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Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatite-microsphere originating from conversion of CaCO3 templates
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 115605 (http://iopscience.iop.org/0957-4484/26/11/115605) View the table of contents for this issue, or go to the journal homepage for more
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Nanotechnology Nanotechnology 26 (2015) 115605 (8pp)
doi:10.1088/0957-4484/26/11/115605
Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatitemicrosphere originating from conversion of CaCO3 templates Qingrong Wei1,3, Jian Lu1, Qiaoying Wang2, Hongsong Fan1 and Xingdong Zhang1 1
National Engineering Research Centre for Biomaterials, Sichuan University, Chengdu 610064, People’s Republic of China 2 School of Engineering & Materials Science, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK E-mail: [email protected] Received 28 November 2014, revised 28 January 2015 Accepted for publication 29 January 2015 Published 26 February 2015 Abstract
Inspired by coralline-derived hydroxyapatite, we designed a methodological route to synthesize carbonated-hydroxyapatite microspheres from the conversion of CaCO3 spherulite templates within a collagen matrix under mild conditions and thus constructed the composite hydrogel of collagen/hydroxyapatite-microspheres. Fourier transform infrared spectroscopy (FTIR) and xray diffraction (XRD) were employed to confirm the successful generation of the carbonated hydroxyapatite phase originating from CaCO3, and the ratios of calcium to phosphate were tracked over time. Variations in the weight portion of the components in the hybrid gels before and after the phase transformation of the CaCO3 templates were identified via thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) shows these composite hydrogels have a unique multiscale microstructure consisting of a collagen nanofibril network and hydroxyapatite microspheres. The relationship between the hydroxyapatite nanocrystals and the collagen fibrils was revealed by transmission electron microscopy (TEM) in detail, and the selected area electron diffraction (SAED) pattern further confirmed the results of the XRD analyses which show the typical low crystallinity of the generated hydroxyapatite. This smart synthesis strategy achieved the simultaneous construction of microscale hydroxyapatite particles and collagen fibrillar hydrogel, and appears to provide a novel route to explore an advanced functional hydrogel materials with promising potentials for applications in bone tissue engineering and reconstruction medicine. Keywords: hydroxyapatite microsphere, CaCO3 template, collagen fibrillogenesis, in situ conversion, composite hydrogel, multiscale structure (Some figures may appear in colour only in the online journal) 1. Introduction
which scaffolds play an essential role as a support matrix for three-dimensional tissue reconstruction. Among the various scaffolding systems, hydrogel, especially composed of natural-based polymers, are the most extensively explored categories due to their compositional and structural similarities to the natural extracellular matrices (ECM) in addition
Over recent decades, intense interest has been focused on tissue engineering and regenerative medicine, in field of 3
Author to whom all correspondence should be addressed.
0957-4484/15/115605+08$33.00
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surfactants [15, 16], most of which are hazardous to human health and environment. Hydrothermic chemical reaction is the most utilized approach for producing coralline HA (CHA) scaffold from calcium carbonate exoskeleton of sea coral calcite [17–19]. This strategy always includes harsh conditions such as high temperature and high pressure which undoubtedly can damage the bioactivity of the biomacromolecules which comprise the hydrogel matrix. In the present study, inspired by the smart route of CHA formation from coral skeletal carbonate, we have developed a CaCO3 template-based method for preparation of carbonated HA spherulites within a collagen matrix and then obtained a composite hydrogel of collagen/HA-microspheres under mild conditions. Based on the previous work [20], this study mainly focuses on the in situ derivation of HA microspheres from CaCO3 templates within a collagen hydrogel matrix by only one procedure. As illustrated in scheme 1, the phase transformation of the inorganic particles from CaCO3 to HA proceeds along with the self-assembly of collagen molecules. After this process, HA microspheres can be obtained in situ from the conversion of the CaCO3 templates within a simultaneously constructed collagen hydrogel. As fabricated, the collagen/HA-microparticle hybrid gels exhibit multiscale structures including nanofibrils of collagen, microscale spherulites of HA and macroscale blocks of the composites. Despite apparent structural differences between these hybrids and nature bone tissue, the significant concept of a multiscale structure for hydrogel materials has been introduced and achieved here. Meanwhile, a novel route to prepare carbonated HA microspheres from the conversion of CaCO3 templates within a bio-macromolecule matrix under mild conditions has been successfully established in this study and would pave the way for designing further smart hydrogel scaffolds for tissue engineering and regenerative medicine.
