Functionalization of octacalcium phosphate for bone replacement

Functionalization of octacalcium phosphate for bone replacement

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Adriana Bigi and Elisa Boanini Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy

3.1

Introduction

The growing occurrence of musculoskeletal disorders due to the increasing average age of the population in evolved countries requires the development of innovative materials which can suitably substitute/repair damaged tissues. Most biomaterials for hard tissues applications involve the employment of calcium orthophosphates (CaPs), due to their chemical and structural similarities with the inorganic phase of the biomineralized tissues of vertebrates. In this field a major role is played by hydroxyapatite (HA), which is the CaP most similar to the mineral component of bone. However, the excellent biocompatibility and bioactivity of HA is shared by other CaPs [1]. In particular, octacalcium phosphate (OCP), which is considered the first phase to deposit in biological environment, is gaining increasing interest for biomedical applications. In fact, it displays osteoconductive properties and converts into HA once implanted in bone defects [2,3]. Moreover, OCP promotion of bone formation has been demonstrated both in in vitro and in vivo studies [4 7]. In recent years, many efforts have been devoted to add specific functionalities to CaPs through enrichment of their composition with additives with the aim to improve their biological performance and/or to provide them with different therapeutic properties [8]. To this purpose the possibility to functionalize CaPs with biologically relevant ions and molecules, amino acids, polyelectrolytes, growth factors, and drugs has been explored [8]. Two different strategies are generally followed to load additives into CaPs: coprecipitation from solution and chemisorption from solution [9]. Coprecipitation usually provides a stronger interaction between additive and CaPs in comparison to chemisorption and generally implies a more sustained release. In fact, coprecipitation implies incorporation of the additive into the CaPs crystals, whereas chemisorption leads to adsorption of the additive just on the surface of the crystals [10]. However, the conditions of synthesis of CaPs do not always allow addition of functionalizing agents in the precipitation environment. Moreover, chemisorption is indeed an easy process and the only possible route when the CaP is synthesized at high temperature and the additive is not thermostable. Although the scientific literature on functionalized OCP is by far less rich than that on HA, the recent attention toward the employment of OCP for the preparation of biomaterials for hard tissue substitution/repair has promoted a number of studies Octacalcium Phosphate Biomaterials. DOI: https://doi.org/10.1016/B978-0-08-102511-6.00003-0 Copyright © 2020 Elsevier Ltd. All rights reserved.

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Octacalcium Phosphate Biomaterials

on the development and characterization of OCP modified with bioactive agents, as reported in the following sections.

3.2

Structure and chemistry of octacalcium phosphate

The first stages of mineralization of the skeletal tissues have been suggested to involve the deposition of OCP, Ca8H2(PO4)6
5H2O (OCP), as a precursor phase of the final, thermodynamically more stable, carbonated apatite [11,12]. This hypothesis is based on different reasons: (1) the plate-like habit of biological apatite crystals, which is not consistent with apatite symmetry; (2) the remarkable similarity between OCP and HA structures; (3) the easy hydrolysis of OCP into HA; and (4) the presence of OCP in calcifying dentine as well as in several pathological calcifications [13 19]. OCP crystallizes in the triclinic space group P-1, with cell parameters ˚ , b 5 9.6300 A ˚ , c 5 6.8750 A ˚ , α 5 89.2800 degrees, β 5 92.2200 a 5 19.8700 A degrees, γ 5 108.9500 degrees (PDF 44 778). The asymmetric unit coincides with the unit formula and the unit cell contains two asymmetric units. The big, plate-like crystals display wide (100) faces and are elongated along the c-axis direction (Fig. 3.1) [17]. In agreement with the peculiar morphology of OCP crystals, the powder X-ray diffraction (XRD) pattern is characterized by the presence of a relatively high-intensity 100 peak at about 4.7 degrees of 2θ. OCP unit cell can be described as an assembly of alternate “apatitic” and “hydrated” layers parallel to the (100) faces. The positions of Ca21 and PO432 ions in the apatitic layer closely resemble those they occupy in the unit cell of HA, whereas the hydrated layer hosts more widely spaced Ca21 and PO432 ions, as well as the structural water molecules (Fig. 3.2) [1]. The apatitic layer is about 1.1 nm thick and contains six Ca21 and two PO432 ions, whereas the 0.8 nm (approx.) thick hydrated layer accommodates two Ca21 and one PO432 ions. The other three phosphate ions are located at the interface

