Calcite and Hydroxyapatite Gelatin Composites as Bone Substitution

Article pubs.acs.org/crystal

Calcite and Hydroxyapatite Gelatin Composites as Bone Substitution Material Made by the Double Migration Technique Benjamin Kruppke,*,† Christiane Heinemann,† Anne Keroué,† Jürgen Thomas,‡ Sina Rößler,† Hans-Peter Wiesmann,† Thomas Gemming,‡ Hartmut Worch,† and Thomas Hanke† †

Max Bergmann Center of Biomaterials and Institute of Materials Science, Technische Universität Dresden, 01069 Dresden, Germany Leibniz Institute for Solid State and Materials Research Dresden, 01069 Dresden, Germany

‡

ABSTRACT: The double migration technique, which is based on mineral formation in an electrical field, was used to synthesize composites of gelatin with calcium phosphate and carbonate, respectively, in order to generate a degradable bone substitute material. Gelatin is integrated in the mineralization process to increase the minerals solubility, in analogy to the formation of the bone mineral dahllite. The mineral formation is affected with respect to the particle size, shown by transmission electron microscopy. Machined polyacrylic barriers increased gelatin stability and allowed investigation of the influence of gelatin barrier position and ion concentrations within the mineralization solutions on mineral morphology and structure by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. During degradation of the mineral in simulated body fluid, the possibility of an adjustable calcium ion concentration could be shown, which might be useful to manipulate the osteoblast/osteoclast ratio.

1. INTRODUCTION Natural bone is well-known to consist of 60−70 wt % dahllitewhich is a carbonate hydroxyapatite (HAp)20− 30 wt % organic matrix and 10% water.1,2 Dahllite consists of a proportion of 4−7% carbonate ions. Both the hydroxide ions and the phosphate ions may be substituted. Since bone is wellknown to be more than just its components, the hierarchical levels across all size ranges determine the mechanical and biological properties.3 Besides mechanical properties the mineral component ensures the release of calcium ions. Thermodynamically stable pure hydroxyapatite is not capable of releasing calcium fast enough. As we assume substitute ions and crystal size as the most important points to influence solubility and degradability, a bone substitute material should consider this structure−property relation. Therefore, the mineralization process has to be understood. Interfibrillar mineralization is carried by amorphous calcium phosphate phases and controlled by amino acids of collagen.4 Noncollagenous acetic proteinsshowing high binding capacity to cationsare supposed to control specific nucleation via inhibition to prevent ectopic crystal growth.5−8 These considerations have led us to a basic materials concept, which might be regarded as conservative referring to its components. It is based on denatured collagen (water-soluble gelatin), calcium, and phosphate ions. This approach has previously been chosen considering mineral morphology and hierarchical structures as important as the chemical composition.9−11 For degradable biomaterials, the immune response of the host after implantation is caused by both the initial material © XXXX American Chemical Society

surface and the material degradation products. Therefore, components of the degradable biomaterial are selected according to their familiarity to the human body. Degradation products might function as bone remodelling material after local resorption by osteoclasts and release in the extracellular space. Additionally degradation causes an ever newly forming surface/interface between bone and substitute material. An important task is therefore the avoidance of chronic inflammation by the constant stimulus of the new interface. The interaction of osteoblasts and osteoclasts is a central mechanism in the bone remodelling process. The calcium ion concentration is one key to influence the cellular response by an implantable material.12−15 In the field of biomaterials the ability of binding calcium and phosphate from surrounding medium is considered as ambivalent. Depletion of calcium without cellular regulation, named dystrophic mineralization, detracts essential ions from the cells. Therefore, a constant or slightly increasing release of ions would be a preferred behavior of biomaterials.16 Therefore, the aim of the present study was the preparation of a mineral or a mixture of minerals, able to release calcium ions in an adjustable degree to compensate or inhibit calcium phosphate precipitation from body fluids. Subsequent to our earlier studies using, on the one hand, model dual membrane diffusion systems based on either collagen I17 or chitosan Received: November 1, 2016 Revised: January 16, 2017 Published: January 17, 2017 A

DOI: 10.1021/acs.cgd.6b01595 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Figure 1. Schematic illustration of the double migration method variable in gelatin position with (a) gelatin membrane, (b) polyacrylic barrier with 2 × 2 long-holes filled with gelatin, and (c) polyacrylic barrier with 4 × 6 cylindric holes filled with gelatin. A drawing (b′) of the barrier with 2 × 2 long-holes and photographs of (b″) the local mineralized gelatin after 7 days from two double migration chambers as well as the polyacrylic 4 × 6 barrier with 10 wt % gelatin (c′) before and (c″) after mineralization.

