Accepted Manuscript Title: Nano-porous calcium phosphate balls Author: Ildyko Kovach Sabine Kosmella Claudia Prietzel Christian Bagdahn Joachim Koetz PII: DOI: Reference:
S0927-7765(15)00319-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.05.021 COLSUB 7094
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
3-2-2015 22-4-2015 13-5-2015
Please cite this article as: I. Kovach, S. Kosmella, C. Prietzel, C. Bagdahn, J. Koetz, Nano-porous calcium phosphate balls, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Nano-porous calcium phosphate card house structures
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Gelatin-chitosan complexes controlling supramolecular ordering
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Dicalcium phosphate platelets building up supramolecular balls
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*Revised Manuscript
Nano-porous calcium phosphate balls Ildyko Kovach, Sabine Kosmella, Claudia Prietzel, Christian Bagdahn, Joachim Koetz* Institut für Chemie, Universität Potsdam, Karl-Liebknecht-Straße 24-25, D-14476 Potsdam,
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Germany
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ABSTRACT
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By dropping a NaH2PO4*H2O precursor solution to a CaCl2 solution at 90°C under continuous stirring in presence of two biopolymers, i.e. gelatin (G) and chitosan (C), supramolecular calcium
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phosphate (CP) card house structures are formed. Light microscopic investigations in combination with scanning electron microscopy show that the GC-based flower-like structure is constructed from very thin CP platelets. Titration experiments indicate that H-bonding between
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both biopolymers is responsible for the synergistic effect in presence of both polymers. Gelatinchitosan-water complexes play an important role with regard to supramolecular ordering. FTIR
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spectra in combination with powder X-ray diffraction show that after burning off all organic components (heating up >600°C) dicalcium and tricalcium phosphate crystallites are formed.
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From high resolution transmission electron microscopy (HR-TEM) it is obvious to conclude, that
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individual crystal platelets are dicalcium phosphates, which build up ball-like supramolecular structures. The results reveal that the GC guided crystal growth leads to nano-porous supramolecular structures, potentially attractive candidates for bone repair.
Keywords: Calcium phosphates, bone repair material, biomineralization, supramolecular ball structure
*Corresponding author at: University of Potsdam, Karl-Liebknecht-Straße 24-25, D-14476 Potsdam, Germany. Tel.: +493319775220; fax: +493319775054. E-mail address: [email protected] (J.Koetz)
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1. Introduction Calcium phosphates (CP) are the most important inorganic components of biological hard tissues. Biologically formed calcium phosphates are often nanocrystals that are precipitated under mild conditions, i.e. near room temperature. Bio-inspired calcium orthophosphates have a great
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importance in material science and biomedicine.
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In the last decades several applications have been developed for calcium phosphates, which can be applied for modifying the surface of bone metal and polymer implants, for artificial bone
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grafts, for dentin regeneration, as injectable cements for bone regeneration, as well as food additives [1-3].
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The calcium orthophosphate family is classified by their calcium phosphate ratio from 0.5 to 2. The major mineral phase of natural hard tissues is hydroxyapatite (HA) with a Ca/P molar ratio 1.67. Besides HA, dicalcium phosphate dihydrate (DCPD, “brushite”, CaHPO4*2H2O), and
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tricalcium phosphates (TCP, Ca3(PO4)2) are the most important biologically relevant calcium orthophosphates. Nevertheless, few members of calcium phosphates can be transformed into
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hydroxyapatite at pH > 5, and can be applied in bone cements [4]. Especially ß-TCP is used in
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bone cements or in combination with HA as a bone-substitution ceramic [5]. From synthetic bone materials TCP has the highest tissue compatibility, and is more soluble than HA. Therefore, it
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provides better access to calcium ions than HA [6]. DCPD is metastable under physiological conditions, is resorbed more easily than HA, and can be used as a precursor for bone mineralization [7,8].
The process of mineralization of hard tissue is guided by organic macromolecules, mainly collagen and non-collagenous water soluble acidic proteins. Collagen associated with acidic proteins serve together as a structural framework with active interface, dividing the areas (spaces) for the nucleation [9]. Based on this, modified associates of collagen, i.e. gelatin, as well as water soluble derivatives of carbohydrates, e.g. chitosan, are of special interest in the field of biopolymer-controlled, bioinspired CP mineralization processes [10-13]. Gelatin is a zwitterionic polypeptide, obtained from collagen through thermal denaturation processes under acidic or basic conditions, classified to A or B type gelatin. The electric belongings of gelatin are pH dependent, it shows cationic properties below its isoelectric point
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(IEP), and anionic ones beyond the IEP. Gelatin has been classified as a safe excipient by the U.S. Food and Drug Administration (FDA), and it is widely used as a component of various biomaterials, e.g. in tissue engineering, drug and gene delivery [14,15]. Chitosan is a modified natural polysaccharide derivative from chitin with a degree of
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deacetylation >70%, soluble in acetic medium in which the glucosamine units (pKa ~6.3) of the chitosan became protonated [16]. Chitosan is the most abundant structural biopolymer applied for
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tissue engineering [17,18]. Furthermore, chitosan is antimicrobial due to positively charged amino groups, which can interact with the negatively charged microbial membrane. The polymer
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can provide osmotic imbalance, and chelate metal ions for hindering the nutrition supply of the microbial cell [19].
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Gelatin and chitosan are biocompatible and biodegradable polymers [20]. Due to their outstanding properties both components were applied already as organic components for the 3-
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dimensional scaffold production, and their hydrogels are used as templates for calcium phosphate growth [21,22]. Zhao et al. have already shown that HA does not retard the formation of the chitosan-gelatin network, and that the polymer network has little influence on the crystallinity of
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hydroxyapatite [23]. Wang et al. detected hydrogen bond complexes in composite
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chitosan/gelatin solutions by using small angle neutron scattering (SANS) in combination with rheology [24]. However, more detailed reports about synergistic effects of both components in
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solution, and their effect on the crystallization process of CP platelets are rather scarce. The aim of our investigations was to study the CP crystallization process in solution in presence of both biopolymers in more detail. In principle the calcium phosphate crystallization conditions have to bring into line with the solution conditions of gelatin and chitosan. The CP precipitation process at room temperature and 90 °C was investigated in absence and presence of chitosan and gelatin in comparison to the appropriate mixture of both components. The morphology of supramolecular CP structures was examined with the help of high resolution scanning electron microscopy (HR-SEM) and light microscopy (LM). The crystalline structure of the CP platelets was investigated by using FTIR spectroscopy in combination with powder X-ray diffraction (PXRD), and high resolution transmission electron microscopy (HR-TEM).
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2. Experimental.
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2.1. Materials.
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All reagents were dissolved in Millipore Milli-Q deionized water.
The low molecular weight chitosan with a degree of deacetylation of 81.2 %, and a moisture
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content ≤ 12 wt%, and NaH2PO4*H2O were obtained from Sigma-Aldrich and used without further treatment. Gelatin (type A) powder, with Bloom number 140, and a moisture content ≤11
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wt%, CaCl2, and glacial acetic acid were purchased from Carl Roth. NaOH is obtained from
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AnalaR Normapure.