Figure 3.1 (A,B) SEM images of OCP crystals. The faces of a typical blade-like crystal are indicated by Miller indices in the B panel. OCP, Octacalcium phosphate. Source: Reprinted with permission from Bigi A, Bracci B, Panzavolta S, Iliescu M, Plouet-Richard M, Werckmann J, Cam D. Morphological and structural modifications of octacalcium phosphate induced by poly-l-aspartate. Cryst Growth Des 2004;4:141 6. Copyright 2004 American Chemical Society.

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Figure 3.2 Schematic of OCP structure that puts into evidence the alternation of hydrated and apatitic layers. The positions of Ca21 and PO432 ions in the apatitic layer closely resemble those they occupy in the unit cell of hydroxyapatite, whereas the hydrated layer hosts the structural water molecules. OCP, Octacalcium phosphate. Source: Reprinted from Boanini E, Gazzano M, Bigi A. Ionic substitutions in calcium phosphates synthesized at low temperature. Acta Biomater 2010;6:1882 94. Copyright 2010, with permission from Elsevier.

Figure 3.3 Structure of OCP where hydrogen-bonding interactions are put into evidence (indicated by pink connecting lines) within the hydrated layers of the original (left) and relaxed (right) structures. OCP, Octacalcium phosphate. Source: Reprinted with permission from Davies E, Duer MJ, Ashbrook SE, Griffin JM. Applications of NMR crystallography to problems in biomineralization: refinement of the crystal structure and 31P solid-state NMR spectral assignment of octacalcium phosphate. J Am Chem Soc 2012;134:12508 15. Copyright 2012 American Chemical Society.

between the two different layers. One of these phosphate ions and the one in the hydrated layer are protonated [11]. The structure of the hydrated layer was suggested as a model to describe “the interface between the apatite crystals and aqueous phase” [13]. A recent refinement of the structure, which was performed through X-ray powder diffraction, density function theory calculation, and solid-state 31P NMR spectroscopy, allowed identification of an extended hydrogen-bonding network within the hydrated layer (Fig. 3.3) [18].

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Octacalcium Phosphate Biomaterials

Figure 3.4 (A) TG DTG plot of OCP. (B) Small-angle region of the powder XRD pattern of OCP: the intensities of the 2θ range 8 12 are magnified by 4. OCP, Octacalcium phosphate; TG DTG, Thermogravimetry/derivative thermogravimetry; XRD, X-ray diffraction. Source: Reprinted with permission from Boanini E, Gazzano M, Rubini K, Bigi A. Collapsed octacalcium phosphate stabilized by ionic substitutions. Cryst Growth Des 2010;10:3612 7. Copyright 2010 American Chemical Society.

The methods of preparation of OCP include hydrolysis of other calcium phosphates, such as brushite [dicalcium phosphate dihydrate (DCPD)] and α-tricalcium phosphate (α-TCP), precipitation from aqueous solution, chemical and physical deposition methods [3,19]. OCP has a tendency to undergo multiple twinning, using the (100) face as a twin plane [20,21]. However, even small variations in the conditions of synthesis can influence the morphology of synthetic OCP [17,21]. In aqueous solution, OCP transforms into the more stable HA according to a process that has been described as in situ hydrolysis and/or dissolution of OCP followed by HA precipitation [13,15,22]. The transition rate is influenced by several factors, including pH, temperature, and the presence of foreign ions and additives [22 25]. Conversion into an apatitic phase occurs also through heat treatment, which causes removal of water molecules from OCP, as evidenced by the two thermal processes present in the thermogravimetric plot between 40 C and 200 C (Fig. 3.4A). Heating at about 170 C provokes a splitting of the low-angle reflection in two separate peaks and the onset of a new reflection at about 10.3 degrees 2θ. On further increase of the temperature greater than 250 C the X-ray pattern no longer shows low-angle reflections, at variance it is consistent with that of a poorly crystalline apatitic phase (Fig. 3.4B) [26].