membranes18 and, on the other hand, the electric field-assisted formation of organically modified hydroxyapatite,19 in this study we followed a biomimetic approach to mineralize gelatin under formation of nanometer sized HAp crystals. These crystals agglomerate to particles in sub-micrometer scale (100− 500 nm) with defined hollow spherical shapes. This selfstructuring process takes place in a double migration chamber based on the double diffusion technique intensively investigated by Rosseeva et al.19 In contrast to the double migration technique, the dual membrane diffusion method,17,18 as well as the double diffusion technique19 are not supported by external electrical fields. Within the double migration chamber a stable gel gelatin barrier is placed and two mineralization solutions containing Na2HPO4 and CaCl2, respectively, are added into the opposing reservoirs. To support and align diffusion controlled movement of the ions and movement due to the concentration gradient, an electric field is applied, starting migration of calcium and phosphate ions. Because of the movement of calcium ions from the cathode to the anode into the gelatin and the opposed movement of the phosphate ions assisted by the potentiostatic electric field, the mineralization method is named the double migration method.20 In this study several variations of the double migration setup were investigated to enhance the amount of mineralized gelatin and to modify the resulting mineral according to its degradation in physiological solutions. The degradable composite of gelatin and calcium phosphate and carbonate is considered to be advantageous due to its variable influence on the calcium ion homeostasis, which makes it an attractive candidate for bone regeneration.

room for gelation. Then, after the plates were removed, a gelatin barrier divided the chamber volume into two reservoirs. This barrier made solely from gelatin is stable at 4 °C and accessible from both sides. The ion concentrations, gelatin barrier position, and pH shown in Figure 1a represent the standard parameters of gelatin mineralization resulting in organically modified hydroxyapatite (ormoHAp) as described by Heinemann et al., where ion concentrations were chosen analogously to the Ca/P ratio of HAp (Ca/P = 1.67).20 A potentiostatic electric field of 1.25 V·cm−1 was applied to accelerate the opposed ion movement (called migration) of calcium and phosphate ions, respectively, into and within the gelatin gel membrane. During 7 days, the mineral was formed. Then the mineralized barrier was detached from the chamber. Finally the mineral was collected by removing the gelatin using multiple steps of heating the gel, centrifugation, and resuspension in deionized water at 50 °C. As a last step of mineral separation, the mineral pellet was resuspended in a small amount of deionized water, frozen at −80 °C, and lyophilized with an Alpha 1-4 freeze-dryer (Martin Christ). Variation of Calcium/Phosphate Ratio. The variability of the system allowed mineralizing gelatin using various ion concentrations and various gelatin modifications. The concentrations of the standard membrane mineralization was altered into a Ca/P ratio of 3.33 by decreasing phosphate ion concentration from 24 mM to 12 mM. The position of the gelatin barrier was changed within the chamber, as well, to change the Ca/P ratio. The moveable gelatin barrier was used to change the reservoir volumes and thus to create a Ca/P ratio of approximately 3.89. Since the Ca/P ratio is more precisely adjustable by changing the ion concentrations in the reservoirs than by the displacement of the membrane position, only one varied positioning is shown as an example. Mineralization was performed, as well, without phosphate ions in the anionic reservoir, which was then filled with pure deionized water (Ca/P = ∞). The increasing oversupply of calcium ions and thus the depletion of phosphate ions were thought to integrate carbonate ions in the mineralization of the gelatin. An overview of the different conditions of mineralization conditions is shown schematically in Figure 2. Polyacrylic Barriers. Besides the standard version of an unsupported gelatin membrane barrier, in one piece, machined multihole polyacrylic barriers were used. The cross sections of the holes show either elongated oval or circular shapes. That way, the initial barrier was subdivided into several smaller strongly supported barriers that are more stable during the mineralization process. The acrylic barriers matched in their dimensions the standard gelatin membrane to fit into the double migration chamber. The barriers were machined with a milling cutter to have external dimensions of 98 mm width, 90 mm in height, and 20 mm thickness. Silicone cords were glued to the polyacrylic barriers to seal the reservoirs. The 2 × 2 longholes (Figure 1b and b′ have a radius at both ends of 13.5 mm and a

