Solution growth of spherulitic rod and platelet calcium phosphate assemblies through polymer-assisted mesoscopic transformations

Materials Science and Engineering C 33 (2013) 2175–2191

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Solution growth of spherulitic rod and platelet calcium phosphate assemblies through polymer-assisted mesoscopic transformations Vassiliki A. Kosma, Konstantinos G. Beltsios ⁎ Department of Materials Science and Engineering, University of Ioannina, Ioannina GR 45110, Greece

a r t i c l e

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Article history: Received 28 June 2012 Received in revised form 3 January 2013 Accepted 17 January 2013 Available online 26 January 2013 Keywords: Apatite assemblies Apatite precursors Gelatin Morphological transformations Chrysalis transformation Reinforcement

a b s t r a c t Solution growth of apatite its precursors in the presence of urea commercial gelatin is found to lead, under appropriate conditions, to a rich spectrum of morphologies, among them high aspect ratio needles in uniform sturdy spherulitic assemblies resulting from a herein documented morphological ‘Chrysalis Transformation’; the latter transformation involves the growth of parallel arrays of high aspect ratio needles within micronscale tablets the formation of a radial needle arrangement upon disruption of tablet wrapping. A different level of gelatin leads to the formation of sturdy platelet-based spherulites through another morphological transformation. We also probe the role of four simple synthetic water-soluble polymers; we find that three of them (poly(vinyl alcohol), polyvinylpyrrolidone and polyacrylamide)) also affect substantially the assembly habits of apatite; the effect is similar to that of gelatin but the attained control is less perfect/complete. The case of poly(vinyl alcohol) provides, through variation of the degree of hydrolysis, insights as regards the chain architecture features that might favor morphological transformations. Morphological transformations of particle assemblies documented herein constitute novel ways of generating dense quasi-isotropic reinforcements with high aspect ratio ceramic particles; it becomes possible to tailor calcium phosphate phases at the structural level of crystal assembly. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Most literature references to apatite (hydroxyapatite), a crystalline ceramic substance (ideally: Ca10(PO4)6(OH)2, stems, directly or ultimately, from the occurrence of a variety sometimes called dahlite as a reinforcing phase of natural biocomposites, such as the bone and the enamel and dentin parts of teeth [1–3]. The components and microstructures of biocomposites can provide important guidelines for the development of man-made materials intended for bio-related uses. Yet other structural applications are also conceivable. For example, as apatite exhibits a modulus of elasticity higher than that of glass (100–130 GPa vs. 70–90 GPa), high aspect ratio apatite particles incorporated in polymer matrices can offer, on a volume fraction basis, enhanced reinforcement compared to chopped glass fibers. While we will not proceed to the development of composite materials in this work we will refer briefly, for future technological consideration, to one important aspect of short-fiber isotropic man-made composites, namely processing. Short glass fibers can be added to polymers individually in a, more or less, random manner; then the achievement of high fiber loadings can be obstructed by the formation of a highly entangled mesh of fibers which enhances greatly effective melt viscosity. There are two common ways out of the latter situation: the short fibers will either have to become locally parallel ⁎ Corresponding author. Tel.: +30 26510 07206; fax: +30 26510 07034. E-mail address: [email protected] (K.G. Beltsios). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.01.041

(this is possible especially in the presence of strong flow fields but the isotropic character of the composite is undermined) or become shorter and shorter (and hence less and less effective as reinforcements) [4–6]. The locally parallel character of short fibers is essentially the outcome of a phase transition from an isotropic to liquid crystalline (lyotropic-type) dispersion but at a scale larger than the molecular one, which, for example, is also the case for rod-like tobacco mosaic virus (TMV) dispersions. For rods with an aspect ratio α (=rod length/rod diameter) [7] lyotropic ordering becomes possible for rod volume fractions on the order of 10/α (this is the typical limit for the so-called athermal systems); flow fields will shift the limit to even lower volume fractions. Consequently, for individual short fibers, which typically exhibit α ≥100, sufficiently isotropic and, at the same times, sufficiently processable dispersions are not possible for fiber loads >10%, while short fiber loads desirable for technologically significant composites usually belong to the 25–50% range. On the other hand, if high aspect ratio particles can be pre-assembled into low aspect ratio (such as spherulitic) arrangements without a loss of the high aspect ratio character of the particles of the assembly, then enhanced composite isotropy can be achieved more easily (as lyotropic ordering is not possible); at the same time the viscosity enhancement during the processing of the composite can be limited. The crystallization of any particular substance might be affected by the presence of the most disparate additives. The effect might pertain to one or more of various processes such as primary nucleation, preferential directional growth, formation of crystalline aggregates

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of regular or quasi-regular morphologies etc. Additional possibilities arise when various stable and metastable crystalline or amorphous phases can form and conversions are observed, as for example in the case of bioceramics such as calcium carbonate and apatite. In the case of apatite, and depending on precipitation conditions, octacalcium phosphate (OCP, Ca8(HPO4)2(PO4)4 5H20, with a crystal structure closely related to that of apatite), the sheet-containing compounds brushite (CaHPO4 2H2O), monetite (CaHPO4) and various other calcium phosphate phases [8,9] may precede or compete [10–13]. Certain proteins are considered capable of affecting strongly the crystallization of apatite. This is a conclusion often deduced indirectly, from the fact that apatite-containing biocomposites such as bone, dentin and enamel are characterized by apatite crystals of precisely tailored dimensions and spatial arrangements within a protein-based matrix [14]. Various attempts to establish the assumed interaction in a detailed manner are also known [15,16] but no unchallenged detailed descriptions have emerged so far. Proteins that might interact with the crystallization of apatite include collagen (cases of bone and dentin) and amelogenins and/or enamelins (case of enamel). In view of the final form of bio-apatite, one might consider a specific correspondence between particular proteins and apatite morphologies; for example, collagen is related to ribbon/platelet apatite forms, while amelogenins are related to apatite rods or ribbons [17]. One further conceptual step is taken frequently: particular biomimetic forms of synthetic apatite might be generated, if in vitro crystallization of apatite is attempted in the presence of appropriate proteins. However an in vivo produced material might not be reproduced (as regards precise composition and/or morphology) in vitro, simply by employing the same key ingredients while disregarding the difference in reaction environment. On the other hand, in vitro routes can be more versatile as regards potentially interesting outcomes in view of the diverse options as regards ingredients and processing. The exploration of the modification of a route yielding apatite and other calcium phosphate particles with technologically interesting shapes (e.g. high aspect ratio needles and platelets, loose or, preferably, in easily processable assemblies) upon addition of a low priced protein such a gelatin (or synthetic low-priced alternatives) remains an attractive subject both for fundamental and applied research. Commercial gelatin results from industrial processing of various collagen-rich sources and can be described approximately as a generic and crosslink-free (and hence soluble) collagen; the fact alone that the exact monomer sequence of collagen chains [18] depends on collagen's origin (at least 28 types of collagen are known so far [19]) implies that, in general, any two commercial gelatin products are not identical as regards composition; in addition, the chains of any single commercial gelatin product might well show some variation as regards the exact monomer sequence. Proteins are natural polymeric substances with a backbone bearing peptidic (amidic) bonds. Research on the in vitro crystallization of apatite in the presence of various amidic substances, not necessarily polymeric ones, has shown that such substances tend to exert a substantial influence on the pertinent crystallization processes. This may be thought as direct evidence that substances bearing amidic bonds are capable of affecting apatite crystallization; again, as regards biocrystallization, in vitro findings are not necessarily related, at least in a straightforward manner, to the formation of biological apatite. Extracted proteins, such as amelogenin from teeth, amides such as formamide and dimethylacetamide [20], urea [10,21] and aspartic acid [22] constitute examples of amidic substances shown to have an in vitro effect, as judged primarily on the basis of the experimental outcome. Also relevant to the amide bond–apatite interaction considerations are the findings from chromatography application studies of beds of preformed apatite particles ([23] and references therein). These experimental studies show that apatite particles exhibit a strong binding capacity for globular proteins and also for various types of collagen. Parallel detailed theoretical considerations by Kawasaki et al. [24] are of related interest.