Scheme 1. Schematic illustration of collagen/HA-microsphere composite scaffold fabrication in situ: (a) Formation of CaCO3 microspheres in an aqueous phase in the presence of collagen molecules. (b) Addition of phosphate into the suspension of collagen molecules and CaCO3 microspheres. (c) Upon elevating the system temperature to 35 °C, fibrillogenesis of collagen molecules into a fibrillar network occurs along with conversion of CaCO3 particles into HA microspheres.
to their desirable framework for cellular proliferation and survival. A natural bone matrix is an organic/inorganic hybrid mainly consisting of hydroxyapatite (HA) and collagen, and it can be characterized by a multiscale distribution of nano-, micro- and macroscale spatial structures [1–3], which have been found to contribute to the physical and biological properties of natural or engineered tissues [4–6]. Accordingly, hydrogel-based scaffolds with structures and compositions similar to those of natural tissues are highly desirable for building appropriate microenvironments for cellular behaviours. Following this principle, extensive efforts have been exerted to develop biomimetic matrices using collagen/HA composite hydrogels or their blends with other inorganic and organic elements, such as magnetite nano-particles, synthetic polymers and biomacromolecules [7–9]. Most of the available composite hydrogels usually comprise single-scale building blocks including nanofibers and nanoparticles. Some biomimetic scaffolds with hierarchical porosity have been developed [10, 11] from the aspect of the pore architecture of the scaffolds. From another angle, however, the bulk material itself comprising the hydrogel-scaffold can be designed as a multiscale distribution in space from the nanoscale to the micro- and macroscales. On the other hand, as the most important inorganic component for bone tissue engineering, HA crystals are generally obtained at the nanoscale rather than at the microscale by using conventional methods such as in situ deposition or mineralization within a biomacromolecular matrix [10–14], because it is comparatively difficult to synthesize microscale HA particles under mild conditions. Traditional methods for fabricating HA microspheres mainly rely on the approaches involving organic solvents and
2. Materials and methods 2.1. Materials
Highly purified collagen (type I) was extracted from calf skin in our laboratory following the procedures described by Miller, with some modifications [21]. Anhydrous Na2CO3, anhydrous CaCl2, Na2HPO4·12H2O and other chemicals (Kermel Ltd, Chengdu, China) were of analytical grade and used as received. The water used in all experiments was super-purified using a Milli-Q system. 2.2. Synthesis of collagen/hydroxyapatite-microsphere hybrid composites
CaCl2 was added dropwise to a 20 ml acidic collagen solution (4 mg ml−1, pH2.5) with gentle stirring to obtain a final concentration of 0.22 M Ca2+. Approximately 21 ml of a 0.22 M Na2CO3 aqueous solution was quickly added into the dispersion of the collagen/Ca2+ under rapid stirring for two minutes. Then, a concentrated aqueous phosphate solution was introduced slowly into the system with stirring. The 2
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amount of phosphate added was calculated from the ratio of calcium to phosphorus (Ca/P ratio) in stoichiometric HA based on the existing amount of Ca2+ in the synthesis system. The suspension of products was then degassed via centrifugation and transferred into beakers, which were then placed in a water bath and incubated at 35 °C for 48-120 h. All experimental procedures above that involved collagen were performed at 4 °C. Along with the occurrence of fibrillogenesis and the gradual construction of a collagen gel initiated by elevating the temperature, the CaCO3 microparticles distributing throughout the fibrillar network of the gel were gradually converted to calcium phosphate (CaP) particles in the presence of ambient phosphate. As obtained, the collagen/HAmicrosphere hybrid gels were thoroughly rinsed in deionized water to remove salt residues and then lyophilized for subsequent uses or analyses.
