPs and b-TCP (for example 19% loading for BCP(HA25– TCP75)), evidenced by SEM and 31P solid state NMR, conversely to the case of CDA(NaOH) and CDA(NH3) (for example 92% loading for CDA(NH3)). It is thus very likely that the observed reactivity of the various CaPs towards Zoledronate and methylene bisphosphonate is general to the O3P–C–PO3 moiety. The evaluation of the biological activity of Zoledronate loaded materials required the development of an in vitro bone resorption model. Various osteoclastic
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2077
VD3, a well known stimulating factor of osteoclastic resorption [46]. We showed that VD3 triggers a 4-fold increase in osteoclastic resorption (Fig. 8d) thereby demonstrating that osteoclasts present in our model exhibit a valuable response to various anabolic or catabolic bone agents.
4. Discussion
Fig. 5. (31P) 31P proton-mediated two-dimensional CP-MAS NMR exchange spectrum (10 kHz) of Zoledronate-modified BCP(HA25TCP75) (Table 1, entry 2). The mixing time was 0.5 s.
models have been previously described [43]. Among these models, previous reports indicate that unfractioned rabbit bone cell model is one of the most convenient [41,44,45]. When cultured on the surface of dentin slices, vitronectin receptor positive cells exhibit typical F-actin rings indicating that bone resorption activity was taking place. This activity is evidenced by a bone resorption assay showing numerous typical resorption pits on the surface of dentin slices. Viewed together, these data indicate that in our culture conditions, the model of unfractioned rabbit bone cells supports the formation and the activity of mature resorbing osteoclasts. Therefore, to assess the biological activity of Zoledronate-loaded materials, subsequent experiments were performed using this model. Quantitative analysis of the resorptive activity of osteoclasts indicated that Zoledronate-loaded BCP(HA25-TCP75) (Table 1—entry 2; Fig. 8a) induced an almost complete inhibition of dentin slice surface resorbed. On the other hand, Zoledronate-loaded CDA(NaOH) (Table 1—entry 4; Fig. 8b) induced a 3fold decrease in the area resorbed when compared with cells cultured in the presence of Zoledronate-free CaPs. To address specifically the biological relevance of this inhibition, we next questioned whether Zoledronate in solution could affect the osteoclastic resorption. As indicated on Fig. 8c, when cells were exposed to 106 mol l1 Zoledronate for 4 days, a 3-fold decrease in dentin slice surface resorbed was evidenced, when compared to untreated cultures. As a control, we tested
In the case of TCP, the concentration of released phosphate increased in direct proportion to the amount of Zoledronate charged. High incorporation ratios (>90%) were observed for the bisphosphonate along with a constant concentration of residual Zoledronate in the supernatant (ca. 120 ppm phosphorus (i.e. B2.103 mol l1 Zoledronate)), that could mirror the solubility of the crystalline Zoledronate derivative forming on the surface of TCP. This assumption was confirmed by suspending various amounts of Zoledronate-loaded b-TCP in water (60, 120 and 200 mg in 1 ml water) for periods ranging from 8 to 72 h. Identical concentrations of released Zoledronate were invariably measured (ca. 120 ppm phosphorus). These data suggest that the chemical process involved in the preparation of the modified b-TCP samples could be the result of a partial dissolution of b-TCP occurring at the liquid– solid interface with concomitant precipitation of a crystalline Zoledronate derivative. The same end-result was achieved with BCPs, although a lower Zoledronate loading (ca. 35%) was observed, presumably because bTCP is more soluble than BCPs [32,47]. Treating CDAs with increasing quantities of Zoledronate (CZoledronate=0.01 to 0.05 mol l1) resulted in a rise of the released phosphate concentration along with a quantitative uptake of the bisphosphonate until a plateau was reached, corresponding to a ca. 10% w/w Zoledronate loading, similar for both of CDAs. The most probable situation is that chemisorption of bisphosphonate takes place on the surface of the calcium deficient hydroxyapatite phases, via polar covalent interactions with calcium atoms and displacement of PO3 species, the plateau noticed for the Zoledronate 4 uptake corresponding to the saturation of the surface. However, it is worth noting that the BET surface area of CDA(NH3) is circa twice as high as the one measured for CDA(NaOH), thereby indicating that the chemical reactivity of the two CDAs is noticeably different. Moreover, we have shown that the Freundlich equation [48] (Qe ¼ Kf Ce1=n ) was well adapted to describe the adsorption of Zoledronate onto CDAs (see Fig. 6 for CDA(NH3)—Table 2). For CZoledronate > 0:05 mol l1, we observed a rise of the amount of Zoledronate incorporated. Indeed a drastic change in the association mode took place, with the formation of a crystalline material onto the surface of CDAs, as evidenced by
ARTICLE IN PRESS S. Josse et al. / Biomaterials 26 (2005) 2073–2080
2078
Qe = (Kf×Ce)1/n
Qe [mol.g-1]
0.2 0.15 0.1 0.05 0 0
2
6
4
8
10
Ce [mol.l-1]
Fig. 6. Isothermal Freundlich curve for Zoledronate adsorption onto CDA(NH3) at 298 K. At equilibrium: Qe: quantity of Zoledronate adsorbed per gram of CDA and Ce: remaining concentration of Zoledronate in solution, measured by a UV-visible-based method (see Materials and Methods). Logarithmic linearization lead to a r2 ¼ 0:98:
Table 2 Experimental data for Zoledronate adsorption onto CDAs at 298 K, described by isothermal Freundlich curves CaP CDA(NaOH) CDA(NH3)
Surface area (m2/g) 66 140
n
Kf 4
6.9 10 3.8 104
3.9 7.3
SEM (Fig. 7a). Concurrently, the shape of the 31P CPMAS NMR signal assigned to the incorporated Zoledronate evolved from a featureless broad line to a gradually resolved signal (Fig. 7b), consistent with the gradual formation of a crystalline Zoledronate complex onto the surface of CDAs. Repeated washings of these materials until the Zoledronate release was under the detection limit, resulted in a disappearance of the crystalline Zoledronate complex confirmed by SEM and 31P CP-MAS NMR, that showed a spectrum similar to that observed for low Zoledronate-loaded CDAs (see Fig. 4). This clearly indicates that in such conditions, the two Zoledronate association modes took place. With regard to the amount of Zoledronate incorporated onto CDA and BCP, the data obtained from the biological evaluation indicate that both materials exhibit differential patterns of Zoledronate release likely involved in the distinct cellular response. Since the inhibition of osteoclastic resorption produced by Zoledronate in solution is similar to that obtained with Zoledronate-loaded CDA, it is reasonable to conclude that Zoledronate-loaded CDA is likely to release approximately 106 mol l1 Zoledronate within 4 days. On the contrary, the bisphosphonate release with the Zoledronate-loaded BCP is controlled by the solubility constant of the crystalline Zoledronate derivative formed onto the surface of the support (amount of released Zoledronate in water: B120 ppm phosphorusE2.103 mol l1 Zoledronate). A large amount of Zoledronate is thus released, resulting in a cytotoxic effect, since this bisphosphonate is known to reduce cell
Fig. 7. (a) SEM image of Zoledronate-modified CDA(NaOH), with a bisphosphonate loading of 13% by weight (magnitude: 12 000). (b) 31P CP-MAS NMR spectrum of Zoledronate-modified CDA(NaOH), with a bisphosphonate loading of 13% by weight.
viability at concentrations in the range of 104 mol l1 [49,50].