2.2. Preparation of polymer stock solutions and precipitation procedure
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Chitosan powder was suspended in 0.1 mol acetic acid, and the resulting 2 wt% polymer stock
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solution was stirred all night. Gelatin powder was dissolved in water and the resulting 2 wt% polymer stock solution kept for 5 min at 40 °C. Gelatin-chitosan (GC) composite solutions were
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obtained by mixing the two above described polymer stock solutions in a 1:1 ratio after stirring 3 days at room temperature (pH=5.5). The polymer stock solutions containing gelatin (G), chitosan (C), or a blend of gelatin and chitosan (GC), respectively, were mixed with an aqueous CaCl 2 solution (0.18 M) under stirring for one hour at 90 °C, under water cooling. The pH value of the resulting calcium-polymer solution was 5.2. Precipitation procedure
The aqueous NaH2PO4*H2O precursor solution (0.45 M) was added drop wise to the calciumpolymer solution up to a molar calcium:phosphate (C:P) ratio of 1:3. By adding NaOH, the pH was adjusted at 4.8. The color changed from transparent to white, immediately. After one hour of continuously gentle stirring the pH of the solution was decreased to 3.8. The precipitation procedure was done at 90 °C in presence of the different polymers, i.e. gelatin, chitosan or gelatin-chitosan mixtures, respectively. After 74 hours altering, the precipitant was washed
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several times and separated through a centrifuge at 13000 rpm, and air-dried for 24 h at 70 °C. In comparison, the precipitation process was performed in absence of a polymer component
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(reference procedure) as well as at room temperature, i.e. 25 °C.
2.3. Characterization Methods
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Supramolecular structures on the µm scale were investigated by using a light microscope (Leica DMLB with a Leica DFC 295 live camera). The crystals were dispersed in water and placed on a
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microscopic slide (76×26 mm) overlaid with micro glass cover (20×20 mm). The images were captured at magnification 10. The bar line on the micrographs is 10 µm. Supramolecular
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structures on the nm scale as well as individual platelets were visualized by using a high resolution scanning electron microscope (HR-SEM) S-4800 from Hitachi. Samples were placed on an aluminium sample holder and sputtered with a platinum layer. Elemental analysis (without
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sputtering) was performed with the Thermo-Noran-SIX energy dispersive X-ray (EDX) detector. The analyses of the calcium phosphate crystals was carried out by using a high resolution-
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transmission electron microscope (HR-TEM) JEM 2200 FS from JEOL, performed at 200 kV
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accelerating voltage in combination with Fast Fourier Transform (FFT). Sample preparation was done by dropping the aqueous CP suspensions on a copper grid. Chemical structures of the
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calcium phosphate were determined with attenuated total reflectance Fourier transform infrared spectroscope (ATR-FTIR). ATR-FTIR spectra were obtained on a NEXUS FT-IR spectrometer (Thermo-Nicolet, Diamond, ATR correction was done via Omnic 8.1.11; Thermo Fischer Scientific Inc). Spectra were recorded from 250 to 4000 cm−1 with a resolution of 2 cm-1. The Xray diffraction patterns were collected using a Bruker D-8 diffractometer with a Cu Kα radiation and step counting at 0.05 degree intervals for 2.5 seconds per data point. For the thermogravimetric analysis 5 mg sample were examined with a Mettler-Toledo TG/SDTA 851e, under N2 at a heating rate of 10 K min-1 from 25 to 900 °C. The precipitation procedure was repeated several times to guarantee the reproducibility. The EM micrographs are characteristic for each sample selected from a pool of micrographs made in our own department. The IR spectroscopy, EDX and TG analysis were checked for reproducibility
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3. Results and discussion The morphology of the precipitated calcium phosphates prepared in absence and presence of polymeric additives, i.e. gelatin, chitosan, and gelatin-chitosan mixture, respectively, were
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investigated systematically by using light microscopy and scanning electron microscopy. 3.1. Morphology of calcium phosphates precipitated in presence of gelatin (G-CP)
In presence of gelatin cube-shaped aggregates can be observed in the light microscope, partly
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flocculated by the polymer (Fig. 1 A.1). Looking into the particle aggregate structure by using SEM (Fig. 1 A.2) thick platelets and rhombic crystals become visible in similarity to calcite
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structures grown under equilibrium conditions [25]. The crystal dimensions are quite different, and individual large rhombic crystals are formed in similarity to the reference system without
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polymer (shown in Fig. S3 A, supplementary part). Taken these results into account one can conclude that on the molecular level gelatin does not control the growth of the platelets, but on
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the supramolecular level gelatin can induce a bridging flocculation between the CP crystals. This flocculation tendency can be related to a polymer adsorption on the crystal surface due to the
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protonated amino acid groups of the biopolymer.
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3.2. Morphology of calcium phosphates precipitated in presence of chitosan (C-CP) LM micrographs show equilateral cubes and aggregates (Fig. 1 B.1). SEM indicates fan-shaped
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aggregates (Fig. 1 B.2) consisting of individually formed very thin (100 nm) crystal platelets arranged in spatial bundles. This highly arranged spatial structure shows similarities with seashell nacre, a morphology of aragonite crystals controlled by chitin [26]. It has to be stated here that the change of the morphology of previously prepared calcium phosphate with macromolecules is of special interest, but reports of direct synthesis of hierarchical structured aggregates with spatial morphology are seldom [27]. One can conclude that chitosan influences the crystal growth of the individual platelets, and induce the bundle formation. Wang et al. have already shown that chitosan can form complexes with metal ions, i.e. Zn2+ or Cu2+[19]. Therefore, one can assume similar structured chitosan-metal complexes with Ca2+ ions, where the metal is arranged as an electron acceptor connected with amino groups and by forming bridges to hydroxyl groups of chitosan. On the one hand these complexes hinder the platelet growth in lateral direction, and on the other hand the complexes induce bundle arrangements.
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3.3. Morphology of calcium phosphates precipitated in presence of gelatin and chitosan (GCCP) By applying gelatin-chitosan blends, the SEM micrographs show flower-like aggregates constructed by thin calcium phosphate platelets with a thickness of about 100nm (Fig. 2 C.2).
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Recently, it was presented that micro-porous calcium phosphate composites were observed when the crystallization process is performed in the iota-carrageenan gel matrix [28]. Compared with
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the densely packed spatial bundles, observed in presence of chitosan (C-CP system), a nanoporous, spatially cross-linked CP structure is observed only in presence of a GC mixture. The
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individual very thin platelets build up a nano-porous card house structure (Fig. 2 C.2). Zeta potential values of [ζ]=15 ± 2mV indicate that the resulting particles are electrostatically
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stabilized.
This more open structure in comparison to the chitosan-based system in absence of gelatin , can be related only to polymer-polymer interactions between chitosan and gelatin. Such phenomena
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of polyelectrolyte complex formation were already described in literature, compare Ref. [29]. Therefore, we have performed polyelectrolyte titrations by using a method combination of
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turbidimetry and conductometry, according to our experiments already published earlier [30].
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The results show non-relevant Coulombic interactions between both biopolymers at the given pH range between 4 and 5. This means only H-bonding and metal complex formation with Ca2+ ions
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can play a role in the given system. DSC investigations of the GC polymer mixture show an unexpected exothermic peak at -20°C (compare Fig. S1, supplementary part). According to our already published DSC experiments with polymer-modified water-in-oil microemulsions [31], this peak can be related to bound or fixed water in polymer complexes. Based on this result one can conclude that phenomena of H-bonding between chitosan and gelatin play an important role with regard to the finally obtained supramolecular structure of the CP platelets. That means gelatin-chitosan-water complexes act as a spacer between the CP platelets resulting in a nanoporous structure.
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3.4. Chemical characterization of calcium phosphates precipitated in presence of gelatin and chitosan (C-CP, G-CP, GC-CP) For a more comprehensive characterization of the calcium phosphates, formed under the given conditions, different additional methods are used, i.e. FTIR spectroscopy, EDX and
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thermogravimetric analysis.