The employment of a specific protein for the in vitro control of the crystal morphology of ceramics occurring in (natural) biocomposites has also been attempted before in the case of other ceramics having biological counterparts. A familiar example is calcium carbonate, which constitutes, in the form of platelets, the reinforcing phase of nacre. Nevertheless, the extraction of proteins by costly processes does not guarantee a highly rewarding structural bioceramic outcome; for example, the control might be substantial as regards to the precise phase that precipitates while the effect on morphology can be unremarkable [25]. In any case, neither successes nor failures exclude the possibility of a substantial effect of non-amidic additives during the in vitro growth of ceramics having biological counterparts. The moderate complexity of the monomer sequence of collagen and the fact that collagen is a non-globular protein (hence the full monomer sequence [18] is not intended to 'simply' guarantee an accurately tailored globule-surface landscape) suggest that collagen in biological environments participates in multiple interactions. Some of the biological interactions of collagen chains are not related to the precipitation of apatite; e.g. there are interactions between chains for the formation of collagen's triple helices. At the same time, some of the biological interactions of collagen might be possible regardless of the exact collagen (>gelatin) chain conformations, while this is not so for globular proteins (though, conceivably, other unintended but interesting interactions might arise for ‘unnatural’ conformations of a given globular protein or a fragment of it). In any case, one should not expect that the combination of a generic mixture of collagens (in the soluble form of commercial gelatin) and a generic precipitated apatite (as various special compositional features of biological apatites are overlooked [26]) hardly duplicates accurately any natural collagen–apatite pair; as a matter of fact additional biological components might also regulate in vivo some important features of what is usually described, in simplified terms, as a natural ‘collagen–apatite’ pair. Yet this work shows that we can take advantage of one or more of the natural apatite (etc.)–collagen (>gelatin) interactions for something technologically attractive, yet not directly related to subjects such as bone regeneration etc. Simply put: we need not use apatite–collagen interactions ‘only as directed’. As a matter of fact, dissolved gelatin is versatile enough to affect in substantial, and possibly non-generic, ways the in vitro precipitation of other bioceramics, such as CaCO3 [27,28] and even SiO2 [28]. Our present work explores the consequences of water-soluble polymer extensions of the solution synthesis of apatite presented by Zhang et al. [10]. The latter authors employ ordinary inorganic sources of calcium and phosphate ions at a stoichiometric ratio, an acidifier (nitric acid) and urea, a small-molecule amidic compound. Apart from the simplicity (and low cost) of the compounds involved and that of the overall synthesis, we adopt (and adapt) the latter route [10] in view of its morphological outcome which is of interest for composites applications: the route leads to individual apatite needles/rods exhibiting a substantial aspect ratio (typically 40–100) and lengths of 50 to 150 μm. The high aspect ratio rod/needle apatite form is not the most common morphological outcome of published in vitro routes, as most of the latter lead to low aspect ratio (ca. 1 to 10) particles (e.g. [29–31]). From the perspective of polymers loaded with ceramic phases, low aspect ratio particles can be best described as fillers (typically raising the elastic modulus of the polymer to a value in the range of 4–9 GPa) and not as substantial reinforcing agents. Of course there is no single morphology that is the optimum one for all possible applications. Our basic polymer modification of the Zhang et al. [10] route involves gelatin as the polymer additive. Further, we take into account that hydrogen bonding is a gentle but rather general and, frequently, rather effective type of interaction in aquatic systems. Such an interaction is, in principle, possible between amidic substances and apatite but appropriate non-amidic substances may also be capable of a related interaction; hence we also consider briefly, as alternatives to gelatin, polyvinyl alcohol (PVOH) and additional water-soluble polymers bearing groups such as -NH2,

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-O- and others. Our work probes the evolution of both phases and morphologies of evolving calcium phosphates and emphasizes the control of the latter; a corresponding materials engineering target is the tailoring of low aspect ratio assemblies of high aspect ratio ceramic particles for reasons already exposed in the first paragraph of the present section.

2. Experimental procedure 2.1. Chemicals and hydroxyapatite synthesis For the polymer-free version the standard aqueous solution contains 0.10 mol/l (NH4)2HPO4 (Reagent grade, Aldrich), 0.167 mol/l Ca(NO3)2 4H2O (Reagent grade, Aldrich) and 0.017 mol/l urea (Reagent grade, Aldrich), all amounts expressed per liter of solvent (distilled water). For all experiments 100 ml of distilled water and 250 ml spherical bottles are employed. Subsequently nitric acid is added dropwise to a pH = 2.34. Reaction is carried under reflux at 95 °C for 48 h (tstandard, sufficient for full conversion to apatite, in the absence of polymers). Products obtained are filtered, washed with distilled water, then ethanol, then again with distilled water and drying follows. All reactions reported are carried for tstandard unless noted otherwise. The issue of stirring requires special attention and is discussed in detail below. For reactions involving a polymer, the polymer is dissolved first at an appropriate temperature (circa 55 °C for gelatin, circa 85 °C for PVOH, circa 30 °C for PEO) and the addition of calcium and phosphorous sources and urea at the aforementioned levels follow. Nitric acid is then added drop-wise under stirring until a clear solution is obtained. Again, the standard conditions correspond to 95 °C for 48 h (=tstandard) under reflux; shorter and longer reaction times are specified in terms of tstandard. The standard amount of commercial gelatin added (standard food-grade, Knox) is 5 g per 100 ml of distilled water. We have also added gelatin at a level of 0.0 g, 0.2 g, 1.0 g, 10.0 g, 15.0 g and 20.0 g per 100 ml of distilled water. The highest amount of added gelatin (expressed per volume of distilled water) differs from the smallest one by a factor of 100. At the 0.2 g/100 ml level the overall effect of gelatin addition is found to be rather weak; hence additions at the b 0.2% level are of no morphological interest. On the other hand at the 20 g/100 ml level a tendency for early inhomogeneity is observed. Hence the full range of dissolved gelatin content exhibiting a potential morphological interest has been probed via discrete steps of reasonable size. We also replace gelatin by the following synthetic water-soluble polymers: (a) 80% polyvinylalcohol (PVOH) hydrolyzed with a nominal molecular weight of Mw = 9000–10000 (Aldrich), (b) 87–89% PVOH hydrolyzed, Mw = 85000–124.000 (Aldrich), (c) Polyethyleneoxide (PEO) with a nominal weight of 400, 1500 and 8000 (Aldrich), (d) Polyvinylpyrrolidone (PVP) with Mw = 29000 (Aldrich), (e) Polyacrylamide, Mw = 10000 (Aldrich). For the structure of gelatin and other employed polymer chains see Fig. 1.

Table 1 Dominant calcium phosphate phases for the of 5% w/w gelatin system at different reaction times. Samples

Temperature °C

Time (h)

Gelatin content %w/w

Calcium phosphate phases

S-0.1 tstand S-0.3 tstand S-0.6 tstand S-0.75 tstand S-0.85 tstand S- tstand S-3 tstand

95 95 95 95 95 95 95

4.8 14.4 28.8 36 40.8 48 144

5 5 5 5 5 5 5

Monetite Monetite Monetite Monetite Monetite Monetite + HAP Monetite

Fig. 1. The polymers which are employed in this work. (a) Gelatin. The dominant aminoacid sequence of gelatin is shown. Gly = glycine (the smallest of aminoacids), while frequently X = proline and Y = hydroxyproline. The precise aminoacid sequence follows no obvious/simple pattern and reflects that of the mother collagen(s). (b) Polyvinylalcohol. The (fully hydrolyzed) homopolymer is shown. We actually employ partially hydrolyzed grades, i.e. random copolymers of vinyl alcohol (\CH2CH(OH)-) and vinyl acetate (\CH2CH(OCOCH3)-) monomers; the higher the degree of hydrolysis the longer, in general, the uninterrupted vinyl alcohol sequences. (c) Polyethyleneoxide. (d) Polyvinylpyrrolidone. (e) Polyacrylamide.