5. Conclusion Osteoarticular disorders associated with increased bone resorption (as observed in osteoporosis, Paget’s disease and osteolytic tumors) often lead to pathological fractures, deformity or bone pain [51]. They are widely treated by the massive systemic administration of bisphosphonates, one of the most potent inhibitors of osteoclast activity [24]. In this context, our preliminary in vitro results suggest that this type of composite material could be suitable for practical application as a local drug delivery system for Zoledronate with release kinetics compatible with the inhibition of bone resorption. Further experiments regarding the association of various BPs to a series of calcium phosphates along with the release profiles of the resulting biomaterials are under intense investigation in our laboratory. This class of materials could be of interest as an alternative to oral treatments in which high doses of BPs are administrated daily or weekly [25], with more or less important gastrointestinal undesired effects. The evaluation of
ARTICLE IN PRESS
BCP
dentin slice surface resorbed (%)
(a)
(c)
* BCP zoledronate
2 1.5 1
*
0.5 0 VEH
Zo
dentin slice surface resorbed (%)
4 3 2 1 0
* CDA
(d)
2079
2.5 2 1.5 1 0.5 0
(b) dentin slice surface resorbed (%)
dentin slice surface resorbed (%)
S. Josse et al. / Biomaterials 26 (2005) 2073–2080
6 5 4 3 2 1 0
CDA zoledronate *
veh
VD3
Fig. 8. Evaluation of the biological activity of Zoledronate–loaded calcium phosphate materials. A total rabbit bone cell preparation was established on dentin slices as described in materials and methods. Cell preparation was then cultured for 4 days either in the presence of BCP or Zoledronateloaded BCP (a), CDA or Zoledronate-loaded CDA (b), 106 mol l1 Zoledronate or its vehicle (VEH) (c) or 108 mol l1 vitamin D3 or its vehicle (d). Dentin slices were next prepared for scanning electron microscopy observation linked to a semi-automatic image analyzer. Resorption activity of osteoclasts is expressed as the percentage of dentin slice surface resorbed. Results are the mean7SEM of three independent experiments. : po0:05 as compared to the respective controls.
these CaP–BP hybrids using in vivo bone resorption models are underway.
Acknowledgements This work was partially supported by the French Ministry of Research (ACI ‘‘Technologies pour la Sante´’’), the C.N.R.S. (Programme ‘‘Mate´riaux Nouveaux—Fonctionnalite´s Nouvelles’’) and the ‘‘Fondation de l’avenir pour la recherche me´dicale applique´e’’. Partial support from the Re´gion Pays de la Loire (CPER ‘‘Biomate´riaux—S3’’) is also acknowledged. We thank Novartis Pharma Research (Basel) for a generous gift of Zoledronate and J.R. Green (Novartis Pharma Research) for fruitful discussion.
References [1] Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. Polymeric systems for controlled drug release. Chem Rev 1999;99:3181–98. [2] Ranade VV. Drug delivery systems 5A. Oral drug delivery. J Clin Pharmacol 1991;31:2–16. [3] Sieminska L, Zerda TW. Diffusion of steroids from sol–gel glass. J Phys Chem 1996;100:4591–7. [4] Sieminska L, Ferguson M, Zerda TW, Couch EJ. Diffusion of steroids in porous sol–gel glass: application in slow drug delivery. Sol–gel Sci Technol 1997;8:1105–9. . [5] Bottcher H, Slowik P, Suss W. Sol–gel carrier systems for controlled drug delivery. J Sol–gel Sci Technol 1998;13:277–81. [6] Falaize S, Radin S, Ducheyne P. In vitro behavior of silica-based xerogels intended as controlled release carriers. J Am Ceram Soc 1999;82:969–76. [7] Santos EM, Radin S, Ducheyne P. Sol–gel derived carrier for the controlled release of proteins. Biomaterials 1999;20:1695–700.