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FTIR spectroscopy
FTIR spectral analysis (shown in Fig. S2, supplementary part) were carried out after drying the
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samples at 70 °C. Characteristic absorption bands relating to water at about 3400 cm-1 are not well observed in the spestrum. In contrast one can find the typical absorption band for the H-O-H
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bending which rises at 1650 cm-1, and a typical P-O-H bending mode at 897 cm-1. However, the absorption band at 1650 can be related to the N-H bending mode of chitosan, too [12]. The absorption band at 1350 cm-1 can be attributed to a C-O-H bending vibration of the polymer
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additives. Characteristic IR bands of PO4 groups can be found at 999, 1066, and 1131 cm−1, respectively, as a result of P-O stretching mode of the PO4 fragment. In this absorption range the C-O stretching vibrations of the chitosan are overlapped [12]. Additional absorptions bands
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appear at 580, 564, and 526 cm-1, as a result of P-O bending of the PO4 fragment. These results,
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in agreement with previous reports of Gashti et al. [32], confirm the formation of brushite in
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presence of gelatin (G-CP), chitosan (C-CP), as well as in presence of a mixture of both components (GC-CP).
EDX analysis
EDX measurements (Fig. S3, supplementary part) show a quite similar element composition in absence and presence of the gelatin-chitosan mixture. This means the calcium phosphate platelets formed have a similar chemical composition, which is in full agreement with the FTIR results. However, in the reference system, that means in absence of the polymer mixture, a densely packed aggregate structure consisting of individual thick CP platelets is formed (compare Fig. S3A), in contrast to a more open nano-porous card house structure in presence of chitosan and gelatin (Fig. S3B.).
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TG analysis Thermogravimetric analyses (shown in Fig. 3) of the chitosan-based systems (C-CP and GC-CP) show 3 main decomposition regions, in comparison to the reference CP system with only one weight loss stage. The first step until 200 °C can be related to the removal of physically adsorbed
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surface water and hydrate water. The weight loss of the G-CP is only marginal. In presence of chitosan, i.e. the more hydrophilic polyelectrolyte, 0.8% is removed. However, the mass loss in
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presence of both polymers is only 0.4%. This behaviour can be explained by the fact that the total chitosan concentration in the mixture is reduced to 50%. The second step between 200 °C and
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400 °C, not observed in the reference system, with a 3% mass loss is more pronounced in the pure chitosan-based (C-CP) system in comparison to the GC-CP system. The weight loss of only
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0.5% in presence of gelatin can be explained with respect to the kind of water adsorption at the polymer surface. Because of its reduced hydrophilicity a less amount of water is adsorbed, whereas in the GC complex a quite higher amount of water is embedded. In that case the water
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molecules are more fixed, which is in accordance with our DSC experiments. Principally, this stage can be related to a charring of organic compounds. The main thermal region is the third step
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between 400 °C and 500 °C where the main mass loss of all systems can be observed. Hereby
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water evaporates from the structure and the crystals are converted into pyrophosphate. The plateau values of the G-CP and C-CP system are in the same order, but about 1% lower than that
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of the reference CP system. The lowest value of about 91.5% shows the GC-CP system. The reason for that is to be seen in the loss of the polymer components, but the interactions between the single polyelectrolyte components on the one side, and the H-bonding complex on the other side with the calcium phosphate particles are different. As explained before, the concentration of the single polymer components in the mixture is reduced to the half. Inspite of that fact, a higher mass amount in the GC-CP system is released because of its more compact gelatin-chitosan complexes incorporated into the CP card house structure. A similar thermal behavior of gelatin modified brushite crystals was detected by Gashti et al. [25, 29]. In comparison to our investigations the weight loss was higher because of a higher polymer concentration used in Ref. [28,32]. A more detailed characterization of the CP crystals was performed in the next chapter after thermal treatment.
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Special interest was given to the gelatin-chitosan modified system because of its unique nanoporous, spatially cross-linked CP structure with high potential to apply as hard tissue repair material. Therefore, the following experiments deal with the characterization of the thermal
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treated form of GC-CP.
3.5. Gelatin-chitosan modified calcium phosphates (GC-CP) after thermal treatment
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The GC-CP sample was heated up to 600 °C to burn out all organic components, as already demonstrated in chapter 3.4. Thermal treated samples were characterized again by using SEM,
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HR-TEM, FTIR, and powder X-ray diffraction (PXRD). The SEM micrograph (Fig. 4.) shows ball-like, nano-porous super structures formed by loosely packed calcium phosphate platelets.
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One can find 5-20 balls of different size on one micrograph (200 μm x 150 μm). Surprisingly the polymers are charred out from the system without ruining the nano-porous structure.
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FTIR spectrum (Fig. 5) presents extra crystalline bands in the phosphate region between 900 and 1250 cm-1, furthermore water and polymer related peaks disappear. An extra peak at 726 cm-1 in combination with a peak at 1212 cm-1 (in agreement with a shoulder in the dried powder curve
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shown in Fig. S2) can be related to pyrophosphate (P2O7 4- ). Absorption bands centered between
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970 and 1180 cm-1 can be assigned to [PO4] groups, in agreement with the weaker bands below 615 cm-1. It should be noted that peaks at 946 and 975 cm-1 are characteristic for β-TCP prepared
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at 800 °C or 900 °C according to [33,34]. However, this is in good agreement with the peaks observed at 938 and 972 cm-1.
The PXRD analysis presents a polydisperse spectrum. Fig. 6 shows similarities with different calcium phosphate types, namely with pyrophosphate Ca2P2O7 according to ref. [35] and β-TCP Ca3(PO4)2 according to ref. [36]. This result confirms the above discussed FTIR results. The lattice parameters of Ca2 (P2O7) and β-TCP Ca3 (PO4)2 are really similar, reflection peaks overlap each other, therefore it is difficult to differ between both crystal types. Furthermore, it is already well known that the peak position depends on the preparation conditions, e.g. temperature, pH value as well as the different record conditions, e.g. counting steps and resolution. However, the detected main reflection peaks of the diffraction patterns observed at 27°, 29°, and 31° can be find in the reference spectrum of Ca2 (P2O7), whereas in the β-TCP spectrum only the main peak at 31° is observed.
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The HR-TEM micrograph in Fig. 7 A shows a highly ordered crystalline structure of one individual platelet with a lattice distance of 3.0 Å. Fast Fourier Transform (FFT) analysis (Fig. 7 B) shows in the crystal lattice the following interplanar spacings at 3.00, 3.03, 3.35, 3.38, and 6.8 Å.
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In Table 1 these data are compared with interplanar spacing of Ca2P2O7 and Ca3(PO4)2. One can find similar interplanar spacings in both cases, but the agreement with the dicalcium phosphate is
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much better. Especially the peak of high intensity at 3.02 (underlined) for the crystal plane {008} and the correspondence between the peak at 3.34 and 6.68 Å for the crystal planes {200} and
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{100}, respectively, indicate that Ca2P2O7 is formed. This result confirms with the X-ray
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diffraction data, discussed before.