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Figure 1. FTIR spectra of (a) pure collagen, (b) collagen/CaCO3microsphere composites, and collagen/HA-microsphere composites derived in situ from collagen/CaCO3-microspheres in phosphate ambience for (c) 72 and (d) 48 h.
2.3. Characterization 2.3.1. Scanning electron microscopy. The microstructure of
Germany) at a heating rate of 10 °C min−1 from 30 °C to 1200 °C under a nitrogen atmosphere.
the collagen/HA-microsphere composites was analysed using a field-emission scanning electron microscope (FESEM, S4800, Hitachi, Japan) with energy dispersive spectroscopy (EDS, Inca Energy 250 with liquid nitrogen cooling, Oxford Instruments, UK). All of the specimens for SEM were sputtercoated with Au, and the specimens for the EDS analysis were applied to a conductive adhesive directly without Au coating.
3. Results 3.1. Functional groups by FTIR
As shown in figure 1, the FTIR spectra show the typical apatite signals together with the signals of CaCO3 and pure collagen.The absorption bands at 713 (v4), 876 (v2) and 1437 cm−1 (v3) were derived from the characteristic vibrations of carbonate in the collagen/CaCO3-microparticle composites (figure 1(b)). After more than 48 h of conversion of CaCO3 to CaP inside the hybrid gels, the bands at 1062 (v3), 604 (v4) and 563 cm−1 (v4) attributed to the PO3− 4 groups of apatite were legibly identified (figures 1(c)–(d)) [22]. The absorption peak at 872 cm−1 (v2) and the bimodal bands at 1421 (v3) and 1469 cm−1 (v3) are evidences for the carbonate substitution in the phosphate B-site of the HA lattice. Compared with the amide vibrational band groups (1654, 1550 and 1239 cm−1 for amide I, amide II and amide III bands, respectively [23, 24]) derived from pure collagen (figure 1(a)), the amide I bands are obviously present in the hybrid gels, with the peak at 1650 cm−1 and 1651 cm−1 for the composite hydrogel of collagen/CaCO3-microparticles and the collagen/HA-microparticles, respectively (figures 1(b)–(d)). The weaker band complexes of the amide II and amide III are absent in the hybrids.
2.3.2. X-ray diffraction. The phase identification of the
composites was performed on an x-ray diffractometer (XRD, X'Pert Pro, PHILIPS) using a Cu-Kα radiation (λ = 1.540 56 Å, 40 kV and 35 mA). Samples were scanned in 0.03° steps from 20° to 70° (2θ) with 0.2 s dwell time per step. 2.3.3. Fourier transform infrared spectroscopy.
For precise chemical composition and structure investigations, Fourier transform infrared spectroscopy (FTIR) was employed running on a NEXUS 670 instrument (Thermo Electron, USA) in the range of 4000 to 400 cm−1 with a resolution of 1.0 cm−1 and 20 scan accumulations. Transmission electron microscopy. The spatial relationship between the collagen fibrils and mineral nanoparticles was revealed using transmission electron microscopy (TEM, FEI Tecnai F20). The samples were crushed into a nanoscale powder in liquid nitrogen with a mortar and pestle. A few small drops of ethanol were then applied to the powder, followed by transferring the slurry to carbon coated copper grids and air drying. Selected area electron diffraction (SAED) was also conducted on the crystals to identify their phase.
2.3.4.