2. MATERIALS AND METHODS 2.1. Mineral Synthesis and Variation. The mineral synthesis was performed within a stable gelatin gel. Porcine gelatin (300 bloom, Gelita) was carboxymethylated with glucuronic acid. For that, 10 wt % gelatin swelled in water at room temperature for at least 10 min under addition of 125 μg of glucuronic acid per 1 mg of gelatin. Storing the gelatin in a water bath at 50 °C for 24 h allowed carboxymethylation according to the Maillard reaction.21 The gelatin gel was prepared as a barrier between two reservoirs of a CaCl2 solution (Roth) and a Na2HPO4 solution (Roth) of various concentrations, respectively. Therefore, two removable plates were placed with 2 cm distance to each other in the double migration chamber. A total of 200 mL of the heated and thus liquefied gelatin was poured between the two plates, and the whole chamber was stored for at least 4 h at 4 °C in a cold B



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semiquantitative method for standardless analysis of the amounts of oxygen, calcium and phosphorus in selected minerals. Degradation in Physiological Liquids. Degradation of the compacted mineral samples was analyzed in Dulbecco’s phosphate buffered saline without Mg2+ and Ca2+ (PBS, Biochrom) and msimulated body fluid (SBF) according to Oyane et al.22,23 Dulbecco’s PBS consists of 153.1 mM Na+, 4.2 mM K+, 139.6 mM Cl−, 8.1 mM HPO42−, and 1.5 mM HPO4−.22 In contrast to this, m-SBF consists of 142.0 mM Na+, 5.0 mM K+, 1.5 mM Mg2+, 2.6 mM Ca2+, 103.0 mM Cl−, 10.0 mM HCO3−, 1.0 mM HPO42−, and 0.5 mM SO42−. It was designed to have a total ion concentration equal to that of blood plasma, except for the concentration of HCO3−, which was set to the saturated level with respect to calcite.23 For disc shaped samples of 5 mm in diameter and about 1.5 mm in height 50 mg of mineral powder was compacted with the hydraulic lever-press (PerkinElmer) and a load of 20 kN. Degradation investigations of three discs each were performed at 37 °C in an incubator over 14 days. The liquid was changed according to the cell culture rhythm after 1, 2, 4, 6, 8, 10, 12, and 14 days. Degradation was characterized by determination of calcium ion concentration in the removed supernatant after the fluid was changed. Therefore, triplicates each were measured with the colorimetric Fluitest CA CPC test kit (Analyticon) and an Infinite 200 Pro microplate reader (Tecan). Loss on Ignition. Loss on ignition was performed to examine the amount of organics in the mineral. Therefore, 200 mg of lyophilized mineral was weighed into melting pots. At least three samples each were pyrolized in a muffle furnace (TC 405/20, Padelttherm) in air atmosphere at a temperature of 1000 °C for 1 h.24 The loss of mass was calculated using a commercial hydroxyapatite after pyrolysis as reference.

Figure 2. SEM image (a) of spherulitic mineral particles under standard conditions (so-called ormoHAp,20 40 mM CaCl2 vs 24 mM Na2HPO4, equal liquid volumes; (b) some rombohedral and lot of spherulitic particles, 40 mM CaCl2 vs 12 mM Na2HPO4 and equal liquid volumes; (c) spherulitic and dumbbell-like particles after gelatin membrane mineralization with 350 mL of 40 mM CaCl2 vs 150 mL of 24 mM Na2HPO4; (d) solely rombohedral particles from mineralization with equal volumes of 40 mM CaCl2 vs water.