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Table 1: Interplanar spacing obtained by FFT analysis in comparison to relevant reference data Interplanar spacing
Interplanar spacing
experimental data
of Ca2P2O7 and corresponding
of Ca3(PO4)2 and corresponding
from FFT analysis
crystal planes (ICDD 04-009-8733)
crystal planes (ICDD 04-014-2292)
3.03 Å
2.97 Å {211}
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3.00 Å
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Interplanar spacing
2.99 Å {210}
3.01 Å {300} 2.88 Å {0210}, {217}
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3.02 Å {008}
3.35 Å
3.34 Å {200}
3.36 Å {122}
3.38 Å
3.38 Å {115}
3.40 Å {211}
6.8 Å
6.68 Å {100}
6.5 Å {104}
Based on the PXRD and HR-TEM we can conclude that the thermal treated GC-CP sample contains individual dicalcium phosphate platelets. However, a final proof is in progress by fitting the experimental data with respective calculated ones.
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Summarizing our results one can say that different CP crystal types are formed. It is already well known that the pH value can influence the crystal type. Therefore, one can assume that during the precipitation process performed here, that means between the starting pH value at 5.5, and the final pH value at 3.8, different CP crystal types are formed, which coexist in the card house
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structure.
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4. Conclusions
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Calcium phosphates precipitated in presence of gelatin and chitosan at 90 °C in a pH range between pH=5.5 and pH=3.8 build up spatially cross-linked card house structures. The individual CP platelets are very thin (100nm) and the flower-like supramolecular structure can be explained
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by polymer-polymer interactions between gelatin and chitosan. It should be noted here that the morphology of calcium phosphate prepared under similar conditions at 25 °C in absence of
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polymers is undefined (formless), in contrast to an adequate gelatin-chitosan system prepared at 25 °C, where we can find already flower-like structures, as to be seen in the supplementary part (Fig. S4). Similar flower-like structures were reported by Mandel et al. [37], investigating
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calcium phosphates prepared in cell culture solutions at 36.5 °C. On the one hand we can conclude from our investigations that the pH, temperature as well as one
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polymer component, i.e. chitosan, has great influence on the formation of single crystals, which leads to sharp rectangular plates. On the other hand gelatin-chitosan polymer mixtures are of relevance with regard to phenomena of structure formation on the supramolecular level. That means well defined flower-like card house structures were obtained only in presence of a mixture of both polymers. Gelatin-chitosan-water complexes located between the calcium phosphate platelets are responsible for the formation of such a more open nanoporous structure. Surprisingly, the flower-like card house structure is stable after a thermal treatment up to 600 °C. This means the gelatin-chitosan-water complexes taking over the role of a spacer between the platelets during the heating process. In the following temperature range up to 900°C, the predominantly formed card house structure still exists, but transformed into macroscopic balls on the supramolecular level. These finally obtained nano-porous calcium phosphate structures should be of special interest as bone repair material.
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Statements The authors declare that they have no competing interests. IK, SK and JK designed the experiments. IK carried out all experiments. SK was responsible for
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the DSC and titration experiments, CB for the IR spectra, and CP for the electron microscopy. IK and JK drafted the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
The authors are grateful to Dr. Brigitte Tiersch for the HR-SEM images. Furthermore we would
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like to thank Björn Gamroth and Sibylle Rüstig for technical assistance.
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[18] A. Di Martino, M. Sittinger, M.V. Risbund, Chitosan: A versatile biopolymer for orthopedic tissue-engineering, Biomaterials 26 (2005) 30, 5983-5990. [19] X. Wang, Y. Du, L. Fan, H Liu, Y. Hu, Chitosan-metal complex as antimicrobial agent: Synthesis, Characterization and Structure activity study, Polymer Bulletin 55
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(2005) 105-113.
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[20] L.Y. Sang, X.H. Zhou, F. Yun, G.L. Zhang, Enzymatic synthesis of chitosan-gelatin antimicrobial copolymer and its characterization, J. Sci. Food Agric. 90 (2010) 1, 58-64.
applications, Biomaterials 24 (2003) 24, 4337-51.
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[22] Y. Huang, S. Onyeri, M. Siewe, A. Moshfeghian, S.V. Madihally, In vitro characterization of chitosan-gelatin scaffolds for tissue engineering, Biomaterials 26
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[23] F. Zhao, Y. Yin, W.W. Lu, J.C. Leong, W. Zhang, J. Zhang, M. Zhang, K. Yao, and
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Preparation
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hydroxyapatite/chitosan-gelatin network composite scaffolds, Biomaterials 23 (2002)
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[24] Y. Wang, D. Qiu, T. Cosgrove, M.L. Denbow, A small-angle neutron scattering and rheology study of the composite of chitosan and gelatin, Colloids and Surfaces B: Biointerfaces 70 (2009) 254-258. [25] S. Mann, The chemistry of form, Angew. Chem. Int. Ed. 39 (2000) 3393-3406. [26] F. Barthelat, Nacre from mollusk shells: a model for high-performance structural materials, Bioinsp. Biomim. 5 (2010) 3, 1-8. [27] X.-Y. Zhao, Y.-J.Zhu, F. Chen, B-Q. Lu, J. Wu, Nanosheet assembled hierarchical nanostructure hydroxyapatite: surfactant-free microwave hydrothermal rapid synthesis, protein/DNA adsorption and pH controlled release, Cryst. Eng. Comm. 15 (2013) 206212.
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[28] M. P. Gashti, M. Stir, J. Hulliger, Synthesis of bone-like micro-porous calcium phosphate/iota-carrageenan composites by gel diffusion, Colloids and Surfaces B: Biointerfaces 110 (2013) 426-433. [29] Y. Yin, Z. Li, Y. Sun, K. Yao, A preliminary study on chitosan/gelatin
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polyelectrolyte complex formation, J. Mater. Sci. (Letter) 40 (2005) 17, 4649-4652.
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[30] J. Kötz, H. Köpke, G. Schmidt-Naake, P. Zarras, O. Vogl, Polyanion-polycation complex formation as a function of the position of the functional groups, Polymer 37
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(1996) 13, 2775-2781.
[31] M. Fechner, M. Kramer, E. Kleinpeter, J. Koetz, Polyampholyte-modified ionic
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microemulsions, Colloid Polym. Sci. 287 (2009) 1145–1153.
[32] M.P. Gashti, M. Bourquin, M. Stir J. Hulliger, Glutamic acid inducing kidney stone
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biomimicry by brushit/gelatin composite, J. Mat. Chem. 1 (2013) 1501-1508. [33] A. Destainville, E. Champion, D. Bernache-Assollante, E. Laborde, Synthesis,
d
characterization and thermal behaviour of apatite tricalcium phosphate, Materials
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Chemistry and Physics 80 (2003) 269-277. [34] S. Meejoo, W. Maneeprakorn, P. Winotai, Phase and thermal stability of
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nanocrystalline hydroxyapatite prepared via microwave heating. Thermochimica Acta 447 (2006) 115-120.
[35] N.C. Webb, The crystal structure of β-Ca2P2O7, Acta Crystallogr. 21 (1996) 942. [36] M. Yashima, A. Sakai, T. Kamiyama, A. Hoshikawa, Crystal structure analysis of βtricalcium phosphate Ca3(PO4)2 by neutron powder diffraction, J. Solid State Chem. 175 (2003) 272-277.
[37] S. Mandel, A.C. Tas, Brushite to octacalcium phosphate transformation in DMEM solution at 36.5°C, Mat. Sci. and Eng. C 30 (2010) 245-254.