Functionalization of octacalcium phosphate for bone replacement

3.3

41

Interaction with carboxylic acids

Mineralized tissues in vertebrates contain matrix macromolecules such as glycoproteins and acidic proteins, which display charged carboxylate groups due to the relevant presence of acidic amino acids [27]. Thus it is conceivable to suppose that calcium phosphates crystal nucleation and growth in vivo may be influenced by the interactions of these proteins with the charged crystal surfaces. Indeed, the results of in vitro experiments carried out on OCP crystallization suggest a specific action of the carboxylate-rich proteins that seem to interact preferentially with the (100) face of OCP structure which corresponds to the hydrated layer [28]. Polymers that contain carboxylate functional groups can mimic in vitro the role of in vivo proteins and affect calcium phosphates deposition. Tsortos and Nancollas demonstrated that polycarboxylic acids can act as crystal nucleators of HA when immobilized on a surface and as inhibitors of crystal growth when free in solution [29]. Similarly, macromolecules containing carboxylic functional groups can affect OCP precipitation in solution, as well as its phase conversion via hydrolysis. OCP synthesized in aqueous solution in the presence of an increasing concentration of polyacrylate ions displayed unchanged lattice constants and no significant polyelectrolyte adsorption on the crystals [30]. However, the presence of the polyelectrolyte inhibited the precipitation of OCP and reduced significantly its crystal growth, as shown by the broadening of the XRD peaks on increasing polyelectrolyte content. The inhibition of the crystal growth was evaluated through calculation of the coherence length of the perfect crystalline domains and was almost isotropic along the directions corresponding to the crystallographic axes, thus indicating no preferential interaction of polyacrylate with the structure of OCP [30]. Furthermore, the morphology of OCP spherulites, consisting of long blade-shaped crystals about 100 μm long, radiating from a common origin, changed on increasing polyacrylate concentration: at low concentration the blades that constitute the spherules exhibited reduced length, whereas higher concentration led to the formation of hollow microspheres with diameters up to 1 mm [21,30]. A similar behavior was also observed in the presence of polyaspartic acid, which provoked an increasing disorder. Moreover, the increasing contribution of the apatitic layer of the OCP structure, which could be related to the observed reduction of thermal stability, suggested a possible interaction of polyaspartic acid with the hydrated layer of the OCP structure [17,21]. This preferential interaction was related to the structural match between the rows of calcium ions parallel to the c-axis direction in OCP structure and the carboxylate groups of the polyaspartic chains in β-sheet conformation [31]. The key role of the (100) hydrated face has been suggested to also control the process of hydrolysis of OCP into HA in the presence of carboxylate-rich macromolecules in solution. Indeed polyacrylate, polyaspartate, and polyglutamate showed to inhibit the phase conversion in a dose-dependent way and allowed to control the shape of the very long, needle-like, final apatitic crystals [32,33]. This behavior could be explained by the hypothesis of carboxylate groups’ adsorption on the (100) face of OCP crystals, which prevented OCP dissolution and promoted the in

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Octacalcium Phosphate Biomaterials

Figure 3.5 AFM images of (100) crystal surface of an OCP crystal (A), and of OCP crystals incubated in solution containing polyacrylate at (B) low and (C) high molecular weight. The complete views of the crystals are reported in the insets (scale-bar 5 3 μm). OCP, Octacalcium phosphate. Source: Reprinted with permission from Bigi A, Boanini E, Cojazzi G, Falini G, Panzavolta S. Morphological and structural investigation of octacalcium phosphate hydrolysis in presence of polyacrylic acids: effect of relative molecular weights. Cryst Growth Des 2001;1:239 44. Copyright 2001 American Chemical Society.