14 mm long parallel part therebetween. This results in a volume of the elongated holes of ca. 20 mL. The polyacrylic barriers were also machined with 4 × 6 = 24 cylindric holes (Figure 1c and c′) of 10 mm diameter as well as 6 × 10 = 60 cylindric holes of 6 mm diameter. The holes of the polyacrylic barriers were sealed at one side with parafilm, filled afterward with heated liquid gelatin, and then stored to gel at 4 °C. The total volume of gelatin for the 2 × 2 barriers is 80 mL, that for the 4 × 6 barrier 43 mL, and for the 6 × 10 barrier 36 mL. The amount of mineral separated from the gelatin for the different barriers was weighted for three different mineralization processes each. Mineralization under N2 Atmosphere. Because every hole might be filled with a different chemical formulation of the gelatin, this kind of barrier allows a higher variability within a single mineralization process. Using the polyacrylic barriers avoids the adverse drying of gelatin in contact with air. Normally, dry parts of the gelatin membrane are formed and hardly separable from the mineral. Because of the new barriers, the gelatin is all the time in contact with the mineralization solutions and that eases the separation process. To investigate the influence of air and especially atmospheric CO2 in the mineral formation, the procedure was performed with previously vacuum degassed CaCl2 solution (40 mM, pH 8, Roth) in the cationic reservoir and deionized water in the anionic reservoir and under N2 atmosphere. The mineral obtained is marked as Ca/P = ∞ hereinafter. 2.2. Characterization. Morphology and Structure Analysis. The morphology of the minerals synthesized with the double migration chambers was analyzed by scanning electron microscopy (Philips XL30 FEG) with 3 kV acceleration voltage. X-ray diffraction data were obtained using a Bruker D8 Discover with scintillation counter and Cu−Kα radiation within the range of 20° ≤ 2Θ ≤ 45° by a step width of 0.04° and 20 s per step. For FT-IR measurements 2 mg of each sample were mixed with 100 mg KBr (Roth). A disk of 25 mg was prepared applying a force of 100 kN with a hydraulic lever-press (PerkinElmer). Infrared transmission was measured with a Spectrum 2000 (PerkinElmer) between a wavenumber of 500−4000 cm−1. Commercially available calcite (Merck) and HAp (InnoTERE) were used as reference material for XRD and FT-IR. Analytical TEM was performed with a Tecnai F30 (FEI) with an accelerating voltage of 300 kV. The mineral samples were removed after separation of the gelatin. The wet mineral paste was resuspended in pure water. Finally, 10 μL of the mineral suspension was dropped on a copper grid and dried at 37 °C. The morphology of the samples was documented by bright field images of typical particles and phases were characterized by energy dispersive X-ray spectroscopy (EDXS). EDXS was applied as a

3. RESULTS 3.1. Influence of Ion Ratio on the Mineral. The ratio of calcium ions to phosphate ions was changed in two ways. On the one hand the concentrations of Ca2+ and PO4+ were changed keeping the volume (250 mL) of both mineralization reservoirs constant. On the other hand the influence of the gelatin barrier position was investigated. Changing the position with constant ion concentrations (that of the standard mineralization procedure) in both reservoirs caused a change in the Ca/P ratio since the volume of the cationic reservoir is increased to 350 mL, and the one of the anionic reservoir is reduced to 150 mL. The standard mineralization parameters (stoichiometric ratio Ca/P = 1.67) resulted in a spherical shaped mineral (Figure 2a). The spheres of 100−500 nm in diameter seemed to be hollow or unfinished as some of the spheres do not have a closed shell. The mineral of Ca/P ratio 3.33 (Figure 2 b) showed a mixture of spherical particles comparable to Ca/P = 1.67 and an amount of larger rhombohedral particles with about 10 μm edge length. An increase of rhombohedral particles with increasing Ca/P was visible. The changed position of the gelatin barrier, leading to a Ca/P ratio of 3.89, led to spherical particles (Figure 2c) of 1 μm in diameter and some elongated particles. For mineralization without phosphate ions in the anionic reservoir (Ca/P = ∞), the mineral consisted exclusively of rhombohedral particles (Figure 2d) with about 10 μm edge length. XRD investigations showed the two mineral structures of calcite and hydroxyapatite, which therefore were used as reference as well (Figure 3). The mineral with Ca/P = 1.67 showed HAp characteristic X-ray diffraction peaks only. These wide broadened peaks were detected for the mineral with Ca/P = 3.33 as well, with decreased intensity. In contrast, the sharp peaks of calcite were increased with an increasing ratio of Ca/P. The minerals Ca/P = 3.89 and Ca/P = ∞ showed exclusively C