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Figures
Fig. 1. LM and SEM micrographs of precipitated gelatin (A.1, A.2) and chitosan (B.1, B.2)
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modified calcium phosphates
Fig. 2. LM and SEM micrographs of precipitated gelatin-chitosan (C.1, C.2) modified calcium phosphates
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Fig. 3. Thermo gravimetric analysis of the different calcium phosphates (CP, G-CP, C-CP, GC-
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CP)
Fig. 4. SEM micrograph of gelatin-chitosan modified calcium phosphates (GC-CP) after thermal treatment up to 600°C
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Fig. 5. IR spectrum of gelatin-chitosan modified calcium phosphates (GC-CP) after thermal treatment up
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to 600°C
Fig. 6. Powder X-ray diffraction (PXRD) pattern of thermal treated GC-CP in comparison to ICDD 04-009-8733 of Ca2P2O7 and ICDD 04-014-2292 of Ca3(PO4)2
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Fig. 7. HR-TEM image (A) and corresponding FFT (B) of a thermal treated GC-CP platelet
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Supplementary Part
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Fig. S1. DSC cooling curve of the aqueous gelatin-chitosan composite solution
Fig. S2. FTIR spectra of gelatin (G-CP), chitosan (C-CP) and gelatin-chitosan (GC-CP) modified calcium phosphates
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Fig. S3. SEM micrographs and corresponding EDX spectra for the reference calcium phosphate
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(CP) system (A) in comparison to the gelatin-chitosan modified calcium phosphate (GC-CP)
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system (B)
Fig. S4. Flower-like SEM image of the GC-CP system prepared at 25°C
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Table 1
Table 1: Interplanar spacing obtained by FFT analysis in comparison to relevant reference data Interplanar spacing
Interplanar spacing
experimental data
of Ca2P2O7 and corresponding
of Ca3(PO4)2 and corresponding
from FFT analysis
crystal planes (ICDD 04-009-8733)
crystal planes (ICDD 04-014-2292)
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Interplanar spacing
2.97 Å {211}
3.01 Å {300}
3.03 Å
2.99 Å {210}
2.88 Å {0210}, {217}
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3.00 Å
3.38 Å
3.38 Å {115}
6.8 Å
6.68 Å {100}
3.36 Å {122}
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3.34 Å {200}
3.40 Å {211} 6.5 Å {104}
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3.35 Å
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3.02 Å {008}
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Figure 1 A
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Figure 1 B
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Figure 2
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Figure 7A
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Figure 7B
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Graphical Abstract (for review)
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S0927-7765(15)00319-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.05.021 COLSUB 7094
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
3-2-2015 22-4-2015 13-5-2015
Please cite this article as: I. Kovach, S. Kosmella, C. Prietzel, C. Bagdahn, J. Koetz, Nano-porous calcium phosphate balls, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Nano-porous calcium phosphate card house structures
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Gelatin-chitosan complexes controlling supramolecular ordering
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Dicalcium phosphate platelets building up supramolecular balls
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*Revised Manuscript
Nano-porous calcium phosphate balls Ildyko Kovach, Sabine Kosmella, Claudia Prietzel, Christian Bagdahn, Joachim Koetz* Institut für Chemie, Universität Potsdam, Karl-Liebknecht-Straße 24-25, D-14476 Potsdam,
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Germany
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ABSTRACT
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By dropping a NaH2PO4*H2O precursor solution to a CaCl2 solution at 90°C under continuous stirring in presence of two biopolymers, i.e. gelatin (G) and chitosan (C), supramolecular calcium
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phosphate (CP) card house structures are formed. Light microscopic investigations in combination with scanning electron microscopy show that the GC-based flower-like structure is constructed from very thin CP platelets. Titration experiments indicate that H-bonding between
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both biopolymers is responsible for the synergistic effect in presence of both polymers. Gelatinchitosan-water complexes play an important role with regard to supramolecular ordering. FTIR
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spectra in combination with powder X-ray diffraction show that after burning off all organic components (heating up >600°C) dicalcium and tricalcium phosphate crystallites are formed.
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From high resolution transmission electron microscopy (HR-TEM) it is obvious to conclude, that
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individual crystal platelets are dicalcium phosphates, which build up ball-like supramolecular structures. The results reveal that the GC guided crystal growth leads to nano-porous supramolecular structures, potentially attractive candidates for bone repair.
Keywords: Calcium phosphates, bone repair material, biomineralization, supramolecular ball structure
*Corresponding author at: University of Potsdam, Karl-Liebknecht-Straße 24-25, D-14476 Potsdam, Germany. Tel.: +493319775220; fax: +493319775054. E-mail address: [email protected] (J.Koetz)
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1. Introduction Calcium phosphates (CP) are the most important inorganic components of biological hard tissues. Biologically formed calcium phosphates are often nanocrystals that are precipitated under mild conditions, i.e. near room temperature. Bio-inspired calcium orthophosphates have a great
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importance in material science and biomedicine.
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In the last decades several applications have been developed for calcium phosphates, which can be applied for modifying the surface of bone metal and polymer implants, for artificial bone
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grafts, for dentin regeneration, as injectable cements for bone regeneration, as well as food additives [1-3].
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The calcium orthophosphate family is classified by their calcium phosphate ratio from 0.5 to 2. The major mineral phase of natural hard tissues is hydroxyapatite (HA) with a Ca/P molar ratio 1.67. Besides HA, dicalcium phosphate dihydrate (DCPD, “brushite”, CaHPO4*2H2O), and
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tricalcium phosphates (TCP, Ca3(PO4)2) are the most important biologically relevant calcium orthophosphates. Nevertheless, few members of calcium phosphates can be transformed into
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hydroxyapatite at pH > 5, and can be applied in bone cements [4]. Especially ß-TCP is used in
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bone cements or in combination with HA as a bone-substitution ceramic [5]. From synthetic bone materials TCP has the highest tissue compatibility, and is more soluble than HA. Therefore, it
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provides better access to calcium ions than HA [6]. DCPD is metastable under physiological conditions, is resorbed more easily than HA, and can be used as a precursor for bone mineralization [7,8].
The process of mineralization of hard tissue is guided by organic macromolecules, mainly collagen and non-collagenous water soluble acidic proteins. Collagen associated with acidic proteins serve together as a structural framework with active interface, dividing the areas (spaces) for the nucleation [9]. Based on this, modified associates of collagen, i.e. gelatin, as well as water soluble derivatives of carbohydrates, e.g. chitosan, are of special interest in the field of biopolymer-controlled, bioinspired CP mineralization processes [10-13]. Gelatin is a zwitterionic polypeptide, obtained from collagen through thermal denaturation processes under acidic or basic conditions, classified to A or B type gelatin. The electric belongings of gelatin are pH dependent, it shows cationic properties below its isoelectric point
Page 3 of 34
(IEP), and anionic ones beyond the IEP. Gelatin has been classified as a safe excipient by the U.S. Food and Drug Administration (FDA), and it is widely used as a component of various biomaterials, e.g. in tissue engineering, drug and gene delivery [14,15]. Chitosan is a modified natural polysaccharide derivative from chitin with a degree of
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deacetylation >70%, soluble in acetic medium in which the glucosamine units (pKa ~6.3) of the chitosan became protonated [16]. Chitosan is the most abundant structural biopolymer applied for
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tissue engineering [17,18]. Furthermore, chitosan is antimicrobial due to positively charged amino groups, which can interact with the negatively charged microbial membrane. The polymer
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can provide osmotic imbalance, and chelate metal ions for hindering the nutrition supply of the microbial cell [19].