situ splitting of OCP crystals along their c-axis to form HA. Furthermore, the interaction of carboxylic-rich macromolecules could be modulated by varying the chain length, that is, the molecular weight: the interaction of crystal surface with a shorter macromolecular chain can be more ordered and results in a regular decrease of the coherence length of the perfect crystalline domains along the c-axis direction, possibly due to a preferential adsorption along this direction. At variance, lying flat on the crystal surface is more difficult for very long chains, which are most likely folded, as suggested by the AFM images of OCP-polyacrylate crystals (Fig. 3.5) [25]. The presence of polyacrylate ions in solution inhibits also the hydrolysis of α-TCP to OCP that takes place through a mechanism of dissolution and reprecipitation. The delayed OCP nucleation and growth in the presence of polyacrylate confirm the great affinity of carboxylate functional group for OCP surface that provokes an anisotropic reduction of the coherence length of the perfect crystalline domains along the a- and c-axis of OCP crystal structure [34]. When Monma and Goto performed the hydrolysis of α-TCP to OCP in the presence of succinate ions in solution, a completely different behavior could be observed: an increase of Ca/P molar ratio and a significant shift of the 100 peak in XRD diffraction pattern, due to an expansion of interplanar distance, suggested a replacement of HPO22 4 ion by succinate within the hydrated layer of the structure [35]. Using this same method, a number of OCP carboxylates, namely, malonate, adipate, suberate, sebacate, fumarate, and malate ions, were prepared and structurally characterized. All these compounds are structurally similar to OCP but display an expanded a-axis dimensions that generally increased with increasing number of carbon atoms in the carboxylate ion [36]. FT-IR spectra of these OCP carboxylates compounds showed the presence of carboxylate groups, changes in water bonding, only slight changes in PO4 environments, and a reduction in HPO4 content, supporting the hypothesis of a partial substitution of HPO422 ion by a dicarboxylate ion within the structure [37]. A solid-state P-31 NMR study confirmed the partial

Functionalization of octacalcium phosphate for bone replacement

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substitution of HPO422 ion in the hydrated layer of OCP with succinate, which eventually altered the dynamics of the water molecules within the structure and caused the higher stability of intercalated OCP to the hydrolysis reaction at high pH conditions with respect to pure OCP [38]. OCP intercalation with dicarboxylate ions was successfully obtained also by direct synthesis in solution from CaCO3 and H3PO4 in the presence of succinic, suberic, or adipic acids [39], and later the same experimental procedure led to the production of OCPs coincorporated with various molar ratios of succinate and suberate ions [40]. When the Suc/(Suc 1 Sub) molar ratio in the starting solution was in the range 0.45 1.0, succinate was preferentially incorporated into the OCP; whereas when the Sub/(Suc 1 Sub) molar ratio was in the range 0.60 1.0, suberate was preferentially incorporated into the OCP crystals. The successful incorporation of the two types of dicarboxylate ions reduced the crystallinity in comparison to pure OCP. The observed angular values of the 100 peak of the OCP carboxylates formed under coaddition of succinate and suberate were between those of succinate-OCP and suberate-OCP. [40]. More recently, enantioselectivity by OCP was demonstrated through the incorporation of (S)-(2)-methylsuccinic acid through direct synthesis, whereas no significant amount of (R)-(1)-methylsuccinic was incorporated under the same experimental conditions. This phenomenon clearly indicates that OCP is able to recognize the structures of guest molecules [41].