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However, the characteristic absorptions of the P−O vibration at 603 cm−1, 564 cm−1 and 960−1085 cm−1 occurred, only with Ca/P = 3.89, 3.33, and 1.67, and they seemed to be very similar to HAp absorptions. TEM investigations of selected samples confirmed ormoHAp spheres (Ca/P ratio: 1.67) to consist of a mineralized shell (Figure 5a and a′). The surface of the spheres consists of needle-shaped particles (Figure 5 a″). Lattice planes indicated by arrows facing in different directions are visible in HR-TEM images showing the presence of small crystallites (l ≈ 10 nm). Spheres are present in Ca/P = 3.33 samples as well (Figure 5b and b′), but here the yield is a mixture of both spheres with a completely mineralized volume and spheres with a mineralized shell only (so-called hollow spheres). EDXS revealed a measured Ca/P ratio in the case of fully mineralized spheres of 1.22 (Figure 5b2) and for the spheres with a mineralized shell of 1.67 (Figure 5b1). In the case of these hollow spheres, needle-shaped particles are visible on their surface, whereas the solid spheres do not show this structural features. They are more sensitive to the electron beam and melt under electron beam irradiation. The mineral with Ca/P = ∞ showed rhombohedra looking at a first glance to be solid crystal (Figure 5c), but gelatin is definitely present since crystallites melt under electron beam irradiation. The edges of the rhombohedron (Figure 5c′) are amorphous in many areas but also contain partly crystalline structures (Figure 5c″, approximately 10 nm) proven by local lattice planes in HR-TEM. Degradation was performed in PBS and SBF. Calcium ion release in PBS, which led to concentrations of less than 0.1 mM, was quite low for all compressed mineral samples. A release during the first hours was measurable for all samples in contrast to incubation in SBF. The release of calcium in PBS was continuously higher for minerals with Ca/P > 1.67 in comparison to standard ormoHAp with Ca/P = 1.67. Storage of compressed ormoHAp samples in SBF (Figure 6a) led to a decrease of calcium ion concentration in comparison to blank SBF. The calcium concentration in SBF after every fluid change is decreased until the next change of liquid to a value of 2.1 mM (after 1 h) and 1.5 mM (after day 14). The crystal mixture of Ca/P = 3.33 caused an initial decrease of calcium in SBF at 1 h (Figure 6 b). Afterward the calcium ion concentration is kept almost constant at the level of the SBF blank. The initial decrease of calcium is lowest for samples made from no-phosphate samples (Ca/P = ∞; Figure 6d). A slight increase of calcium ion concentration up to 4 mM at day two was measured. The calcium concentration was between 1 and 14 days higher than the SBF blank with a measured initial concentration of 2.61 mM. An even higher calcium concentration was measured for samples made of mineral with Ca/P = 3.89 (Figure 6c). After an initial decrease after 1 h, the calcium concentration was increased above 5 mM at 1 and 2 days. Afterward a decrease was measured to the level of SBF blank. 3.2. Variants of the Gelatin Barrier. The standard process of gelatin mineralization was performed with a 2 cm thick gelatin membrane. To ease handling and increase gelatin stability polyacrylic barriers with different holes arrangements were used (f.i. Figure 1b′ and c′). One barrier variant (Figure 1b′) allows the local mineralization of gelatin in 2 × 2 long holes. The mineralized gelatin is shown in Figure 1b″. Two barriers have cylindrical shaped holes of 10 mm and 6 mm in diameter arranged in an array of 4 × 6 (Figure 1c′) and 6 × 10

Figure 3. Mineral structure analyzed by XRD in dependence of the Ca/P ratio with calcite and hydroxyapatite reference peak positions.

calcite peaks. Here, the peaks were much sharper for the Ca/P = ∞ mineral. For FT-IR studies a reference spectrum of the gelatin in the initial state, which was used for the mineralization, was measured besides calcite and HAp as reference (Figure 4).

Figure 4. Experimental FT-IR spectra of different minerals, nonmineralized gelatin, and commercially available calcite and hydroxyapatite.

The major characteristic vibrational bands of gelatin occurred at 3330 cm−1 (amide A), 3070 cm−1 (amide B), 1660 cm−1 (amide I), 1555 cm−1 (amide II), and 1205/1281 cm−1 (amide III).25,26 In addition, the O−H vibration emerged clearly at 3340 cm−1. This broad absorption band occurred in all minerals. The absorption bands of the three minerals with the Ca/P ratios of 1.67 and 3.33 are very similar to the HAp reference. However, the O−H absorption in the range of 1650 cm−1 and the C−O−-absorptions at 1422 and 1457 cm−1 are considerably more pronounced for the produced minerals. In this case, an overlap of the O−H absorption in the range of 1650 cm−1 with the amide I vibration occurred as well. The characteristic absorption bands of calcite increased with increasing Ca/P ratio. For example, the νcomb2 band at 2516 cm−1 and especially the ν2 (C−O) band at 871 cm−1 appeared with increasing clarity. The occurrence of the characteristic bands of calcite are also visible in the samples with Ca/P = 3.89 and Ca/P = ∞. Both the combination vibrations νcomb1 (1795 cm−1) and νcomb2, as well as the basic vibrations ν3 (1450 cm−1), ν2 (871 cm−1) and ν4 (712 cm−1) were observed. Unlike the mineral with Ca/P = ∞ and the calcite reference, the mineral with Ca/P = 3.89 had an absorption at the amide I band in the FT-IR spectrum. D



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Figure 5. (HR-)TEM series of magnifications (a), (a′), and (a″) of ormoHAp particles from mineralization with Ca/P 1.67, (b) and (b′) of spherical particles from Ca/P 3.33, and (c), (c′), and (c″) of rhombohedral particles from Ca/P ∞. Asterisks indicating so-called hollow spherical ormoHAp particles, while arrow heads indicate full mineralized spherical particles. Hash indicates rhombohedral particle with arrows pointing in directions of lattice planes. EDXS analysis of (b1) hollow and (b2) full mineral particles from mineralization with Ca/P 3.33 (values are semiquantitatie).