2.2. Characterization A scanning electron microscopy (SEM) was used to observe sample morphology and microstructure and for the Energy Dispersive Spectra (EDS) analysis. All samples were sputter coated with gold and examined using a JEOL JSM-6400 V at 20 kV. X-ray diffraction (XRD) patterns were collected on a D8 Advance Bruker powder X-ray diffractometer and the samples were scanned from 2θ = 10 0 to 2θ = 60 0. Fourier transform infrared spectra were recorded in a spectral range of 4000–400 cm −1 at a resolution rate of 2 cm −1, by using a FT-IR 8400 Shimadzu, employing a KBr pellet method. 3. Results and discussion 3.1. The system of phases Three calcium phosphate solid phases are encountered during the course of the reaction; monetite (Ca/P atom ratio = 1.00), octacalcium phosphate (OCP, Ca/P atom ratio= 1.33), apatite (Ca/P atom ratio → 1.67). Monetite blocks precipitate first, while high aspect ratio particles (ribbons, platelets or needles) of OCP and, eventually, apatite follow. OCP and apatite exhibit related crystal structures; the structure of OCP can be described as an alternation of apatite-type and hydrated layers [9]. Also frequently, the morphology of OCP domains dictates the morphology of the final apatite domains and OCP conversion to apatite can be a gradual one. While three calcium phosphate phases are pertinent to the system, a simpler, two-phase, description (precursor monetite vs. final OCP or HAP) will be adopted; the distinction between OCP and HAP will be considered only when it is pertinent to important morphological details. 3.2. Reference system: Morphological evolution in the absence of polymer Our reference, gelatin-free, system is falling within the range of systems explored by Zhang et al. [10], who report both monetite and OCP intermediates. The latter work should be consulted for a discussion of occurring reactions and an analysis of IR spectra. The aim of

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the latter authors was the formation of individual (non-assembled) apatite needles and they report, under optimized conditions, lengths of 50–150 microns, diameters ca. 0.1–0.15 microns and aspect ratios of 40 to 100. We observe similar trends though we detect a somewhat richer morphology (3 families of needles, 1 family of ribbons and some spherulites); minor morphological differences might result from the precise mode of stirring. We will discuss the precise morphology and interpret its origin in a later section, as mechanisms can be better understood following detailed consideration of the 5% gelatin system (see below). 3.3. Basic modified system: 5% w/w gelatin content The dominant morphological feature is that of spherulitic apatite (Fig. 2a). Apatite is found to form simple spherulites, i.e. without branching, a situation which actually is common for inorganic spherulites, while ordinary polymeric spherulites are heavily branched. Also the apatite spherulites observed are of high perfection and appear to exhibit a relatively narrow size distribution with a diameter of ca. 30 microns. The individual spikes/needles have a width in the 500–700 Å range (Fig. 2a-inset).The width of the needles combined

with a length of ca. 15 microns, leads to a needle aspect ratio on the order of 200. As spherulitic aggregates of needles are produced in the presence of gelatin, while similar but distinct needles are produced under the same conditions in the absence of gelatin, the simplest possible hypothesis is that needles continue to grow in a similar manner but now emanate in groups from nucleation centers involving gelatin. However, a detailed probing of structure evolution reveals a substantially different and surprising spherulite formation mechanism which involves a morphological ‘Chrysalis Transformation’ (see below). Another morphological feature is that of tablets with sharp edges, a continuous wrapping and an internal morphology of bundles of rods/ needles or ribbons; tablets constitute a minor portion (ca. 1%) of the final 5% gelatin sample (Fig. 2b). The basic structural features of a tablet with rods, i.e. both the wrapping and the emerging rods, are preserved after firing at 550 °C in the air (not shown); hence wrapping should be, at least predominantly, an inorganic one; an EDS examination of few wrappings empty of needles indicates a Ca/P ratio of ca. 0.92, i.e. a ratio comparable to that for monetite. In addition the full geometry of the final (spherulitic) assembly is not affected unless subjected to a prolonged heating at 850 °C; under the latter conditions slow coarsening of needle branches is observed. While temperature elevation modifies gradually the inorganic phase (which is so for plain hydroxyapatite as well) the assembly morphology persists and, hence, does not rest heavily upon an organic part. The present authors are not aware of any similar morphological feature (tablets with bundles of rods or ribbons as an interior) produced via crystallization of manmade materials, though somewhat related morphologies are found in certain natural biocomposites. For example, Lowenstam and Wiener ([1], Fig. 6.5) present a mollusk-shell image of cylindrical bundles (ca. 0.5–1 μm wide) of parallel calcite prisms within a protein-based wrapping (‘sheath’); nevertheless, our wrapping is, predominantly at least, an inorganic one. In addition, the ‘packaging’ in the aforementioned biological example is stable, while in our case when the development of needles is completed the needles leave the wrapping in a coordinated manner, not unlike an insect undergoing a metamorphosis; as it will be discussed in more detail below, tablets with needle-bundle interior (such as those in the inset of Fig. 2b) and spherulites (such as those in Fig. 2a) are closely related as the former are the immediate precursors of the latter. Finally the mild morphological similarity between our needlebearing tablets and the aforementioned example presented by Lowenstam and Wiener should not be taken as a strong indication of similarity as regards formation mechanisms. This is because similar morphologies can be produced by rather disparate mechanisms and, as a matter of fact, the present work documents such a case: in addition to Fig. 2a-type spherulites we also observe, sometimes within the same sample, similarly sized spherulites resulting from ordinary spherulitic growth. 3.4. Precipitate evolution as a function of time

Fig. 2. Morphologies at t = tstandard for the standard experiment (5% w/w gelatin) (a) Spherulitic aggregates of needles which emanate in groups from nucleation centers involving gelatin. The individual spikes appear to have a width in the 500–700 Å range (inset). Fragments of the tablet wrappings can be seen in the periphery of the central group of spherulites. (b) A different sample area: the upper triangle which includes top-left corner of the micrograph is dominated by spherulites (such as those of Fig. 2a), while the lower triangle is dominated by a cluster of tablets; the inset shows two tablets with an internal morphology in the form of bundles of parallel growing rods or ribbons.

The reaction in the presence of 5% w/w gelatin in our pristine solution was performed at different fractions of standard time (=48 h) as shown in Table 1 in order to observe intermediate products and understand the origin of the attractive final morphologies presented in Fig. 2. Fig. 3a depicts the FT-IR spectra of the samples formed the different fractions of standard time. Before tstandard we observe the bands assigned to the stretching and deformation vibrations of phosphate and acidic phosphate groups, whereas HAP bands are missing; with progressing time the area attributed to acidic phosphate group vibration decreases, an indication of conversion of precursor monetite crystals to HAP crystals. At tstandard we observe the bands at 563, 605, 1054 and 1095 cm −1 of PO43− and 873 cm −1 which is attributed to ΗΡΟ42−, as well as the band at 3571 cm −1 due to the stretching

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exception of a brief comment at the end of the present section we will limit our further considerations to times up to tstandard. Morphological study using SEM/EDS suggests the following:

Fig. 3. a, b: FT-IR data and XRD patterns respectively of the samples in the presence of 5% w/w gelatin at different fractions of standard time. The stretching band at 3571 cm−1 originating from OH− groups is visible at tstandard, whereas the libration band at 635 cm−1 is missing; the sample at tstandard is HAP, but not a well crystallized one. Τhe ν3 band at 1095 and 1054 cm−1 and the ν4 bands at 563 and 605 cm−1 are characteristic of PO43− ions and they are clearly visible in all samples. The sharp absorption band of HPO4 2− at 873 cm−1 is due to P\O(H) stretching vibrations and is visible even at tstandard. The two techniques are in good agreement and suggest that HAP evolves from monetite largely at a time >0.85tstandard while upon substantially longer processing (such as for t= 3 tstandard) the system can revert to monetite.

vibrations of OH− ions. However we must mention that the band at 635 cm−1 due to the librational motion of OH− was not observed, indicating that the sample received at 48 h was not well crystallized HAP. For t = 3 tstandard the FT-IR spectrum is, roughly speaking, to spectra for t b tstandard. Fig. 3b shows the XRD patterns of the aforementioned precipitants. In all samples before tstandard the XRD patterns were characteristic of monetite. However there is a peak at 2θ = 32.9° which appears in all samples and is attributed to HAP. We observe that the peak intensity is raised upon raising reaction time, so the products at 48 h are equivalent to HAP with a fraction of monetite (: peaks at 2θ = 26.5°and 2θ = 30.5°). As with the case of FT-IR spectra, the XRD spectra (based on ICDD PDF 24–33 and 34–10) suggest that composition-wise the conversion for t = tstandard is higher than that for t = 3tstandard. In addition, morphology (geometry of crystals and their assemblies) is equally important for this work and cursory examination has not revealed any morphological benefit from continuation of the standard experiment for times beyond tstandard; hence with the