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Gelatin and chitosan are biocompatible and biodegradable polymers [20]. Due to their outstanding properties both components were applied already as organic components for the 3-
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dimensional scaffold production, and their hydrogels are used as templates for calcium phosphate growth [21,22]. Zhao et al. have already shown that HA does not retard the formation of the chitosan-gelatin network, and that the polymer network has little influence on the crystallinity of
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hydroxyapatite [23]. Wang et al. detected hydrogen bond complexes in composite
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chitosan/gelatin solutions by using small angle neutron scattering (SANS) in combination with rheology [24]. However, more detailed reports about synergistic effects of both components in
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solution, and their effect on the crystallization process of CP platelets are rather scarce. The aim of our investigations was to study the CP crystallization process in solution in presence of both biopolymers in more detail. In principle the calcium phosphate crystallization conditions have to bring into line with the solution conditions of gelatin and chitosan. The CP precipitation process at room temperature and 90 °C was investigated in absence and presence of chitosan and gelatin in comparison to the appropriate mixture of both components. The morphology of supramolecular CP structures was examined with the help of high resolution scanning electron microscopy (HR-SEM) and light microscopy (LM). The crystalline structure of the CP platelets was investigated by using FTIR spectroscopy in combination with powder X-ray diffraction (PXRD), and high resolution transmission electron microscopy (HR-TEM).
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2. Experimental.
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2.1. Materials.
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All reagents were dissolved in Millipore Milli-Q deionized water.
The low molecular weight chitosan with a degree of deacetylation of 81.2 %, and a moisture
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content ≤ 12 wt%, and NaH2PO4*H2O were obtained from Sigma-Aldrich and used without further treatment. Gelatin (type A) powder, with Bloom number 140, and a moisture content ≤11
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wt%, CaCl2, and glacial acetic acid were purchased from Carl Roth. NaOH is obtained from
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AnalaR Normapure.
2.2. Preparation of polymer stock solutions and precipitation procedure
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Chitosan powder was suspended in 0.1 mol acetic acid, and the resulting 2 wt% polymer stock
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solution was stirred all night. Gelatin powder was dissolved in water and the resulting 2 wt% polymer stock solution kept for 5 min at 40 °C. Gelatin-chitosan (GC) composite solutions were
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obtained by mixing the two above described polymer stock solutions in a 1:1 ratio after stirring 3 days at room temperature (pH=5.5). The polymer stock solutions containing gelatin (G), chitosan (C), or a blend of gelatin and chitosan (GC), respectively, were mixed with an aqueous CaCl 2 solution (0.18 M) under stirring for one hour at 90 °C, under water cooling. The pH value of the resulting calcium-polymer solution was 5.2. Precipitation procedure
The aqueous NaH2PO4*H2O precursor solution (0.45 M) was added drop wise to the calciumpolymer solution up to a molar calcium:phosphate (C:P) ratio of 1:3. By adding NaOH, the pH was adjusted at 4.8. The color changed from transparent to white, immediately. After one hour of continuously gentle stirring the pH of the solution was decreased to 3.8. The precipitation procedure was done at 90 °C in presence of the different polymers, i.e. gelatin, chitosan or gelatin-chitosan mixtures, respectively. After 74 hours altering, the precipitant was washed
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several times and separated through a centrifuge at 13000 rpm, and air-dried for 24 h at 70 °C. In comparison, the precipitation process was performed in absence of a polymer component
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(reference procedure) as well as at room temperature, i.e. 25 °C.
2.3. Characterization Methods
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Supramolecular structures on the µm scale were investigated by using a light microscope (Leica DMLB with a Leica DFC 295 live camera). The crystals were dispersed in water and placed on a
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microscopic slide (76×26 mm) overlaid with micro glass cover (20×20 mm). The images were captured at magnification 10. The bar line on the micrographs is 10 µm. Supramolecular
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structures on the nm scale as well as individual platelets were visualized by using a high resolution scanning electron microscope (HR-SEM) S-4800 from Hitachi. Samples were placed on an aluminium sample holder and sputtered with a platinum layer. Elemental analysis (without
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sputtering) was performed with the Thermo-Noran-SIX energy dispersive X-ray (EDX) detector. The analyses of the calcium phosphate crystals was carried out by using a high resolution-
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transmission electron microscope (HR-TEM) JEM 2200 FS from JEOL, performed at 200 kV
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accelerating voltage in combination with Fast Fourier Transform (FFT). Sample preparation was done by dropping the aqueous CP suspensions on a copper grid. Chemical structures of the
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calcium phosphate were determined with attenuated total reflectance Fourier transform infrared spectroscope (ATR-FTIR). ATR-FTIR spectra were obtained on a NEXUS FT-IR spectrometer (Thermo-Nicolet, Diamond, ATR correction was done via Omnic 8.1.11; Thermo Fischer Scientific Inc). Spectra were recorded from 250 to 4000 cm−1 with a resolution of 2 cm-1. The Xray diffraction patterns were collected using a Bruker D-8 diffractometer with a Cu Kα radiation and step counting at 0.05 degree intervals for 2.5 seconds per data point. For the thermogravimetric analysis 5 mg sample were examined with a Mettler-Toledo TG/SDTA 851e, under N2 at a heating rate of 10 K min-1 from 25 to 900 °C. The precipitation procedure was repeated several times to guarantee the reproducibility. The EM micrographs are characteristic for each sample selected from a pool of micrographs made in our own department. The IR spectroscopy, EDX and TG analysis were checked for reproducibility
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3. Results and discussion The morphology of the precipitated calcium phosphates prepared in absence and presence of polymeric additives, i.e. gelatin, chitosan, and gelatin-chitosan mixture, respectively, were
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investigated systematically by using light microscopy and scanning electron microscopy. 3.1. Morphology of calcium phosphates precipitated in presence of gelatin (G-CP)
In presence of gelatin cube-shaped aggregates can be observed in the light microscope, partly
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flocculated by the polymer (Fig. 1 A.1). Looking into the particle aggregate structure by using SEM (Fig. 1 A.2) thick platelets and rhombic crystals become visible in similarity to calcite
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structures grown under equilibrium conditions [25]. The crystal dimensions are quite different, and individual large rhombic crystals are formed in similarity to the reference system without
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polymer (shown in Fig. S3 A, supplementary part). Taken these results into account one can conclude that on the molecular level gelatin does not control the growth of the platelets, but on
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the supramolecular level gelatin can induce a bridging flocculation between the CP crystals. This flocculation tendency can be related to a polymer adsorption on the crystal surface due to the
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protonated amino acid groups of the biopolymer.
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3.2. Morphology of calcium phosphates precipitated in presence of chitosan (C-CP) LM micrographs show equilateral cubes and aggregates (Fig. 1 B.1). SEM indicates fan-shaped
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aggregates (Fig. 1 B.2) consisting of individually formed very thin (100 nm) crystal platelets arranged in spatial bundles. This highly arranged spatial structure shows similarities with seashell nacre, a morphology of aragonite crystals controlled by chitin [26]. It has to be stated here that the change of the morphology of previously prepared calcium phosphate with macromolecules is of special interest, but reports of direct synthesis of hierarchical structured aggregates with spatial morphology are seldom [27]. One can conclude that chitosan influences the crystal growth of the individual platelets, and induce the bundle formation. Wang et al. have already shown that chitosan can form complexes with metal ions, i.e. Zn2+ or Cu2+[19]. Therefore, one can assume similar structured chitosan-metal complexes with Ca2+ ions, where the metal is arranged as an electron acceptor connected with amino groups and by forming bridges to hydroxyl groups of chitosan. On the one hand these complexes hinder the platelet growth in lateral direction, and on the other hand the complexes induce bundle arrangements.