3.2. Phase identification by XRD spectrometry
The evolvement of the mineral phase transformation inside the composite gel was demonstrated by XRD patterns (figure 2). It is shown that the inorganic component of the collagen/CaCO3-microparticle composite hydrogel comprises CaCO3 vaterite and calcite (figure 2(a)). The characteristic
Thermogravimetric analysis. Thermogravimetric analyses (TGA) of the lyophilized hybrids were carried out using a Thermal Analysis Instrument (STA 49C, Netzsch,
2.3.5.
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microspheres (figure 2(b)). This information suggests the successful conversion of CaCO3 to a crystalline HA phase. The broaden peaks of the XRD patterns imply weak crystallinity in the apatite.
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3.3. Analysis of the Ca/P ratio evolving process
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vaterite
As shown in the graph of the EDS analyses (figure 3), the final Ca/P ratio of the collagen/CaP-microsphere composites is 2.08 ± 0.14 at 25 °C and 1.53 ± 0.06 at 37 °C. This difference suggests that the process of the conversion of CaCO3 to CaP is mainly affected by temperature rather than by reaction time and that higher temperatures are favourable for achieving the phase transformation. Additionally, the Ca/P ratio shows small fluctuations after 72 h at 37 °C, and finally, it settles approximately 1.5, indicating that the phase transformation reaction is almost complete after 72 h. The final Ca/P ratio of the inorganic component of the collagen/HA-microsphere composites is slightly lower than that of stoichiometric HA and is similar to biologically inspired HA. Such carbonated apatite possesses the bioactivity of providing a ‘bone bond’ capability in vivo [25].
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3.4. Microstructures and morphologies of hybrid gels containing inorganic microspheres
Figure 2. XRD patterns of (a) collagen/CaCO3-microsphere composites and (b) collagen/HA-microsphere composites derived in situ from collagen/CaCO3-microspheres in phosphate ambience for (a) 48 and (b) 72 h.
Figure 4 presents the microstructures of the composite hydrogels of collagen/inorganic-microparticles. Morphological features reveal that these hybrids possess multiscale structures comprising collagen nanofibrils and inorganic microspheres and that the collagen fibrils and the inorganic microspheres interpenetrate. The CaCO3 spherulites, as the templates for fabrication of the HA microspheres, are present throughout the collagen fibrillar network and further composed of nanocrystal particles (figure 4(a)). After undergoing the phase transformation in phosphate ambience, the CaCO3 nanoparticles were thoroughly substituted with platy or needle-like HA nanocrystals either forming porous HA microspheres with ‘cauliflower’ structures or aggregating into clusters that enwrap the collagen fibrils (figure 4(b)). TEM analyses were performed to depict the CaP minerals and their distribution relationship to the collagen fibrils in great detail. The inorganic crystals existing in clusters or as individual needle-like particles attach to the surface of the collagen fibrils (figure 5). From the SAED pattern (figure 5 insert), the mineral content has been identified as HA with low crystallinity, which further confirms the results of the XRD analyses. Figure 6 shows the macroscopic morphology of the collagen/HA-microsphere composites. The shape of the hybrid hydrogel (figure 6(a)) is mouldable because the mixture suspension produced from this synthesis strategy are initially injectable. A sponge-like scaffold material (figure 6(b)) can be obtained from the hybrid gel after lyophilisation.
Figure 3. EDS analyses of the evolving Ca/P ratios of the composite gels over the conversion reaction time.
diffraction peaks of apatite became evident when the reaction time reached 48 h, and the positions of these diffuse peaks are located at 2θ = 26.3° (002), 32.3° (211), 40.3° (310), 47.0° (222) and 49.6° (213) for the hybrid gel of collagen/CaP4
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Figure 4. SEM images showing the morphology of the composite hydrogel scaffolds of (a) collagen/CaCO3-microspheres and (b) collagen/ HA-microspheres derived in situ from collagen/CaCO3-microspheres in phosphate ambience for 48 h.