(a) For t = 0.1tstandard, block elements 1made of monetite according to Fig. 3b and exhibiting various sizes appear (small dimension mostly in the 2–10 micron range, long dimension mostly ca. 30 microns but varies from 10 to 50 or more). Blocks are often found in radial (cylindrical) arrangements ca. 30–50 microns in diameter (Fig. 4a); helicity is present in some other arrangements while fully spherical block aggregates are practically absent. Apparently the scale for the long dimension of the final product is largely defined already as the long dimension of the blocks is comparable to the length of the final spherulite spikes; X-ray study of the 0.1 tstandard product reveals that the dominant phase is monetite. (b) For t = 0.3 tstandard we observe blocks that, on an individual basis, have similar dimensions to those for t = 0.1 tstandard but some larger aggregates along with some more haphazardly assembled ones are also detected (not shown). (c) For t = 0.6 tstandard we observe blocks in arrangements comparable to those for t = 0.3 tstandard and, in addition, we observe some ‘spongy’ (more precisely: finely structured) objects (Fig. 4b). The latter objects: (a) have a long dimension of 30–50 μm, while a width on the order of 10–20 μm can be distinguished in some cases, (b) include the remains of a flat and relatively compact surface, (c) exhibit an imperfectly radiating microstructure with each object having its own center, (d) can either occur in aggregates or as isolated objects, (e) very rarely can start growing even as early as t = 0.3tstandard. It appears that the ‘spongy’ aggregates result from the OCP to HAP transformation of blocky aggregates. Nucleation is a surface event but some material from the underlying block might be consumed (Fig. 5a). Still these spongy objects might serve as precursors of some of the urchin-like spherulites observed at t = tstandard, provided that ribbons (Fig. 5b) split and straighten by t = tstandard. (d) Arrangements of blocks continue to prevail at t = 0.75 tstandard (36 h). A minority of material (possibly not exceeding 10% of the total precipitate) has already adopted spherulitic forms directly related to those observed at t = 0.6 tstandard (Fig. 6a). In addition, at t = 0.75 tstandard we detect the earliest, still very sparse, events of conversion of the interior of compact close-packed tablets to stacks of parallel needles (Fig. 6b, main and inset). At longer times the latter conversion will be completed and the type of spherulites dominating the final structure will be obtained upon operation of the morphological ‘Chrysalis Transformation’ (see below). Finally, in the same figure (Fig. 6b) we see another tablet to the left of the one 1 Blocks and tablets. We use the term ‘block’ to refer to units which are originally made of monetite; block thickness values in the range of ca. 4 to 25 μm have been measured at different times and/or areas of the sample. Blocks are already present at t = 0.1 tstandard, some can still be detected at t= 0.85 tstandard and none is present at t = tstandard. We use the term ‘tablet’ to refer to units made of calcium phosphate precursors with a composition progressing towards that of HAP; tablets exhibit a thickness in the range of ca. 0.5 to 3.3 μm (i.e. they are thinner than our ‘blocks’) and the thicker of them eventually undergo the ‘Chrysalis transformation’ (see Section 3.5). Tablets are absent at t = 0.3tstandard, they exhibit a limited presence at t = 0.6tstandard, a massive presence at t = 0.75 tstandard and undergo the ‘Chrysalis Transformation’ except for a minority which exhibits a subcritical thickness as regards the latter morphological transformation. Blocks appear to split, possibly because of the stress from conversion, into tablets during the progress of compositional changes of monetite (ultimately towards formation of HAP) in the solid state; yet, the solution phase also plays a role as it contributes species necessary for compositional changes and, more generally, constitutes the vehicle for dissolution and precipitation events throughout the period of the experiment. The above description applies strictly to the 5% gelatin system which has been explored in detail, while qualifications might be necessary for some of the explored varieties.

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Fig. 4. (a) Morphology at t = 0.1 tstandard, (b) Morphology at t = 0.6 tstandard.

transforming to a stack of parallel needles and attached to it; this additional domain apparently exhibits a thickness below the critical one for transformation into a stack of parallel needles. On the basis of numerous observations for t = tstandard the critical thickness for the latter transformation appears to be ca. μm. The, apparently, subcritical tablet of Fig. 6b begins to split into ribbons while subcritical tablets appear to remain morphologically intact when encountered at t = tstandard. Furthermore, the first generation of spherulites, that resulting from surface nucleation, will be a minority, as packing prevents the majority of blocks from having a large free surface; in addition, the branches of the same type of spherulites begin to straighten while some kind of lobes/subdomains is also observed (Fig. 6a). The final form of the first generation spherulites in consideration might not be immediately distinguishable from the second generation of spherulites which constitutes the majority of the t = tstandard product. Nevertheless for other reaction conditions the two populations can be easily distinguished both during early and late stages of structure evolution. (e) At t = 0.85tstandard (40.8 h) the morphological features (not shown) are almost the same as that for t = 0.75tstandard, so the massive conversion of the blocks to tablets with needles, then to fans and finally to second generation spherulites (Fig. 2) occurs during the final 7 h of the reaction. (f) At t = 3tstandard the only peculiar morphological feature is the presence of some relatively round objects with a smooth surface (not shown); accidental surface defects reveal that the latter

Fig. 5. Details pertinent to 0.6 tstandard ‘sponges’: (a) Nucleation of a ‘spongy’ object on the surface of a block; the inset shows fully expanded ‘spongy’ objects with a radius of ca. 30 μm, (b) ‘Spongy’ objects actually consist of approximately-radiating ribbons with a thickness of less than 0.1 μm and a width of 0.3–0.5 μm.

objects exhibit a grainy fine structure. From the study of the product for t = 3tstandard and also for selected intermediate times (tstandard b t b 3tstandard; pertinent data are not reported herein) it appears that a (local) morphological optimum is attained at t ≈tstandard. 3.5. Ordinary spherulitic formation and the Chrysalis transformation On the basis of our observations needle-type spherulites appear to evolve by two distinct mechanisms at least in the case of the standard experiment: I. Ordinary spherulitic formation. A precursor calcium phosphate compact object, favors the formation (nucleation), at its large surfaces (if free), of a spherulitic assembly of elongated objects (approximately ribbon-like, emanating from a common nucleus area and arranged in an approximately radial manner). Matter for such spherulites can be transported from the liquid phase or can be directly drawn from the substrate or both. In view of their dimensions, the ribbons at some further stage of the transformation split into straightened single crystal apatite needles with approximately the same thickness as the ribbons. Portions of spherulites or full spherulites result from this process. II. Chrysalis transformation. First we will present briefly the reasoning behind some new terminology. The terms ‘transformation’ (common in materials science)

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Fig. 6. Details pertinent to 0.75 tstandard structures: (a) First generation spherulites with branches that are more straightened (inset) compared to those seen for t = 0.6 tstandard, (b) Two tablets. The upper tablet begins to split into finer tablets/ribbons while bundles of parallel needles evolve in the interior of the lower tablet (see also inset); a new spherulite on its lower and free surface of the lower tablet without obvious consumption of substrate material.

and ‘metamorphosis’ (common in entomology) are literally nearly equivalent ones; here we encounter a striking mesoscopic transformation of the morphology of a particle assembly following the disruption of a wrapping. We suggest the term ‘Chrysalis transformation’2as a description resting both on materials science and entomology languages; we will also borrow an additional term from entomology (‘ecdysis’) for the description of an important step of the Chrysalis (/Chrysallis) transformation.

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(c) array of short needles within the tablet contour, (d) array of needles that may start extending beyond the tablet contour, while the wrapping is about to disrupt. When apatite needles fully evolve, the wrapping is disrupted (the ‘ecdysis’ stage; Fig. 7), apparently because of the accumulated stress, and the released 1-D stack of needles gradually spreads out in a fan-like manner, initially yielding a quasi-2-D arrangement of needles and then a 3-D arrangement, nearly indistinguishable from an ordinary spherulite (or a sector of it); i.e. one resulting from radial growth from a center. The latter dimensionality change of the stack of needles is a continuing stress-release process. That is, the process does not necessitate addition or molecular-level rearrangement of material; if the latter process involved a substantial extent of molecular transport of material the stack would have coarsened to a more compact shape. The long dimension of the tablets is frequently equal to the spherulitic radius, though a limited redistribution or even further deposition of material might take place during the final stage of evolution of the spherulitic aggregate mass. One end of the original parallel stack of needles behaves as a fixed center holding the needles together and sometimes has the form of a stem, while the spread of needles is maximized at the other end of the stack. Depending on the original arrangement of neighboring tablets, distinct spherulite segments, butterfly-like pairs, quadruplets or full spherulites can be obtained at the completion of the transformation. Near-flat surfaces sometimes observed in clusters of spherulitic type of material represent areas where 3-D spreading has not been completed. This is because either expansion is slow and an intermediate stage is captured or because full expansion was prevented as a result of overcrowding and/or adjacent stacks meeting at high angles (e.g. 90°). Furthermore very thin tablets (thickness below ca. 1 μm) may not transform to needles (either massively or at all). Here we have focused on the understanding, through exhaustive SEM observations, of the initially highly puzzling morphological part of the transformations; as a matter of fact the ‘Chrysalis Transformation’ can be best described as a morphological transformation at the mesoscopic level. Loosely a transformation leads to a new distinct spatial arrangement of structural units [32]. Here we deal with a mesoscopic morphological transformation as the rearrangement does not take place at the level of atoms or molecules but at the level of nanorods (or platelets in the case of the alternative ‘Van Gogh Transformation’; see below). The Chrysalis Transformation is directly collective and coordinated at the (intra)block-spherulite scale while it is indirectly collective at a larger scale through ‘communication’ via the fluid reaction medium; all blocks of parallel stacks of rods transform

A precursor calcium phosphate eventually forms thin (with a thickness of ca. 1–3 μm) compact tablets; each tablet gradually evolves into a structure consisting of a stack of parallel apatite needles in the interior and a compact wrapping; as discussed previously (Section 3.4d) the formation of the 1-D stack of needles becomes possible when tablet thickness is in excess of ca. 1 μm. Formation of needles is initiated at the lateral, frequently unobstructed, small sides of the tablets and progresses parallel to the long tablet sides. Numerous SEM observations (with some representative micrographs presented in the figures of this work) suggest the following possible succession of surface morphologies of lateral sides perpendicular to the needle direction: (a) array of speckles (= ‘nuclei’), (b) array of quasi ribbon-like objects, 2 We employ the widely recognizable spelling ‘chrysalis’ but the etymologically correct spelling ‘chrysallis’ (from Latin chrysallis, from Greek χρυσαλλις) is preferable.