3.4

Interaction with biological molecules

The role of OCP as precursor phase of poorly crystalline apatite in calcified tissues has raised interest about its physicochemical behavior in biological environment. Applicative studies have tried to combine OCP with biological molecules in order to obtain biomaterials useful in orthopedic surgery [42,43]. A nano micro structured OCP/collagen composite coating was obtained on titanium substrate by using an electrochemically induced deposition technique, with the aim to provide metallic implants with a biomimetic surface. As a result of the interaction of collagen with the crystals growing on the metallic surface, the XRD pattern of the composite coating displayed a significant reduction of the relative intensities of the 100 reflection compared to pure OCP coating deposited under the same conditions, in agreement with the reduced crystals dimensions and, as a consequence, much smaller (100) faces observed by SEM [42]. A step-by-step approach allowed to obtain a coating on Ti surface of OCP functionalized with antimicrobial peptides. Large crystals of OCP were deposited by electrolytic deposition in the absence of antimicrobial peptides that were later immobilized on the coating through adsorption from solution on the mineral phase [43]. Simple soaking led to loading of up to 9 mg/cm2 of antimicrobial peptides on a 7 μm thick porous OCP coating, without affecting its crystalline structure. The peptide loaded coatings displayed a strong bactericidal effect against both Gram-positive and Gram-negative bacteria and were also biocompatible with osteoblast-like cells [43].

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The adsorptions of human serum albumin and basic protein lysozyme on crystallographic planes of OCP have been studied by molecular simulations. The results show that adsorption interaction energy of basic lysozyme is higher than that of acidic albumin, which indicates that lysozyme could be preferentially adsorbed onto OCP surface in comparison to albumin [44]. This behavior is consistent with other studies carried out on different calcium phosphates, such as HA, biphasic calcium phosphate, and beta TCP [45]. Variation of absorption behavior on different OCP crystallographic planes must be attributed to differences of their surface properties that may include surface energy and planar density of individual atoms. The calculations showed that the OCP (001) surface has the highest interaction energy with both proteins, followed by planes (111), (110), and (100). At variance, the strain energy strongly depended on the orientations of the proteins before absorption but weakly on crystallographic planes [44]. An experimental study on adsorption onto OCP of bovine serum albumin, a strong inhibitor of the crystal growth of calcium phosphates in vitro, was described by a Langmuirian model, without further clarifying any structural interaction [46]. Microscopy studies have shown that proteins with an abundance of carboxylated amino acids affect OCP crystal morphology differently than phosphorylated proteins as a consequence of face-specific adsorption for these molecules [47]. In particular, polyaspartic acid and phosphophoryn have been suggested to adsorb specifically on the (100) and (010) faces of OCP, respectively. Their effects on crystal growth were examined at a constant level of supersaturation where both polymers were adsorbed on the substrate following a Langmuirian model and the maximum adsorbed amount for phosphophoryn was 100-fold less than that for polyaspartic acid. The latter inhibited OCP growth by 20% when only 1% of the (100) face was covered, whereas the former had to reach over 200% (010) face coverage before a similar level of crystal growth inhibition was obtained (either multilayering occurred or only a segment of phosphophoryn was attached to the crystal). This difference in inhibitory effect may indicate that growth at the (100) face is rate controlling for crystal growth [47]. The kinetics of the (100) surface growth of OCP in the presence of amelogenin C terminus peptides were studied inside a fluid cell of an atomic force microscope, where synthetic OCP crystals were placed as seed substrates in a supersaturated solution. It was verified that a controlled aggregation of primary amelogenin nanoparticles takes place to form nanorods, which further selfassemble into elongated nanostructures. These organized and elongated amelogenin peptide nanofibers are able to attach onto the OCP (100) crystal surface to induce the final formation of organic inorganic composite nanorods. As a result, the crystallographic c-axis of OCP is aligned with the long axes of the peptide assemblies [48]. Further studies on the interaction between tooth proteins and OCP are reported in different chapters of this book. The ability of OCP surfaces to bind to proteins was demonstrated by incubating crystals in rat serum. Extraction of the adsorbed proteins and identification through mass spectrometry verified that a total of 138 proteins were detected from OCP and 103 proteins were detected from HA with similar surface area, so there were proteins that were adsorbed only on OCP surfaces [49].