Figure 6. Calcium concentrations during degradation investigations by storage of compressed mineral samples (Ca/P (a) = 1.67; (b) = 3.33; (c) = 3.89; (d) = ∞) in SBF and PBS for 14 days. Note the two different ordinate scales for PBS (blue) and SBF (red). Furthermore, the blue columns represent an increase in Ca2+-concentration over zero, whereas the red ones represent decrease or increase around the initial Ca2+-concentration in SBF.

Figure 7. Required volume of 10 wt % gelatin for the various barriers and the therefrom obtained amount with indicated standard deviation of separated lyophilized minerals after 7 days of mineralization (n = 3).

3.4. Loss on Ignition. Incorporation of gelatin within the mineral was analyzed by loss on ignition. Prior to pyrolysis, ormoHAp contains an amount of about 34 wt % gelatin, which is calculated from its loss on ignition compared to commercial available hydroxyapatite (Figure 8). Commercial available calcium carbonate was pyrolized as well and revealed almost the same loss on ignition of 40 wt % as ormoHAp.

(not shown) holes, respectively. The 4 × 6 barrier was filled with gelatin (Figure 1c′), and mineralization occurred homogeneously across the barrier (Figure 1c″). The standard gelatin barrier had a ratio of separated and lyophilized mineral to initial used gelatin of 2.0 mg/mL (Figure 7). Using the polyacrylic barriers leads to increased mineral to gelatin ratio of 4.3 mg/mL, 7.3 mg/mL, and 8.8 mg/mL for 2 × 2 long-hole, 4 × 6 hole, and 6 × 10 hole barriers. 3.3. Mineralization in N2 atmosphere. Double migration in N2 atmosphere without phosphate ions in the cationic reservoir and without CO2 did not cause gelatin mineralization. Instead the mineral separation process led to a tiny amount of insoluble gelatin without any mineral characteristics in FT-IR and SEM (data not shown).

4. DISCUSSION Owing to the flexibility of the procedure, mineralization of gelatin gel membranes by double migration allows a targeted variation of the produced minerals. To use this possibility, it was necessary to investigate the variation of different parameters in terms of its influence to the resulting minerals. 4.1. Polyacrylic Barriers. Application of the different polyacrylic barriers led to an increased ratio of separated mineral to gelatin. That is probably due to a local increase of the electrical field. The insulating polyacrylic barrier increases E



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incorporation of organic macromolecules seemed to be suitable. The increased solubility is intended to increase the extracellular calcium ion concentration after the bone substitute material has been implanted. There are hints that the osteoblastic activity is directly increased14,29 or indirectly influenced via impairment of osteoclasts.30 The manipulation of the Ca/P ratio in the chamber to larger values than in case of regular HAp lead to calcium-deficient HAp31,32 For increased Ca/P ratios, the mineral production within the double migration chamber resulted in calcite. The actual aim of gelatin mineralization with a partly carbonated HAp, in terms of a solid solution, was not achieved. Calcium phosphate and calcium carbonate phases remain separated. In that way, a mixture of ormoHAp and calcite crystals with an adjustable proportion between both was synthesized. The sharp XRD peaks of calcite within the minerals with Ca/P = 3.33, and ∞ indicate an almost undisturbed crystal structure of calcite. The finding is confirmed by SEM images, showing the characteristic rhombohedral morphology of calcite crystals. However, TEM images visualized a high degree of crystallographic defects within the mineral particles, especially of the fully mineralized particle fraction of the Ca/P = 3.33 sample and the edges of calcite rhombohedra. The decrease of the measured Ca/P ratio of 1.22 in those particles of the Ca/P = 3.33 sample that are fully mineralized indicates a calcium deficient HAp. The low crystal size and the high degree of disturbance in the edge region of the calcite rhombohedron observable in HR-TEM images also points to an impairment of the crystal growth by the surrounding gelatin even though the particle size of calcite rhombohedra of 10 μm edge length shown in SEM did not support this suggestion. 4.3. Changed Barrier Position. The mineral formed with the off center gelatin barrier position leading to a Ca/P ratio of 3.89 is of special importance. In this case the morphology of the mineral reminds us of an early stage of the dumbbell minerals synthesized by the double-diffusion technique by Kniep et al. and Tlatlik et al.33,34 They presented a model of gelatin modified mineral formation, which was supported by intense characterization including TEM by Simon et al.10 These particles of Ca/P = 3.89 show a characteristic calcite absorption in FT-IR with a clear amide I absorption indicating the incorporation of gelatin in the mineral. This absorption was not detected for the rhombohedral crystallites of Ca/P = ∞ and the calcite reference. Additionally, the Ca/P = 3.89 mineral showed P−O absorption, which was only detected for HAp and ormoHAp containing minerals. XRD of the Ca/P = 3.89 mineral showed diffraction characteristic for calcite only, but the peak intensity was quite low and the peaks were slightly broadened. This again indicated a disturbed crystal structure and/or nanocrystallinity of the mineral, presumably due to gelatin and/or phosphate ion incorporation. 4.4. Degradation and Bioactivity of Minerals. Bioactivity of a material is its ability to form a mineralized hydroxyapatite layer on the surface. High bioactivities of bone substitutes are published to be critical for osteoblasts as a result of an nonphysiological calcium ion decrease due to the mineral layer deposition.35,36 Therefore, the materials development is dealing with calcium releasing minerals that are able to keep the calcium level in the extracellular space constant or even increase it. That has been shown to be an adequate stimulus for osteoblastic cells.14,15 For that a controlled dissolution of the material is necessary, which has to be in balance with the