Fig. 7. Standard experiment. Spherulites from the Chrysalis transformation with an ecdysis event captured at the forefront.

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into spherulites at (approximately) the same time. There is also a size requirement (a critical tablet thickness exists) but this is hardly unusual even for various structural transformations involving rearrangements at the atomic/molecular scale. We have not attempted to determine the phase corresponding to the intermediate ribbons of mechanism (I) (= ordinary spherulitic formation). Literature pertinent to the formation of apatite from calcium phosphate precursors and the case of more standard versions (absence of additional components affecting, at least, morphology) contains in some cases suggestions for intermediate amorphous phases. Xin et al. [33] propose a more complex answer which, nevertheless, is based on observations of transformation induced through electron irradiation; hence the conclusions are not necessarily relevant to irradiation-free cases. Also we have not assessed the precise role of gelatin (or that of other polymers, see below), which can be a multiple one; the fact that certain simple synthetic polymers (see Section 3.8) appear to exert part-only of the overall effect of gelatin is compatible with the multiplicity-of-roles hypothesis for gelatin. Keeping in mind the complexity of the precise range of interactions between collagen and apatite during bone formation we will not opt for a hastened answer for the gelatin-apatite case explored herein. TGA (not shown) suggests that only a minor part of the gelatin added to the solution is incorporated in the precipitated mass. Nevertheless on the basis of the structural outcome we believe that gelatin has a role in forming the centers of primary spherulitic nucleation in mechanism (I) and the centers of fanning in mechanism (II) (= Chrysalis transformation), as loose assemblies of needles prevail when gelatin is omitted. Also the protein affects the lateral dimension of the spherulite branches (either directly or indirectly, e.g. through promotion of lateral association of needles). Finally, the wrapping (and the flaky debris from its rupture) of the tablets in mechanism (II) while it might contain some polymer it is, as it has been mentioned, largely an inorganic one with a Ca/P ratio comparable to that for monetite. 3.6. Further probing of the 5%w/w gelatin case 3.6.1. The role of urea In order to probe the contribution of urea to the precipitation process in consideration we replaced urea with an equal weight of gelatin. The outcome is the type of uncharacteristic morphology presented in Fig. 8a; nothing comparable (as regards the fineness of morphology) to the outcome from the combination of urea and gelatin is produced. The two substances have different roles and, hence, they cannot be interchanged. One aspect of the later claim is further verified by the findings of Section 3.8; the contribution of gelatin is that of a polymer (while urea is not a polymer) exhibiting short but uninterrupted sequences of monomers capable of hydrogen bonding. Distinct needles are produced when urea alone is employed, while the polymer controls the formation of assemblies. Another experiment involved the employment of an excess amount of urea, specifically three times that of the basic experiment. The morphological features are almost the same with that of the basic experiment, as we observe the same types of first and second generation spherulites (Fig. 8b). Finally, the replacement of urea by DMF leads to relatively coarse monetite aggregates (Fig. 8c). In brief, urea is necessary while the enhancement of its amount leads to no obvious morphological benefit. 3.6.2. Replacement of the liquid phase In the case of our basic experiment, the process was interrupted at t = 0.6 tstandard, fluid was carefully removed via a syringe, replaced by an equal volume of distilled water at 95 °C and the experiment was continued for a total time equal to tstandard. In order to avoid the disturbance of the domain arrangement, a small portion (ca. 2–3%) of the original fluid was preserved; hence, in practice, the species still

Fig. 8. Effect of urea. a) When urea is replaced by gelatin the product has the form of irregularly shaped chunks, b) first and second generation spherulites upon using threefold urea and c) the replacement of urea by DMF leads to coarse monetite aggregates.

dissolved in the aqueous liquid phase (calcium species, phosphate species, gelatin etc.) were instantaneously reduced by a factor of ca. 30 to 50. As the Ca/P ratio of the solid at t = 0.6 tstandard is substantially different from that of the solid at t = tstandard, the replacement of the liquid phase might undermine the sequence of events leading to the conversion of calcium phosphate precursors to hydroxyapatite. The basic question to be answered is whether the species found dissolved in the aqueous phase affect the outcome only at the early

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stage of reaction (i.e. primarily up to the stage of precursor precipitation) or if interaction between the dissolved species and the precipitant continues at later stages. In view of the observed substantial differences in final morphology (see below) it appears possible that the interaction in consideration remains substantial at later stages and this is true both for first stage and second stage spherulites. Nevertheless as the replacement of liquid also leads to a disturbance of the Ca/P balance a safer conclusion should be described in broader terms: the species found in the liquid phase continue to affect substantially the final outcome even beyond t = 0.6 tstandard. In particular we do not detect second generation spherulites, despite the fact that a needle-type fine structure begins to evolve, at a slower pace, in the interior of the tablets (Fig. 9a,b). The transformation of precursor material to tablets enveloping needles is clearly seen to proceed as a sharp front (Fig. 9a) from a small lateral surface to the interior in a direction parallel to the large faces of the tablets. On the basis of examination of numerous tablets as regards the extent of the progress of the transformation of the compact interior into needles, we conclude that the liquid replacement in consideration reduces conversion rate by a factor on the order of 5 to 10. Surface-nucleated first generation spherulites are also clearly visible (an example is seen on the right side of the tablet of Fig. 9b); yet it should be recalled that first generation spherulites are already present at t = 0.6 tstandard (Section 3.4). Finally a portion of the sample remains in the form of unconverted blocks (Fig. 10). When liquid is replaced at a much earlier stage, i.e. at t = 0.1 tstandard, precursor vegetation-type agglomerates dominate at t= tstandard

Fig. 9. a, b) Details of needle-type growth within tablets for t = tstandard but upon liquid replacement at t = 0.6 tstandard.

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Fig. 10. An overall view of the precipitant for t = tstandard but upon liquid replacement at t = 0.6 tstandard. First generation spherulites are obvious, especially at the magnifications presented in the insets.

(Fig. 11a). Surface-nucleated first generation spherulites appear sporadically in the periphery of block aggregates (Fig. 11b), while we do not detect second generation spherulites.

Fig. 11. Structure at t=tstandard but upon liquid replacement at t=0.1 tstandard. (a) Large agglomerates of precursor blocks dominate the central perpendicular zone of the image, while material of more fluffy appearance (see Fig. 11b) appears more frequently on the two sides. (b) ‘Fluffy material’: Almost fully mature spherulites consisting of straight polyhedral needles with diameters from under 0.1 to 0.5 μm range.

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In practical terms it is not found to be morphologically beneficial to proceed to liquid replacement either at an early (0.1 tstandard) or at late (0.6 tstandard) stage of the reaction.

reveals that the morphology is also different at the mesoscopic level. Upon continuous stirring, the basic structural entities observed are: (a) Nearly cubic blocks and low aspect ratio plates with a large dimension of ca. 5 μm (Fig. 13a), (b) fine needles with a length

3.6.3. Delayed introduction of gelatin at 0.3 tstandard The structure is substantially different from that achieved when gelatin is introduced from the start; this suggests that gelatin is incorporated to the blocks before 0.3 tstandard. We find that surface nucleation is more massive; possibly gelatin absorbed at the surface of precursor blocks facilitates nucleation of first generation spherulites which are small and at places numerous (Fig. 12a). Tablets (more generally: thin domains) are rare (i.e. the blocks do not split massively into thinner domains) while some blocks develop directly (i.e. without splitting into tablets) needles but in a slow and, apparently, somewhat haphazard fashion (Fig. 12b). We conclude that for a successful ‘Chrysalis transformation’ it is essential to include gelatin from the start (or, at least, introduce it at a very early stage). 3.6.4. Continuous mechanical stirring for the full duration of the 48 h reaction When mechanical stirring is applied for the full duration of the 48 h (= tstandard) reaction, morphologically different products are obtained. At the macroscopic level the continuous stirring product has the appearance of coarsely crumbled chalk, while the product obtained through the limited-stirring process (our standard process) has the appearance of a finer powder. Scanning electron microscopy

Fig. 12. Final products for delayed introduction of gelatin. (a) First generation spherulites are nucleated massively on block surfaces, (b) aggregates of blocks; the two insets document (at successive magnifications) the slow evolution of needles within blocks.