Functionalization of octacalcium phosphate for bone replacement

3.5

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Ion-substituted/doped octacalcium phosphate

The deposition of the mineral phase in biological tissues occurs in an environment rich in ions, which play a role on the biomineralization processes and, as a consequence, on the relative stability of calcium phosphates. The study of ionic substitutions provides useful information for a better understanding of the biomineralization processes and allows for the development of materials that combine the very good bioactivity of calcium phosphates with the specific biological role of the substituent ions. Although most of the literature on ionic substitutions in calcium phosphates is focused on HA, the interest is high also toward other phases for their possible involvement in the deposition of the mineral phase of bone and teeth, as well as their use for the preparation of biomaterials for hard tissues substitution/repair [1]. The most investigated ions include strontium that displays antiosteoporotic properties, magnesium for its beneficial action on osteoblast/osteoclast activity and bone growth, zinc for its involvement in a number of biological functions, silicon for it has been reported to enhance bone repair at the wounded sites, fluoride for its promoting role on osteoblast differentiation in vivo, and carbonate for its relative high presence in biological apatites [50]. The presence of foreign ions during the synthesis, as well as the hydrolysis of OCP, was reported to influence the crystallization process and the rate of conversion into HA [51 55]. According to the results of first principle calculations, the formation energies of several substitutional foreign ions at a number of Ca sites are much smaller in OCP than in HA, suggesting an easier incorporation of these ions in OCP than in HA structure [56,57]. On the contrary, experimental data indicate that OCP can generally accommodate a smaller amount of foreign ions than HA [1,26]. As an example, strontium can substitute for calcium in the structure of HA in the whole range of composition [50], while it can be incorporated into OCP just in limited amounts [26,58,59]. Strontium substitution for calcium in OCP structure is generally reported not to exceed 10 atom% [26,58]. At variance, greater strontium contents, up to about 23 atom%, have been reported by Shi et al. [59]. However, the relative XRD patterns of these samples show the presence of broad diffraction peaks, which suggest the presence of a poorly crystalline apatite/amorphous phase and hinder a proper evaluation of the lattice constants of OCP [59]. Other studies indicate that syntheses carried out in the presence of Sr21 concentrations in solution higher than 15 atom% result in the precipitation of apatite as a second phase, whereas no precipitation at all can be obtained for concentrations higher than 60 atom% [26]. Substitution causes an enlargement of the unit cell of OCP, coherent with the greater ionic radius of strontium than that of calcium ion [26,59]. Several studies report results of in vitro tests, indicating a beneficial effect on bone cells of strontium association with OCP, which has been shown to enhance viability, proliferation, and differentiation of mouse bone mesenchymal stem cells, human mesenchymal stromal cells, and osteoblast-like MG63 [59 61]. Magnesium exerts an even more destabilizing effect than strontium on OCP structure. Similarly to what reported for strontium, increasing magnesium concentration

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in solution provokes the precipitation of a secondary phase, namely, DCPD (brushite), up to complete inhibition of precipitation. Mg21 substitution to Ca21 into OCP structure was found to amount to just about 1 atom%, causing a slight, but significant, reduction of the dimensions of the unit cell [26]. Both Mg21 and Sr21 affect OCP crystal morphology, provoking a reduction of the mean size of the long blade-like crystals and an increase of disorder in the direction perpendicular to the (h00) planes. Moreover, the destabilizing role of these two ions on OCP structure is supported also by the reduced thermal stability of the functionalized materials. The temperatures of the thermal processes associated to the removal of water molecules from OCP structure shift to lower temperature in the order OCP . Sr-substituted OCP . Mg-substituted OCP [26]. Thermal treatment causes conversion into a poorly crystalline apatitic phase, which occurs through the onset of a new crystalline phase, “collapsed OCP.” Features suggesting a collapsed structure were first observed during dehydration of OCP by Nelson and McLean [62], who hypothesized a modification toward a structure “more similar to apatite but with a double a-axis dimension.” More recently, it was possible to determine that this new phase exhibits a slight reduced a-axis, a wider γ angle, and a different inclination of the unit cell in comparison to OCP (Fig. 3.6). The presence of the foreign ions lowers the temperatures of the thermal transitions [26]. Ionic substitution in OCP has been proposed as a tool to introduce magnesium and strontium ions in the composition of α-TCP-based cements. Both Mg-OCP and Sr-OCP were demonstrated to reduce the setting times to enhance the wetting compressive strength and yield a denser microstructure of the cement [63,64].