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3.3. Morphology of calcium phosphates precipitated in presence of gelatin and chitosan (GCCP) By applying gelatin-chitosan blends, the SEM micrographs show flower-like aggregates constructed by thin calcium phosphate platelets with a thickness of about 100nm (Fig. 2 C.2).
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Recently, it was presented that micro-porous calcium phosphate composites were observed when the crystallization process is performed in the iota-carrageenan gel matrix [28]. Compared with
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the densely packed spatial bundles, observed in presence of chitosan (C-CP system), a nanoporous, spatially cross-linked CP structure is observed only in presence of a GC mixture. The
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individual very thin platelets build up a nano-porous card house structure (Fig. 2 C.2). Zeta potential values of [ζ]=15 ± 2mV indicate that the resulting particles are electrostatically
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stabilized.
This more open structure in comparison to the chitosan-based system in absence of gelatin , can be related only to polymer-polymer interactions between chitosan and gelatin. Such phenomena
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of polyelectrolyte complex formation were already described in literature, compare Ref. [29]. Therefore, we have performed polyelectrolyte titrations by using a method combination of
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turbidimetry and conductometry, according to our experiments already published earlier [30].
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The results show non-relevant Coulombic interactions between both biopolymers at the given pH range between 4 and 5. This means only H-bonding and metal complex formation with Ca2+ ions
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can play a role in the given system. DSC investigations of the GC polymer mixture show an unexpected exothermic peak at -20°C (compare Fig. S1, supplementary part). According to our already published DSC experiments with polymer-modified water-in-oil microemulsions [31], this peak can be related to bound or fixed water in polymer complexes. Based on this result one can conclude that phenomena of H-bonding between chitosan and gelatin play an important role with regard to the finally obtained supramolecular structure of the CP platelets. That means gelatin-chitosan-water complexes act as a spacer between the CP platelets resulting in a nanoporous structure.
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3.4. Chemical characterization of calcium phosphates precipitated in presence of gelatin and chitosan (C-CP, G-CP, GC-CP) For a more comprehensive characterization of the calcium phosphates, formed under the given conditions, different additional methods are used, i.e. FTIR spectroscopy, EDX and
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thermogravimetric analysis.
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FTIR spectroscopy
FTIR spectral analysis (shown in Fig. S2, supplementary part) were carried out after drying the
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samples at 70 °C. Characteristic absorption bands relating to water at about 3400 cm-1 are not well observed in the spestrum. In contrast one can find the typical absorption band for the H-O-H
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bending which rises at 1650 cm-1, and a typical P-O-H bending mode at 897 cm-1. However, the absorption band at 1650 can be related to the N-H bending mode of chitosan, too [12]. The absorption band at 1350 cm-1 can be attributed to a C-O-H bending vibration of the polymer
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additives. Characteristic IR bands of PO4 groups can be found at 999, 1066, and 1131 cm−1, respectively, as a result of P-O stretching mode of the PO4 fragment. In this absorption range the C-O stretching vibrations of the chitosan are overlapped [12]. Additional absorptions bands
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appear at 580, 564, and 526 cm-1, as a result of P-O bending of the PO4 fragment. These results,
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in agreement with previous reports of Gashti et al. [32], confirm the formation of brushite in
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presence of gelatin (G-CP), chitosan (C-CP), as well as in presence of a mixture of both components (GC-CP).
EDX analysis
EDX measurements (Fig. S3, supplementary part) show a quite similar element composition in absence and presence of the gelatin-chitosan mixture. This means the calcium phosphate platelets formed have a similar chemical composition, which is in full agreement with the FTIR results. However, in the reference system, that means in absence of the polymer mixture, a densely packed aggregate structure consisting of individual thick CP platelets is formed (compare Fig. S3A), in contrast to a more open nano-porous card house structure in presence of chitosan and gelatin (Fig. S3B.).
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TG analysis Thermogravimetric analyses (shown in Fig. 3) of the chitosan-based systems (C-CP and GC-CP) show 3 main decomposition regions, in comparison to the reference CP system with only one weight loss stage. The first step until 200 °C can be related to the removal of physically adsorbed
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surface water and hydrate water. The weight loss of the G-CP is only marginal. In presence of chitosan, i.e. the more hydrophilic polyelectrolyte, 0.8% is removed. However, the mass loss in
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presence of both polymers is only 0.4%. This behaviour can be explained by the fact that the total chitosan concentration in the mixture is reduced to 50%. The second step between 200 °C and
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400 °C, not observed in the reference system, with a 3% mass loss is more pronounced in the pure chitosan-based (C-CP) system in comparison to the GC-CP system. The weight loss of only
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0.5% in presence of gelatin can be explained with respect to the kind of water adsorption at the polymer surface. Because of its reduced hydrophilicity a less amount of water is adsorbed, whereas in the GC complex a quite higher amount of water is embedded. In that case the water
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molecules are more fixed, which is in accordance with our DSC experiments. Principally, this stage can be related to a charring of organic compounds. The main thermal region is the third step
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between 400 °C and 500 °C where the main mass loss of all systems can be observed. Hereby
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water evaporates from the structure and the crystals are converted into pyrophosphate. The plateau values of the G-CP and C-CP system are in the same order, but about 1% lower than that
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of the reference CP system. The lowest value of about 91.5% shows the GC-CP system. The reason for that is to be seen in the loss of the polymer components, but the interactions between the single polyelectrolyte components on the one side, and the H-bonding complex on the other side with the calcium phosphate particles are different. As explained before, the concentration of the single polymer components in the mixture is reduced to the half. Inspite of that fact, a higher mass amount in the GC-CP system is released because of its more compact gelatin-chitosan complexes incorporated into the CP card house structure. A similar thermal behavior of gelatin modified brushite crystals was detected by Gashti et al. [25, 29]. In comparison to our investigations the weight loss was higher because of a higher polymer concentration used in Ref. [28,32]. A more detailed characterization of the CP crystals was performed in the next chapter after thermal treatment.
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Special interest was given to the gelatin-chitosan modified system because of its unique nanoporous, spatially cross-linked CP structure with high potential to apply as hard tissue repair material. Therefore, the following experiments deal with the characterization of the thermal
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treated form of GC-CP.
3.5. Gelatin-chitosan modified calcium phosphates (GC-CP) after thermal treatment
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The GC-CP sample was heated up to 600 °C to burn out all organic components, as already demonstrated in chapter 3.4. Thermal treated samples were characterized again by using SEM,
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HR-TEM, FTIR, and powder X-ray diffraction (PXRD). The SEM micrograph (Fig. 4.) shows ball-like, nano-porous super structures formed by loosely packed calcium phosphate platelets.
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One can find 5-20 balls of different size on one micrograph (200 μm x 150 μm). Surprisingly the polymers are charred out from the system without ruining the nano-porous structure.
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FTIR spectrum (Fig. 5) presents extra crystalline bands in the phosphate region between 900 and 1250 cm-1, furthermore water and polymer related peaks disappear. An extra peak at 726 cm-1 in combination with a peak at 1212 cm-1 (in agreement with a shoulder in the dried powder curve
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shown in Fig. S2) can be related to pyrophosphate (P2O7 4- ). Absorption bands centered between
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970 and 1180 cm-1 can be assigned to [PO4] groups, in agreement with the weaker bands below 615 cm-1. It should be noted that peaks at 946 and 975 cm-1 are characteristic for β-TCP prepared
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at 800 °C or 900 °C according to [33,34]. However, this is in good agreement with the peaks observed at 938 and 972 cm-1.