Fig. 13. The morphological outcome for the continuous stirring case. a) Aggregates of nearly cubic blocks mixed with low aspect ratio plates. These may be ultimately a consequence of the breaking action of mechanical stirring on precursor blocks, b) cocoons of needles; these needles are under 0.1 μm thick and short (typical length: 3–5 μm), while their cocoons occasionally engulf spherulites having bundles of needles as branches, c) engulfing cocoons may either start from 2-D needle meshes or from loose but sticky needle-type material entangled around clusters of other entities. Spherulites nucleate on compact solid surfaces or on 2-D needle-meshes.

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of few microns and width often in the range of a few hundred Å (Fig. 13b). Mixed structures from the continuous mixing version should be compared to the substantially uniform product obtained through our gentler ‘standard’ mixing method. In retrospect, as the stacking mode and the size of domains of intermediate phases strongly affect final morphology, it is not surprising that mechanical stirring upsets the final outcome and leads to a more mixed and less interesting aggregate morphology. Apart from the gelatin component and the hydroxyapatite product, the avoidance of continuous vigorous stirring during material growth might also be viewed as a bio-inspired choice characterizing this work. Overall, the ‘Chrysalis transformation’ appears to be a delicate morphological transformation; nevertheless, the final morphological outcome (spherulitic assemblies of high aspect ratio needles) is a mechanically sturdy one. 3.7. Systematic variation of the gelatin content As the new states of aggregation of apatite crystals appear to result from the incorporation of gelatin in solution, it is of interest to probe the effect of the added amount of gelatin; the gelatin content is varied within a range spanning 2 orders of magnitude (0.2% to 20.0%). The same time of total reaction is adopted (48 h), though the gelatin level affects the time required for precursors to convert to apatite, as suggested by the phases reported in Table 2. The crystallographic phase is deduced from a combination of XRD and FT-IR data which are presented in Fig. 14a,b. The combination of the two techniques leads to the conclusion that low gelatin contents for example 0.2 and 1% w/w favor OCP and HAP respectively; in the case of 1% gelatin, both IR and XRD data reveal the precipitation of well crystallized HAP crystals. In the sample with 5% w/w gelatin content, which corresponds to our basic experiment, HAP and monetite crystals coexist, as we have already discussed. A further raised gelatin level slows down conversion to the point that monetite prevails at t = tstandard. The formation of monetite (or other precursors) instead of apatite might be viewed as a weakness if the research goal is limited to the direct formation of apatite; however there are, for example, bio-related applications for which a precursor is more attractive than the final product (apatite). Even for non-bio-related applications a high aspect ratio precursor in attractive forms (e.g. platelet-based spherulites as in the case of 10% gelatin; see below) might still be of interest (e.g. as a reinforcing phase of a composite) either in the as-produced form or following additional, composition-modifying, processing. The subsections that follow provide additional information as regards the outcome for t = tstandard and various gelatin contents. 3.7.1. 0.2% w/w gelatin A low, 0.2%, gelatin content leads to incomplete 2-D agglomerates of fine needles and loose fine needles. The finest of the needles are under 1000 Å wide but usually they are found clustered in groups of nearly parallel units. The needles appear to derive from blocks/tablets that usually start disintegrating from the surface (Fig. 15a) first into

Table 2 Dominant calcium phosphate phases for standard reaction time (48 h) and different levels of gelatin content. Samples

Temperature °C

Time (h)

Gelatin content %w/w

Calcium phosphate phases

S-0 % S-0.2% S-1% S-5% S-10% S-20%

95 95 95 95 95 95

48 48 48 48 48 48

0 0.2 1 5 10 20

HAP OCP HAP HAP + monetite Monetite Monetite

Fig. 14. a, b: Good agreement between the two techniques for samples with different levels of gelatin in solution. Τhe ν3 band at 1095 and 1054 cm−1 and the ν4 bands at 563 and 605 cm−1 which constitute the characteristic bands due to PO43− ions are clearly observable in all samples, whereas the stretching band at 3571 cm−1 and libration band at 635 cm−1 originating from OH− groups is only visible in the case of the sample with 1% w/w gelatin, confirming that the latter is well crystallized HAP. All other samples exhibit the HPO42− absorption band at 873 cm−1; the latter stems from the presence of monetite.

stripes and then into near parallel needles; hence, most needles are not 3-D aggregated. A minority of 3-D agglomerates (spherulites) of thicker needles is also present (Fig. 15b). These spherulites apparently correspond to the spherulites of 5% w/w gelatin through ordinary transformation. Also in the same sample one can see some arrangements of blocks which possibly correspond to material yet unconverted to apatite. Overall, the low (0.2% w/w) gelatin level does not lead to a morphological outcome deviating substantially from that for the gelatin-free case; still gelatin slows down somewhat the conversion and OCP rather than apatite is the dominant phase for t = tstandard. 3.7.2. 1% w/w gelatin Upon raising the gelatin content to 1% w/w we observe spherulites that compared to the 5% w/w gelatin case (basic experiment) are larger (ca. 70 μm on the average) though substantially less uniform (Fig. 16a). In addition, the spikes (rods) are thicker (diameter of ca. 1 μm) and tend to exhibit a hexagonal cross-section (six-sided cross-sections prevail) while the needle tips are flat; for some of them, a grainy fine structure is present at the scale of ca. 150–200 Å but, overall, tips are much more smooth than those found in the 5%

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Fig. 15. Structural features of the 0.2% gelatin product at t = tstandard. (a) Needles appear to derive from blocks/tablets. The inset corresponds to a block that has started to surface-transform into parallel domains which gradually split further into parallel needles. This frequent surface-initiated disintegration reduces the occurrence of fans/ cones and corresponding spherulites, (b) occasional 3-D agglomerates (first generation spherulites) with their needles exhibiting a six-sided cross-section but with a symmetry usually lower than a 6-fold one (inset).

case. Fig. 16b shows a spherulite with a flat surface/basis, a feature suggesting that the underlying formation mechanism for first generation spherulites is ordinary surface nucleation. Overall, the 1% w/w gelatin level does lead to structural features qualitatively similar to those for the 5% w/w gelatin case but the outcome is more perfect in the latter case. 3.7.3. 10% w/w gelatin Upon raising gelatin content to 10% w/w we observe three types of agglomerate structures: (a) stacks of roughly parallel platelets which frequently exhibit a gradual collective bending; also platelets are partially merged, (b) spherulites with platelet-type (aspect ratio ca. 40) (Fig. 17a), (c) lacy spherulites (Fig. 17b). The first two structures are the dominant features, while type (c) aggregates are rare. Again, firing at 550 °C in the air does not affect the integrity of the spherulites (no cracked spherulites are observed) or that of individual platelets (they neither crack nor split into fibrils); i.e. both platelets and their spherulitic assemblies are sturdy ceramic entities. The thickness of platelets of (a) aggregates is comparable-to or larger-than that of the platelets of spherulites (b), while the large dimensions of the (a) and (b) aggregates are comparable (often in the 30–50 μm range). Numerous observations including occasionally captured intermediates (e.g. Fig. 17b, left side) suggest that (a) and (b)

Fig. 16. Aggregates for 5% gelatin. (a) A cluster of second generation spherulites; at the top band a number of cones/spherulite sectors document more directly the operation of the Chrysalis transformation, (b) first generation spherulites.