Figure 3.6 Schemes of OCP (A) and “collapsed” OCP (B) structures along the c-axis The comparison between the unit cell of OCP (blue) and that of “collapsed OCP” (red) is also reported (C). OCP, Octacalcium phosphate. Source: Reprinted with permission from Boanini E, Gazzano M, Rubini K, Bigi A. Collapsed octacalcium phosphate stabilized by ionic substitutions. Cryst Growth Des 2010;10:3612 7. Copyright 2010 American Chemical Society.

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Zn-doped OCP was synthesized using a coprecipitation method up to a Zn21 content of a few atom%. The presence of zinc slows down the hydrolysis reaction into apatite and promotes the formation of amorphous material [65]. Both carbonate and fluoride have been reported to suppress the growth of OCP along the c-axis direction and to promote OCP conversion into HA [3,24,66]. In particular, low doses of fluoride inhibit the precipitation of OCP in favor of HA where it can replace OH ions [66]. Strontium, zinc, silicon, and magnesium and their combination were utilized to develop multilayered porous beads of ion-doped OCP using wet chemical reactions promoted by aspartic acid. The presence of the doping ions, which provokes slight variations in the values of OCP lattice constants, modulates beads dimensions and stability, as well as ion release, and accelerates the growth of appositional bone growth in the early stage after implantation into the rat femur defect model [67].

3.6

Octacalcium phosphate for drug delivery

Calcium phosphates functionalization with therapeutic agents aimed to develop drug-delivery systems for the treatment of bone pathologies has been mainly focused on calcium phosphate nanoparticles and HA nanocrystals, as well as on apatitic cements, coatings, and porous scaffolds [8,68 70]. On the other hand the relevant literature on the functionalization of OCP with drugs is still limited. Most of the researches in this field have been addressed to functionalization of OCP with bisphosphonates (BPs) [9]. BPs include a number of molecules, characterized by the presence of two phosphonate groups bound to a central carbon atom, which binds two further side chains. The chemistry of these side chains influences the affinity of the molecule for calcium ions in general, and for apatite in particular, as well as their mechanism of action toward osteoclast [71]. All these drugs are powerful antiresorptive agents, especially the so-called nitrogen-BPs (N-BPs), which contain an N atom in their side chain, such as alendronate and zoledronate. In fact, BPs are the most employed drugs for the treatments of alteration of bone metabolism characterized by excessive bone resorption [72]. However, their prolonged systemic use can cause several adverse side effects that are the reason of the increasing interest toward BPs delivery systems [73]. As in the case of HA, BPs functionalization of OCP has been performed through two different strategies: chemisorption on presynthesized OCP and coprecipitation from aqueous solution. Soaking of OCP in aqueous solutions at increasing alendronate concentration provoked the quantitative precipitation of tiny rod-like crystals of calcium alendronate monohydrate, C4H11NO7P2Ca
H2O, or CaAL
H2O, at the expense of OCP, which could be completely digested and utilized as Ca21 source [74]. In vitro coculture tests of the products constituted of OCP and CaAL
H2O demonstrated that the composite materials display an inhibiting role on osteoclastogenesis and osteoclast differentiation, whereas they enhance osteoblast proliferation and activity, even more than pure CaAL
H2O [74,75]. Moreover, alendronate-loaded OCP exhibited a greater