[8] Ahola M, Kortesuo P, Kangasniemi I, Kiesvaara J, Yli-Urpo A. Silica xerogel carrier material for controlled release of toremifene citrate. Int J Pharm 2000;195:219–27. [9] Ahola M, S.ailynoja ES, Raitavuo MH, Salonen JI, Yli-Urpo A. In vitro release of heparin from silica xerogels. Biomaterials 2001;22:2163–70. [10] Otsuka M, Tokumitsu K, Matsuda Y. Solid dosage form preparations from oily medicines and their drug release. Effect of degree of surface-modification of silica gel on the drug release from phytonadione-loaded silica gels. J Control Release 2000;67:369–84. [11] Khan AI, Lei L, Norquist AJ, O’Hare D. Intercalation and controlled release of pharmaceutically active compounds from a layered double hydroxide. Chem Commun 2001;2342–43. [12] Passuti N, Daculsi G, Rogez JM, Martin S, Bainvel JV. Macroporous calcium phosphate ceramic performance in human spine fusion. Clin Orthop 1989;248:169–76. [13] Daculsi G, Bagot d’Arc M, Corlieu P, Gersdorff M. Macroporous biphasic calcium phosphate efficiency in mastoid cavity obliteration: experimental and clinical findings. Ann Otol Rhinol Laryngol 1992;101:669–74. [14] Piattelli A, Scarano A, Mangano C. Clinical and histologic aspects of biphasic calcium phosphate ceramic (BCP) used in connection with implant placement. Biomaterials 1996;17: 1767–70. [15] Ransford AO, Morley T, Edgar MA, Webb P, Passuti N, Chopin D, Morin C, Michel F, Garin C, Pries D. Synthetic porous ceramic compared with autograft in scoliosis surgery: a prospective, randomized study of 341 patients. J Bone Joint Surg Br 1998;80:13–8. [16] Cavagna R, Daculsi G, Bouler JM. Macroporous calcium phosphate ceramic: a prospective study of 106 cases in lumbar spinal fusion. J Long Term Eff Med Implants 1999;9:403–12. [17] Delecrin J, Takahashi S, Gouin F, Passuti N. A synthetic porous ceramic as a bone graft substitute in the surgical management of scoliosis: a prospective, randomized study. Spine 2000;25(5):563–9. [18] Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed 2002;41:3130–46. [19] Guicheux J, Gauthier O, Aguado E, Heymann D, Pilet P, Couillaud S, Faivre A, Daculsi G. Growth hormoneloaded macroporous calcium phosphate ceramic: in vitro
ARTICLE IN PRESS 2080
[20]
[21]
[22]
[23]
[24] [25] [26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35]
S. Josse et al. / Biomaterials 26 (2005) 2073–2080 biopharmaceutical characterization and preliminary in vivo study. J Biomed Mater Res 1998;40(4):560–6. Obadia L, Amador G, Daculsi G, Bouler JM. Calcium-deficient apatite: influence of granule size and consolidation mode on release and in vitro activity of vancomycin. Biomaterials 2003;24:1265–70. Slosarczyk A, Szymura-Oleksiak J, Mycek B. The kinetics of pentoxifylline release from drug-loaded hydroxyapatite implants. Biomaterials 2000;21:1215–21. Netz DJA, Sepulveda P, Pandolfelli VC, Spadaro ACC, Alencastre JB, Bentley MVLB, Marchetti JM. Potential use of gelcasting hydroxyapatite porous ceramic as an implantable drug delivery system. Int J Pharm 2001;213:117–25. Burgos AE, Belchior JC, Sinisterra RD. Controlled release of rhodium (II) carboxylates and their association complexes with cylcodextrins from hydroxyapatite matrix. Biomaterials 2002;23:2519–26. Fleich H. In: Bisphosphonates in bone disease, from the laboratory to the patients. San Francisco: Academic Press; 2000. Bisphosphonates for osteoporosis. Drug and Therapeutics Bulletin 2001;39:68–72. Doggrell SA. Zoledronate once-yearly increases bone mineral density—implications for osteoporosis. Expert Opin Pharmacother 2002;3:1007–9. . Denissen H, Van Beek E, Lowik C, Papapoulos S, Van der Hooff A. Ceramic hydroxyapatite implants for the release of bisphosphonate. Bone Min 1994;25:123–34. Denissen H, Van Beek E, Van den Bos T, De Blieck J, Klein C, Van den Hooff A. Degradable bisphosphonate-alkaline phosphatase-complexed hydroxyapatite implants in vitro. J Bone Miner Res 1997;12:290–7. Denissen H, Van Beek E, Martinetti R, Klein C, Van der Zee E, Ravaglioli A. Net-shaped hydroxyapatite implants for release of agents modulating periodontal-like tissues. J Periodont Res 1997;32:40–6. Denissen H, Martinetti R, Van Lingen A, Van den Hooff A. Normal osteoconduction and repair in and around submerged highly bisphosphonate-complexed hydroxyapatite implants in rat tibiae. J Periodont 2000;71:272–8. Denissen H, Montanari C, Martinetti R, Van Lingen A, Van den Hooff A. Alveolar bone response to submerged bisphosphonatecomplexed hydroxyapatite implants. J Periodont 2000;71:279–86. LeGeros RZ. Calcium phosphates in oral biology. In: Myers HM, editor. Monographs in oral science. Basel: Karger; 1991. Weiss P, Gauthier O, Bouler JM, Grimandi G, Daculsi G. Injectable bone substitute using a hydrophilic polymer. Bone 1999;25(Suppl. 2):67S–70S. Daculsi G, Weiss P, Bouler JM, Gauthier O, Millot F, Aguado E. Biphasic calcium phosphate/hydrosoluble polymer composites: a new concept for bone and dental substitution biomaterials. Bone 1999;25(Suppl. 2):59S–61S. Gauthier O, Boix D, Grimandi G, Aguado E, Bouler JM, Weiss P, Daculsi G. A new injectable calcium phosphate biomaterial for immediate bone filling of extraction sockets: a preliminary study in dogs. J Periodontol 1999;70:375–83.