Figure 8. Determination of gelatin amount and standard deviation in ormoHAp by loss on ignition of ormoHAp and commercially available hydroxyapatite and calcite.

the field strength within its holes, where ions are increasingly forced to migrate through the gelatin, where they finally form the mineral. A major advantage of the polyacrylic barriers is the reduced contact of gelatin with air during the 7 days of mineralization. Air contact led to a desiccation of the corresponding gelatin volumes. The desiccated parts got hard and were hardly suspendable again during the washing process. Additionally, the gelatin within the holes ensures greater stability; this allows the use of higher field strengths, leading to faster ion migration. The polyacrylic barriers caused a slight decrease of mineral mass almost linearly correlating with the decrease of gelatin needed for the holes compared with the total barrier volume. The reduced amount of gelatin and the better separation of gelatin from the mineral after 7 days represent a useful technical optimization of the double migration system. 4.2. Variation of Calcium/Phosphate Ratio. The mineral formation within the gelatin membrane revealed spherical HAp particles in the range of 100−500 nm in diameter under standard conditions. Those particles, the so-called hollow spheres, consist of nanocrystals in the rim and an amorphous predominantly gelatin containing core proved by TEM investigation. The disordered crystalline structure, amorphous parts and nanocrystallinity, respectively, are indicated by the broadened HAp XRD peaks and proven by the HR-TEM image (Figure 5a″). The gelatin incorporated in ormoHAp was detected by FT-IR, since the C−O- and amid I absorptions were considerably pronounced for Ca/P = 1.67, and 3.33 in comparison to the HAp reference. Depending on the pH, amino acids of gelatin offer binding sites for cations. The gelatin used was digested under acidic conditions and provides a pH of 5.2 ± 0.5 in solution. To increase the number of these binding sites at gelatin, lysine εamino groups were carboxylated by glucuronic acid according to the Maillard reaction.21 As a result an increased amount of mineral of same morphology and structure was produced.20 A comparable self-assembly of calcium phosphate nanoparticles into hollow spheres was published by Hagmeyer et al. as well as by Liu et al., who used synthetic polymer hydrogels as templates for mineralization, limiting mineral size and providing nucleation sites for the formation of apatite containing ca. 60 wt % organics.27,28 Both groups did not deal with soluble solids of calcium carbonate and phosphate as a bone substitute material, which was an intention of the present study. In order to provide an appropriate solubility of the bone substitute material, substitution of OH− and PO43− by carbonate ions and the F