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enhancement of osteoblast differentiation markers when compared to HA loaded with similar amount of alendronate, suggesting that OCP can be considered a better support for BP local delivery [76]. The alendronate-loading capacity of oriented OCP nanobelts synthesized in presence of cetyltrimethylammonium bromide (CTAB) was compared with that of HA obtained through hydrolysis/thermolysis of the nanobelts. The results demonstrated a higher loading capacity of OCP than HA, which was ascribed to the less negative zeta potential of OCP and consequent lower energy barrier for the interaction between the inorganic phase and the BP [77]. It has been further showed that the addition of very low concentration (2 8 μM) of alendronate during the synthesis of OCP in the presence of CTAB stabilizes the calcium phosphate inhibiting its hydrolysis into HA, whereas slightly higher concentrations promotes the precipitation of HA [78]. In the absence of surfactants the synthesis of OCP in the presence of increasing amounts of BPs in solution yielded functionalized materials containing relatively high drug amounts. Functionalized OCP crystals containing up to 3.5 and 5.2 wt.% BP were obtained in the case of zoledronate and alendronate, respectively [79]. Functionalization provoked slight structural and morphological modifications suggesting a preferential interaction of the BPs with the (0k0) faces of OCP structure (Fig. 3.7) and prevented its hydrolysis into HA. In vitro test were carried out on osteoblast, osteoclast, and endothelial cells assembled in a triculture model in order to mimic bone microenvironment. The results indicated that the functionalized materials promoted the osteoblast production of collagen type I and osteocalcin, inhibited monocyte viability and

Figure 3.7 Suggested preferential interaction of bisphosphonates (alendronate and zoledronate) with the (0k0) faces of OCP structure. OCP, Octacalcium phosphate. Source: Reprinted from Forte L, Torricelli P, Boanini E, Gazzano M, Fini M, Bigi A. Antiresorptive and anti-angiogenetic octacalcium phosphate functionalized with bisphosphonates: an in vitro tri-culture study. Acta Biomater 2017;54:419 28, ©(2017), with permission from Elsevier.

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differentiation in osteoclast, reduced endothelial cell viability, and downregulated vascular endothelial growth factor, suggesting that the BPs imbue functionalized OCP with antiresorptive and antitumor properties [79]. Spherical OCP/HA porous granules were proposed as drug carriers of a different drug molecule with bone anticancer properties, namely, methotrexate. Functionalization was performed on OCP/HA porous granules prepared through hydrolysis of α-TCP. Results showed that release of loaded methotrexate from the biphasic spheres continues for at least 48 h, provoking a significant inhibition of human osteosarcoma cells [80]. OCP crystals with a high aspect ratio prepared through a modified hydrothermal synthesis in the presence of urea and glutamic acid was used as supports to load relative high amounts of the antiinflammatory agent ibuprofen (IBU) [80]. Addition of IBU during the synthesis of OCP yielded functionalized materials containing more than 25 wt.% of the drug, which displayed a sustained release described by a Higuchi model. Interestingly, complete release required longer times (36 h) than those previously reported for calcium phosphates with different morphologies [81]. Functionalization of OCP was also proposed as a tool to develop systems with antibacterial properties. Bacterial colonization is among the main causes of failure of orthopedic implants [82], and the problem is aggravated by the increasing antibiotic resistance of clinically relevant bacteria [83]. Silver nanoparticles (AgNPs) are widely employed in biomedical applications, thanks to their known antimicrobial properties against a number of pathogens [84,85]. Loading of AgNPs on OCP crystals was performed using polydopamine (PDA) as functionalizing and reducing agent [7]. The oxidative polymerization of dopamine on the surface of OCP crystals provided a stable PDA film on their surface, which triggered the reduction of silver ions. AgNPs loading up to about 8.2 wt.% provided systems able to support osteoblast viability and differentiation and to inhibit the growth of relevant sensitive and multidrugs-resistant bacteria [7].

3.7

Concluding remarks

The role of foreign ions on the properties of OCP, although investigated by a limited number of studies, has been addressed for some time now and has provided useful information to clarify the biomineralization processes of the hard tissues of vertebrates. On the other hand, the development of OCP functionalized with biological relevant ions, as well as with amino- and poly-amino acids, proteins, and drugs, with the aim to imbue the inorganic phase with specific properties and improve its biological performance, is a relatively young field. However, the growing interest toward the use of OCP as biomaterials for applications in the musculoskeletal system suggests a promising future for the development of researches in this field. Extension of the studies toward further functionalizing agents and enrichment of the characterization with in vitro and in vivo tests would give a considerable boost to the development of OCP-based biomaterials for local delivery of therapeutic agents.

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