are strongly related: aggregates (b) can be obtained from a morphological transformation of (a) through substantial collective twists. The twists can be induced from te stresses of microscopic structural transformation. Aggregates (a) are apparently the equivalents of precursor blocks of the 5% gelatin samples and conversion proceeds initially though formation of thinner plates. Further conversion increases stresses; hence twists are enhanced and, simultaneously, plates split into thinner and narrower platelets which form the final arrangement (aggregates (b)). Overall the latter process replaces the Chrysalis transformation which we have observed in our basic (5% gelatin) experiment (also in the 1% gelatin case); the present process is simpler as there is no fine structure (needle) evolution within tablets. The field of botany can certainly suggest a name for the present morphological transformation; yet as the platelet arrangements considered herein are reminiscent of certain groups of strokes seen in Van Gogh paintings we opt for the term ‘Van Gogh Transformation’ for the set of processes leading to the formation of twisted platelet assemblies such as those documented in Fig. 17. In terms of morphology the platelets are reduced-size versions of the original monetite plates that we observe in our basic (5% gelatin) experiment at shorter reaction times. Also because of packing restrictions here the platelets often reach the assembly center through thinner stems. While the enhanced amount of gelatin in solution appears to be responsible for the preservation of the platelet shape (i.e. there is no splitting into needle-like entities), we have not been able to assess the pertinent mechanism at the molecular level. What

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Fig. 17. (a) A representative view of the 10% gelatin outcome. Stacks of platelets and platelet-based spherulites dominate, (b) a fully twisted platelet-based spherulite (upper right side), an intermediate between a stack of platelets and a full- twisted platelet-based spherulite (lower left side); a lacy spherulite is attached to the latter entity and is presented in more detail in the inset.

is impressive is that the whole process is a ‘military’ transformation at the mesoscopic level (while martensitic is an example of a ‘military’ transformation at the lattice level); again the whole rearrangement to platelet spherulites is accomplished with limited-only debris. Another population of spherulites is also observed; they are spherulites with an intricate lacy structure Fig. 17b (inset). While esthetically intriguing, these spherulites result from a more ordinary process; they are the surface-nucleating analogs of the first generation spherulites found to coexist with the second generation spherulites that result from the ‘Chrysalis transformation’.

3.7.4. 20% w/w gelatin Here nearly compact blocks in arrangements with statistical spherical, cylindrical or intermediate symmetry are observed; some macroporosity is present in some blocks (Fig. 18). Blocks display sharply defined edges, while the dimensions of blocks exhibit substantial variation, both in terms of absolute magnitude and aspect ratio. The blocks are made of monetite and, for example, Fig. 18c can be compared to Fig. 4a (inset), which pertains to 5% gelatin and t = 0.1 tstandard; i.e. the main consequence of the employment of an excessive amount of gelatin (such as 20% vs. 5%) is the marked slowing down (by about an order of magnitude in terms of time) of the reaction progress.

Fig. 18. Representative entities observed for 20% gelatin and t= tstandard. Blocks in spherical, cylindrical and related arrangements.

Overall the findings of Section 3.7 suggest that the most interesting outcomes result for gelatin contents on the order of 5–10%. 3.8. Effect of other water-soluble polymers Conceivably Chrysalis and related transformations of calcium phosphate particles can be induced by additives other than gelatin or by totally different means. In this section we consider the replacement of gelatin by selected commercial water-soluble synthetic

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polymers: partially hydrolyzed poly(vinyl alcohol) (PVOH; two products with different degrees of hydrolysis), poly(ethyleneoxide) (PEO; three products with different molecular weights), polyvinylpyrrolidone (PVP) and polyacrylamide. First we consider the case of 80% hydrolyzed PVOH. Fig. 19a and b depict the FT-IR spectra and the XRD patterns of the precipitants. The two techniques reveal that the product for t= tstandard and 5% w/w PVOH (80% hydrolyzed) is well crystallized HAP, whereas a substantially increased polymer content, such as 15% w/w PVOH, leads to monetite crystals (which is also the case for an excessive level of gelatin as a polymeric additive; see Section 3.7). 5% w/w PVOH (80% hydrolyzed) leads to a structure (Fig. 20a) dominated by needle-spherulites, while there is also a minority population of individual rods/needles. It follows that the addition of 5% w/w PVOH (80% hydrolyzed) has some notable morphological impact, as spherulites are not the dominant morphology for the same recipe in the absence of polymer. On the other hand: (a) spherulites are highly heterogeneous, i.e. their branches exhibit widths from the 1 μm range to the 10 μm range, (b) tablets appear to be gradually disintegrating rather than disrupted and collectively emptied and they also bear surface striations (Fig. 20a, inset) suggestive of forthcoming splitting into individual rods; hence a morphological transformation such as the ‘Chrysalis transformation’ cannot operate at least systematically and/or with precision. Overall, 5% w/w PVOH (80% hydrolyzed) does have a morphological effect but the control exerted by

Fig. 20. Morphologies from the addition of PVOH (80% hydrolyzed) and t= tstandard. a) 5% PVOH: a spherulite made of thick rods appears in the foreground while a gradually disintegrating and surface-striated tablet appears in the background; inset shows a set of spherulites with rods of various diameters, (b) 15% PVOH: compact blocks of monetite in radial to quasi-spherulitic arrangements.

Fig. 19. a, b) Representative FT-IR spectra and XRD patterns for two samples prepared via the employment of 80% hydrolyzed PVOH at the levels of 5% and 15%; for 5% PVOH the sample is HAP (note for example the stretching band at 3571 cm−1 and the libration band at 635 cm−1 originating from OH− groups) while for 15% PVOH the sample is monetite, as the conversion reaction is slowed down markedly.

the polymer is not high especially at the tablet-precursor level. A substantially higher level of PVOH (80% hydrolyzed), simply slows down conversion (Fig. 20b; 15% w/w PVOH, 80% hydrolyzed). Subsequently we consider the addition of 87–89% hydrolyzed PVOH at the level of 5% w/w. Compared to the corresponding 80% hydrolyzed PVOH case, the overall conversion is slowed down as an enhanced portion of the sample appears in the form of precursor blocks, while on the surface of some blocks few first generation (surface-nucleated) spherulites of apatite needles with polyhedral cross-sections are observed (Fig. 21a). The observed slowing down of conversion compared to the 80% hydrolyzed PVOH case is not surprising since the chains of the 87–89% hydrolyzed PVOH are longer by one order of magnitude (see below for the effect of polymer molecular weight). A minority of material exhibits the morphology shown in (Fig. 21b). Detailed probing of the latter morphology reveals radial or quasispherulitic arrangements of 0.1 to 0.5 μm-thick ribbon-like crystallites with a typical length on the order of 10 μm and a typical width of ca. 1–2 μm. Imperfect tablet precursors are also detected; they exhibit, at their surfaces (Fig. 21c-inset), the beginnings of the aforementioned ribbons that subsequently group into second generation spherulites and related assemblies. Despite differences, the case of 87–89% hydrolyzed PVOH is the one that compares best, among the synthetic polymers studied in this section, to the case of the gelatin additive as regards the type of morphological consequences especially at the tablet

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Fig. 22. Morphologies for t = tstandard and additives: (a) 5% w/w PEO (Mw = 400), (b) 5% w/w polyacrylamide, (c) 10% w/w PVP. Fig. 21. Products for a 5% w/w PVOH, 87–89% hydrolyzed. (a) First generation (surfacenucleated) spherulites of apatite needles at t=tstandard, (b) Radial or quasi-spherulitic arrangements of 0.1 to 0.5 micron-thick ribbon-like crystallites with a typical length on the order of 10 microns and a typical width of ca. 1–2 microns; t=tstandard. (c) A quasi-spherulitic cluster of ribbons found in the area shown Fig. 21b; a tablet precursor is presented in the inset.

precursor level. It should be noted that in the case of 87–89% hydrolyzed PVOH the uninterrupted sequences vinyl alcohol monomer are longer compared to the case of 80% hydrolyzed PVOH. In Fig. 22a we present the morphological outcome for t = tstandard and 5% polyethyleneoxide (PEO, Mw = 400); isolated needles dominate

and there is no substantial morphological benefit from the addition of the polymer; the only unusual morphological characteristic is the rare occurrence of cylindrical rod aggregates (Fig. 22a, inset). In the cases of polyvinylpyrrolidone (PVP) and polyacrylamide while we have not obtained systematically second-stage spherulites (such as those obtained from morphological transformations observed for 5% and 10% gelatin) we do find instances of precursor structures reminiscent of the needles within tablets seen in the case of 5% gelatin. In Fig. 22b (5% w/w polyacrylamide) we see growing needles within an irregularly shaped pocket while in Fig. 22c (10% w/w PVP) and Fig. 21c we see