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bioactivity driven mineral deposition from physiological solutions. In the case of all gelatin modified minerals produced by double migration, during degradation in PBS only a slight increase of calcium concentration in the solution appeared. The highest one of 0.13 mM was obtained for spherical calcite mineral (Ca/P = 3.89). The high initial phosphate concentration of 9.6 mM in PBS probably comes to an enhanced reprecipitation compared to SBF with a basic phosphate concentration of ca. 1.0 mM only. This reprecipitation in PBS is confirmed by the decrease of calcium ion concentration from 8 h to 1 days, where the liquids were not changed but all mineral samples showed a decrease of calcium in the liquid. The calcium ion concentration in SBF was decreased due to the bioactivity of ormoHAp (Ca/P = 1.67). After every change of SBF (periods cf. Materials and Methods), the mineral samples caused a decrease of calcium concentration in the just added SBF, leading to an overall decrease of calcium ion concentration during the whole time of incubation. Precipitation of calcium phosphates from SBF is thus probably favored by ormoHAp itself, acting as a nucleation germ, and increased over time by earlier precipitated mineral phases acting as nucleation germs as well. In contrast to ormoHAp, the rhombohedral calcite mineral (Ca/P = ∞) showed a dissolution in SBF. From day one on, the calcium concentration is always above the SBF blank. The mixture of ormoHAp and rhombohedral calcite, which is produced by Ca/ P = 3.33, showed an influence on calcium ion concentration exactly in between ormoHAp and calcite alone. The Ca/P = 3.33 mineral kept the calcium ion concentration almost constant after an initial decrease after 1 h of incubation. Thus, a balance between calcite dissolution and mineral precipitation became apparent. The Ca/P = 3.89 mineral showed the highest calcium ion release of all minerals. After an initial decrease after 1 h, the calcium concentration is increased up to 5.3 mM at day two. Afterward the calcium concentration was only slightly increased above (until day six) or decreased below (until day 14) the level of calcium in the SBF blank. This high release for the first days of incubation is probably due to the restriction in crystal growth with Ca/P = 3.89 and its reduced crystallinity as it is apparent from SEM images and XRD measurements. 4.5. Mineralization under N2 Atmosphere. The source of carbonate ions for the mineralization process was investigated after intense calcite formation for Ca/P = 3.33, 3.89, and ∞. There are two sources, gelatin and HCO3−-anions formed by hydration of dissolved CO2 and its subsequent dissociation in water. Since no mineral formation was observed when a phosphate-free solution in the cationic reservoir and the mineralization under N2 atmosphere were combined, the source of carbonate ions is certainly the atmospheric CO2. As the change of position as well as the decrease of phosphate ion concentration increases the amount of calcium ions in the gelatin gel facing a deficit of phosphate ions, the reaction of calcium with carbonate ions occurred. In contrast to phosphates, the formation of calcite is not limited, as CO2 is constantly supplied by the surrounding atmosphere and its equilibrium with dissociated carbonic acid.

quantitative ratio of separated mineral to the initially used gelatin could be increased by factor four in comparison to the standard gelatin barrier. In addition, the stability of the gelatin could be increased and the contact of gelatin to air could be avoided, which facilitates the process of mineral separation from nonmineralized gelatin. These investigations are the basis for using polyacrylic barriers for the internal mineralization of gelatin infiltrated macroporous scaffolds. The change of the Ca/P ratio by variation of the ion concentrations in the mineralization solutions or by changing the gelatin barrier position allows the synthesis of various minerals and mineral mixtures, which differ in morphology and crystal structure. Thus, it was possible to increase the calcium ion concentration of simulated body fluid. The two calcite forming conditions of Ca/P = 3.89, and Ca/P = ∞ enable an increase in the calcium concentration to values of about 3 mM over a period of 6 and 14 days, respectively. Upcoming experiments will focus on osteoblast proliferation and differentiation in vitro. The goal of further studies is to use self-organized inorganic/ organic minerals to produce a biodegradable, load-bearing bone substitute using the double migration method. Of main interest in the following are mineral density, strength, and degradation, and the cellular response of both major bone cells osteoblasts and osteoclasts. The variability of the system allows us to mineralize gelatin with various ion combinations (Ca2+, Sr2+ vs PO43−, and CO32−) to influence the cells directly with the minerals degradation products.

5. CONCLUSIONS AND OUTLOOK In conclusion, the present study demonstrates the variability of the double migration chamber to produce gelatin mineral composites. By introduction of polyacrylic barriers the

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +49 351 463 42762; fax: +49 351 463 39401; e-mail: [email protected]. ORCID

Benjamin Kruppke: 0000-0002-0659-0238 Funding

We gratefully acknowledge the Deutsche Forschungsgemeinschaft (DFG Collaborative Research Centre TRR 79/SP M3 and Z2) for financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Axel Mensch for XRD investigations.

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ABBREVIATIONS:

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REFERENCES

HAp; hydroxyapatite; EDXS; high resolution transmission electron microscopy; HR-TEM; energy dispersive X-ray spectroscopy; FT-IR; Fourier transform infrared spectroscopy; XRD; X-ray diffraction; ormoHAp; organically modified hydroxyapatite; PBS; phosphate buffered saline; SBF; simulated body fluid

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Calcite and Hydroxyapatite Gelatin Composites as Bone Substitution

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