[36] Gauthier O, Bouler JM, Weiss P, Bosco J, Daculsi G, Aguado E. Kinetic study of bone ingrowth and ceramic resorption associated with the implantation of different injectable calcium-phosphate bone substitutes. J Biomed Mater Res 1999;47:28–35. [37] Bourgeois B, Laboux O, Gauthier O, Obadia L, Aguado E, Daculsi G, Bouler JM. Calcium-deficient apatite: a first in vivo study concerning bone ingrowth. J Biomed Mater Res 2003;65A(3):402–8. [38] Wilder L, Jaeggi KA, Glatt M, Muller . K, Bachmann R, Bisping M, Born A, Cortesi R, Guiglia G, Jeker H, Klein R, Ramseier U, Schmid J, Schreiber G, Seltenmeyer Y, Green JR. Highly potent geminal bisphosphonates. From pamidronate (Aredia) to zoledronic acid (Zometa). J Med Chem 2002;45:3721–38. [39] Ishikawa K, Ducheyne P, Radin S. Determination of the Ca/P ratio in calcium deficient hydroxyapatite using X-ray diffraction analysis. J Mater Sci Mater Med 1993;4:165–8. [40] Ames BN. Assay of inorganic phosphate, total phosphate and phosphatases. In: Colowick SP, Kaplan NO, editors. Methods in enzymology, Vol. 8. Orlando: Academic Press; 1966. p. 115–8. [41] Guicheux J, Heymann D, Rousselle AV, Gouin F, Pilet P, Yamada S, Daculsi G. Growth hormone stimulatory effects on osteoclastic resorption are partly mediated by insulin-like growth factor I: an in vitro study. Bone 1998;22:25–31. [42] Schmidt-Rohr KW, Spiess H. In: Harcourt Brace and Company, editors. Multidimensional solid-state NMR and polymers. New York: Academic Press, 1994. [43] Heymann D, Guicheux J, Gouin F, Passuti N, Daculsi G. Cytokines, growth factors and osteoclasts. Cytokine 1998;10(3):155–68. [44] David JP, Neff L, Chen Y, Rincon M, Horne WC, Baron R. A new method to isolate large numbers of rabbit osteoclasts and osteoclast-like cells: application to the characterization of serum response element binding proteins during osteoclast differentiation. J Bone Miner Res 1998;13(11):1730–8. [45] Tezuka K, Sato T, Kamioka H, Nijweide PJ, Tanaka K, Matsuo T, Ohta M, Kurihara N, Hakeda Y, Kumegawa M. Identification of osteopontin in isolated rabbit osteoclasts. Biochem Biophys Res Commun 1992;186(2):911–7. [46] Roodman GD. Advances in bone biology: the osteoclast. Endocr Rev 1996;17(4):308–32. [47] Bouler JM, LeGeros RZ, Daculsi G. Biphasic calcium phosphates: influence of three synthesis parameters on the HA/betaTCP ratio. J Biomed Mater Res 2000;51:680–4. [48] Freundlich H. In: Hatfield HS, editor. Colloid and capillary chemistry. London: Methuen, 1926. [49] Li EC, Davis LE. Zoledronic acid: a new parenteral bisphosphonate. Clin Therapeut 2003;25:2669–708. [50] Reinholz GG, Getz B, Pederson L, Sanders ES, Subramaniam M, Ingle JN, Spelsberg TC. Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts. Cancer Res 2000;60:6001–7. [51] Consensus Development Conference, ‘Diagnosis, prophylaxis and treatment of osteoporosis’, conference report. Am J Med 1993;94(6):646–50.