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ribbons or platelets growing within a tablet. It is possible that optimization as regards precipitation conditions, polymer (PVP or polyacrylamide) concentration and polymer molecular weight can also lead to completed morphological transformations. On the basis of the results from the employment of either gelatin or the other water-soluble polymers considered in this section we recognize that the addition of polymers in the aqueous phase has two general types of effects on the solution growth of apatite: I. Overall slowing down of apatite formation kinetics Slowing down of apatite formation kinetics upon addition of polymer is true for all water-soluble polymers employed (gelatin, PVOH 80%, PVOH 87–89%, PEO, PVP and polyacrylamide). For instance, the employment of PEO of different molecular weights (400, 1500 and 8000) clearly shows that the slowing down of reactions is stronger when the molecular weight of the polymer is higher. This is a generic effect that amounts to the enhancement of resistance to the exchange of inorganic species between the liquid and precipitant phases. Added polymer enhances solution viscosity (and for a given polymer and concentration the viscosity is higher for a higher polymer molecular weight) and might reduce diffusion coefficients in the liquid phase; in addition, it is possible that chains adsorbed at the liquid–solid interface create a barrier to the transport of inorganic species between the solution and the calcium phosphate solid precursors. Here we note the precipitant weight for the same reaction time (48 h) and 5% PEO of different molecular weights. The relative precipitant weights were: 1 (MW: 400), 0.97 (MW: 1500), 0.91 (MW: 8000); i.e. increasing molecular weights lead to decreasing reaction rates towards apatite formation. As regards the effect of the amount of polymer, the inclusion of different amounts of gelatin was probed by X-ray, EDS/SEM and precipitant weight measurements for the same reaction time (48 h), as before. The relative precipitant weights were: 1.00 (0% gelatin), 0.87 (1% gelatin), 0.76 (5% gelatin), 0.58 (20% gelatin). Qualitatively, the lower the precipitant weight the more limited the progress of reactions towards apatite formation; hence, according to our data, the larger the amount of gelatin the slower the progress of the reaction. The latter trend is also found for PVOH 80%; apparently the trend in consideration is a general one. II. Morphological guidance of intermediate and final ceramic structures through mesoscopic transformations This is a chain-specific effect with proper chains acting on the bulk of the solid phases. One way for the overall effect to be realized is the presence of monomer units bearing sites capable of hydrogen bonding; this hypothesis is compatible with the trends observed in the cases of gelatin and PVOH and also suggested by the cases of PVP and polyacrylamide. Further, for a morphological control at the tablet precursor level it might be necessary for the chains to exhibit uninterrupted sequences of 5–10 monomer units bearing sites capable of hydrogen bonding; this is suggested from the study of the consequences of employment of PVOHs characterized by substantially different degrees of hydrolysis (80% vs 87–89%). Obviously the latter is only a rough guide as, for example, the monomers of different polymers are characterized by different lengths and, in addition, PEO has no clear effect; also our suggestion cannot be extended simply to encompass the case of globular proteins as chain conformations are fundamentally different. Finally, additives having different chemistries might be appropriate as well. Delayed gelatin introduction experiments and experiments of solution phase replacement by distilled water at various reaction times for the 5% gelatin system, suggest that gelatin guiding the mesoscopic transformation is incorporated in the solid ceramic precursor at times no later than 0.3 tstandard, that is well before any mesoscopic signs of the morphological transformations are perceptible (starting at ca. 0.7 tstandard). Polymer chains incorporated in the precursor blocks are not molecularly inserted as we do not observe

new crystallographic spacings or amorphization of the calcium phosphate precursors. Diffractograms show no evidence for systematic insertion of gelatin chains between crystallographic planes of the precursor; hence gelatin is incorporated at a larger scale. We might note here the case of natural apatite–collagen composites: collagen chains guide apatite development at the molecular scale (at least in part) but the outcome is not some form of mixed compound. The precise scale at which chains are inserted during precipitation in our case has not been determined and it might even shift with the progress of inorganic reactions; nevertheless, a scale comparable to that of final needle width is a reasonable educated guess. Finally, as collagen (and, hence, gelatin, the most successful polymeric additive of this work) is not a homopolymer it is possible, at least in principle, that compositionally different short sequences of the protein play some substantially different roles in the mesoscopic transformations in consideration; in the latter case the full range of roles cannot be duplicated/imitated by a homopolymer or imitated well enough by a [synthetic] random copolymer.

3.9. Application potential of the new forms of apatite particle assemblies Among spherulitic types obtained, the following are the most interesting ones: (a) spherulites of radiating needles with an aspect ratio α ≈200 and resulting from a mesoscopic morphological ‘Chrysalis transformation’ and (b) spherulites of platelets with an aspect ratio α ≈40 and resulting from a mesoscopic morphological ‘Van Gogh transformation’. Aspect ratios of 200 (case of needles; see (a)) most certainly suffice for the unleashing of the full reinforcement potential of short fibers [4–6]. Also, on the basis of the principles exposed in the Introduction, any loads of α≈200 loose apatite needles in excess of ca. 5% will tend to exhibit lyotropic ordering; hence, for substantially higher loads (e.g. 25–40%) the composites will be much more isotropic if the α≈200 needles are added in the form of the herein developed spherulites (a schematic 2-D comparison of the two alternatives is presented in Fig. 23). Interestingly, the occurrence of the mesoscopic Chrysalis

Fig. 23. Liquid crystalline arrangement of individual long apatite needles (a) vs. quasi-isotropic arrangement when the long apatite needles are pre-assembled in the form of spherulites (b). Lines represent long apatite needles while the space between needles will be occupied by a polymer matrix. Limited spherulite interpenetration can result from polymer matrix contraction during solidification.

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Transformation during needle fabrication prevents the occurrence of another mesoscopic transformation (in this case an undesirable one), that of liquid crystalline ordering of needles during composites processing. An aspect ratio of 40 (case of platelets; see (b)) might not offer maximum possible reinforcement for asymmetric particles; yet a novel potential solution to another problem encountered during processing of certain composites is suggested. In the case of man-made composites with platelet dispersions the platelets frequently aggregate to form dense arrangements which exhibit very low aspect ratios (e.g. aspect ratios of 1 to 3) and host little or no matrix material between the platelets; in the latter case essentially all reinforcement stemming from the asymmetry of individual particles is lost. Consequently, it is attractive to develop platelet assemblies which guarantee that the platelets in a composite are permanently spaced apart, thus allowing for the easy insertion of matrix material between them. It might be noted that in the case of layered materials such as clays the dispersion of asymmetric particles for the accommodation of matrix material between the latter is pursued via chemistry choices that allow for exfoliation [34]. Both needle and platelet based spherulites have practical interest as ceramic reinforcements of polymeric matrices (for quasi-isotropic composites with moderate elastic modulus values) and we intend to study the applicability of pertinent concepts (outlined in the Introduction) as part of follow up research. Of course this does not preclude the consideration of composites aiming specifically at biomedical applications. 4. Conclusions (i) The gelatin modification of a solution route yielding apatite needles slows down the conversion reactions of the precursor calcium phosphate phases but also allows, for properly selected gelatin levels, for the formation of sturdy low aspect ratio (ca. 1) assemblies of high aspect ratio (ca. 200) needles or moderately high aspect ratio (ca. 40) platelets through mesoscopic morphological transformations, such as the herein documented ‘Chrysalis Transformation’ (for 5% gelatin) and ‘Van Gogh Transformation’ (for 10% gelatin). Limited-only stirring is crucial for the coordination necessary for a fruitful ‘Chrysalis transformation’. (ii) Slowing down of the conversion reactions of the precursor calcium phosphate phases is also observed when various synthetic water-soluble polymers are used in place of gelatin but slowing down alone does not guarantee the polymer capacity to assist mesoscopic morphological transformations; slowing down of conversion is more intense for higher concentration and higher molecular weight of the polymer. (iii) Evidence from the employment of PVOHs (and secondarily from the employment of PVP and polyacrylamide) suggests that water-soluble polymeric additives capable of supporting morphological transformations related to those documented herein for gelatin should exhibit uninterrupted arrays of a minimum of 5–10 monomer sites capable of hydrogen bonding; this might be a necessary but not a sufficient condition and/or might pertain only part of the effect of gelatin, in case of a multiplicity of roles of the latter in the transformations in consideration.

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(iv) Low aspect ratio assemblies of high aspect ratio particles of apatite and related ceramics, such as those presented herein, are attractive reinforcement candidates for easily processable quasi-isotropic composites with a moderate elastic modulus. Acknowledgments Ms. Chr. Roussa is thanked for the preliminary experimental data described in her Diploma Thesis (UOI, Greece, 2007) which was supervised by the present authors. Professors Liao-Ping Cheng and Dar-Jong Lin (Tamkang University, Taiwan) have kindly provided valuable insights and data about the morphology and composition of some of the products considered herein. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]

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