The PXRD analysis presents a polydisperse spectrum. Fig. 6 shows similarities with different calcium phosphate types, namely with pyrophosphate Ca2P2O7 according to ref. [35] and β-TCP Ca3(PO4)2 according to ref. [36]. This result confirms the above discussed FTIR results. The lattice parameters of Ca2 (P2O7) and β-TCP Ca3 (PO4)2 are really similar, reflection peaks overlap each other, therefore it is difficult to differ between both crystal types. Furthermore, it is already well known that the peak position depends on the preparation conditions, e.g. temperature, pH value as well as the different record conditions, e.g. counting steps and resolution. However, the detected main reflection peaks of the diffraction patterns observed at 27°, 29°, and 31° can be find in the reference spectrum of Ca2 (P2O7), whereas in the β-TCP spectrum only the main peak at 31° is observed.
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The HR-TEM micrograph in Fig. 7 A shows a highly ordered crystalline structure of one individual platelet with a lattice distance of 3.0 Å. Fast Fourier Transform (FFT) analysis (Fig. 7 B) shows in the crystal lattice the following interplanar spacings at 3.00, 3.03, 3.35, 3.38, and 6.8 Å.
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In Table 1 these data are compared with interplanar spacing of Ca2P2O7 and Ca3(PO4)2. One can find similar interplanar spacings in both cases, but the agreement with the dicalcium phosphate is
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much better. Especially the peak of high intensity at 3.02 (underlined) for the crystal plane {008} and the correspondence between the peak at 3.34 and 6.68 Å for the crystal planes {200} and
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{100}, respectively, indicate that Ca2P2O7 is formed. This result confirms with the X-ray
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diffraction data, discussed before.
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Table 1: Interplanar spacing obtained by FFT analysis in comparison to relevant reference data Interplanar spacing
Interplanar spacing
experimental data
of Ca2P2O7 and corresponding
of Ca3(PO4)2 and corresponding
from FFT analysis
crystal planes (ICDD 04-009-8733)
crystal planes (ICDD 04-014-2292)
3.03 Å
2.97 Å {211}
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3.00 Å
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Interplanar spacing
2.99 Å {210}
3.01 Å {300} 2.88 Å {0210}, {217}
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3.02 Å {008}
3.35 Å
3.34 Å {200}
3.36 Å {122}
3.38 Å
3.38 Å {115}
3.40 Å {211}
6.8 Å
6.68 Å {100}
6.5 Å {104}
Based on the PXRD and HR-TEM we can conclude that the thermal treated GC-CP sample contains individual dicalcium phosphate platelets. However, a final proof is in progress by fitting the experimental data with respective calculated ones.
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Summarizing our results one can say that different CP crystal types are formed. It is already well known that the pH value can influence the crystal type. Therefore, one can assume that during the precipitation process performed here, that means between the starting pH value at 5.5, and the final pH value at 3.8, different CP crystal types are formed, which coexist in the card house
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structure.
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4. Conclusions
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Calcium phosphates precipitated in presence of gelatin and chitosan at 90 °C in a pH range between pH=5.5 and pH=3.8 build up spatially cross-linked card house structures. The individual CP platelets are very thin (100nm) and the flower-like supramolecular structure can be explained
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by polymer-polymer interactions between gelatin and chitosan. It should be noted here that the morphology of calcium phosphate prepared under similar conditions at 25 °C in absence of
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polymers is undefined (formless), in contrast to an adequate gelatin-chitosan system prepared at 25 °C, where we can find already flower-like structures, as to be seen in the supplementary part (Fig. S4). Similar flower-like structures were reported by Mandel et al. [37], investigating
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calcium phosphates prepared in cell culture solutions at 36.5 °C. On the one hand we can conclude from our investigations that the pH, temperature as well as one
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polymer component, i.e. chitosan, has great influence on the formation of single crystals, which leads to sharp rectangular plates. On the other hand gelatin-chitosan polymer mixtures are of relevance with regard to phenomena of structure formation on the supramolecular level. That means well defined flower-like card house structures were obtained only in presence of a mixture of both polymers. Gelatin-chitosan-water complexes located between the calcium phosphate platelets are responsible for the formation of such a more open nanoporous structure. Surprisingly, the flower-like card house structure is stable after a thermal treatment up to 600 °C. This means the gelatin-chitosan-water complexes taking over the role of a spacer between the platelets during the heating process. In the following temperature range up to 900°C, the predominantly formed card house structure still exists, but transformed into macroscopic balls on the supramolecular level. These finally obtained nano-porous calcium phosphate structures should be of special interest as bone repair material.
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Statements The authors declare that they have no competing interests. IK, SK and JK designed the experiments. IK carried out all experiments. SK was responsible for
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the DSC and titration experiments, CB for the IR spectra, and CP for the electron microscopy. IK and JK drafted the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
The authors are grateful to Dr. Brigitte Tiersch for the HR-SEM images. Furthermore we would
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like to thank Björn Gamroth and Sibylle Rüstig for technical assistance.
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Figures
Fig. 1. LM and SEM micrographs of precipitated gelatin (A.1, A.2) and chitosan (B.1, B.2)
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modified calcium phosphates
Fig. 2. LM and SEM micrographs of precipitated gelatin-chitosan (C.1, C.2) modified calcium phosphates
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Fig. 3. Thermo gravimetric analysis of the different calcium phosphates (CP, G-CP, C-CP, GC-
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CP)
Fig. 4. SEM micrograph of gelatin-chitosan modified calcium phosphates (GC-CP) after thermal treatment up to 600°C
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Fig. 5. IR spectrum of gelatin-chitosan modified calcium phosphates (GC-CP) after thermal treatment up
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to 600°C
Fig. 6. Powder X-ray diffraction (PXRD) pattern of thermal treated GC-CP in comparison to ICDD 04-009-8733 of Ca2P2O7 and ICDD 04-014-2292 of Ca3(PO4)2
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Fig. 7. HR-TEM image (A) and corresponding FFT (B) of a thermal treated GC-CP platelet
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Supplementary Part
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Fig. S1. DSC cooling curve of the aqueous gelatin-chitosan composite solution
Fig. S2. FTIR spectra of gelatin (G-CP), chitosan (C-CP) and gelatin-chitosan (GC-CP) modified calcium phosphates
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Fig. S3. SEM micrographs and corresponding EDX spectra for the reference calcium phosphate
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(CP) system (A) in comparison to the gelatin-chitosan modified calcium phosphate (GC-CP)
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system (B)
Fig. S4. Flower-like SEM image of the GC-CP system prepared at 25°C
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Table 1
Table 1: Interplanar spacing obtained by FFT analysis in comparison to relevant reference data Interplanar spacing
Interplanar spacing
experimental data
of Ca2P2O7 and corresponding
of Ca3(PO4)2 and corresponding
from FFT analysis
crystal planes (ICDD 04-009-8733)
crystal planes (ICDD 04-014-2292)
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Interplanar spacing
2.97 Å {211}
3.01 Å {300}
3.03 Å
2.99 Å {210}
2.88 Å {0210}, {217}
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3.00 Å
3.38 Å
3.38 Å {115}
6.8 Å
6.68 Å {100}
3.36 Å {122}
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3.34 Å {200}
3.40 Å {211} 6.5 Å {104}
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3.35 Å
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3.02 Å {008}
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Figure 1 A
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Figure 1 B
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Figure 2
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Figure 4
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Figure 7A
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Figure 7B
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Graphical Abstract (for review)
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