78.8%, 79.3% and 79.9% for composites of collagen/HAmicrospheres-48 h (figure 7(d)), collagen/HA-microspheres72 h (figure 7(e)) and collagen/HA-microspheres-96 h (figure 7(f)), respectively. Very small difference between the weight percentage of the inorganic residues at 48 and 96 h indicates that the conversion of CaCO3 to a crystalline HA phase is almost complete after two days of reaction time in phosphate ambience.
4. Discussion It has been demonstrated that homogeneous isotropic crystal growth on tiny crystal nuclei yields a spherical shape and a uniform distribution of porous CaCO3 spheres under strong stirring [27, 28]. In the present work, this conventional synthesis route for CaCO3 particles has been introduced into an aqueous collagen solution to obtain the templates of CaCO3 microspheres within a biomacromolecular matrix. The inorganic component of the collagen/CaP-particle hybrid gel mainly consisted of carbonate-substituted apatite, which is thought to more closely resemble biologically inspired HA such as bone mineral [29–31]. As most of the calcium phosphate phase in biomimetic scaffolds for bone tissue engineering [32, 33], the apatite microspheres derived from CaCO3 templates show the typical ‘cauliflower-like’ morphology. Such carbonated apatite with poorly crystallized feature is significant for reabsorption and remodelling processes in tissues [34, 35]. From the absence of the absorption peaks of the amide II and amide III for the hybrid gels, it has been hypothesized that there would exist a certain interaction between the inorganic minerals and the collagen molecules, which could exert some constrain on the movements of stretching and bending of the N-H and C-N bonds in the collagen molecules. However, the fibrillogenesis behaviour of the collagen molecules received no any significant effect from this interaction. Nature bone consists of both inorganic and organic ECM. The organic matrix is mainly collagen type I which is an important structural protein in ECM. Collagen macromolecules are characterized by self-assembling into fibrillar network of hydrogel in vitro under suitable conditions. The inorganic ECM, namely natural bone mineral, is not pure and
Figure 5. TEM micrograph showing a typical mineralized collagen
fibril from a collagen/HA-microsphere composites derived in situ from collagen/CaCO3-microspheres in phosphate ambience for 48 h; the inset shows the SAED pattern of this mineralized fibril.
3.5. Depiction of the components of the hybrid gels via TGA
TGA was used to analyse the weight loss of the composites under elevated temperatures (figure 7). The sharpest decreases in weight were extrapolated to be at approximately 283 °C and 752 °C for pure collagen (figure 7(a)) and pure CaCO3 microparticles (calcite, figure 7(c)), respectively, while at approximately 282 °C and 710 °C for the components of collagen and CaCO3, respectively, in the collagen/CaCO3microsphere composites (figure 7(b)). Calcite is more thermodynamically stable than vaterite [26]. And additionally, the presence of collagen macromolecules could result in some defects in the CaCO3 crystal lattice. Consequently, the decomposition temperature of the CaCO3 in the collagen/ CaCO3-microparticle hybrids is slightly lower than that of pure CaCO3. The onset of the decomposition temperature of collagen in the collagen/HA-microsphere composites was recorded near 330 °C. In the range of 670–770 °C, a weight loss was recorded for the composites (figures 7(d)–(f)), and this phenomenon could be contributed to the presence of trace CaCO3 components. The residue degrees at 1000 °C are 5
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Figure 6. Images of macroscale morphology recorded by a camera showing (a) a composite hydrogel of collagen/HA-microspheres in a glass beaker and (b) a sponge-like scaffold block obtained from the same hybrid gel after lyophilisation.
pressures have to be needed in the exchanging process. So, based on the idea of fabricating CHA constructs, we synthesized micro-scale calcium carbonate component within collagen matrix and further obtain HA products from these CaCO3 templates by inorganic phase transformation in situ. In our previously described work [20], the hybrid collagen hydrogels containing CaCO3 microspheres were fabricated at the first step, then the gels were soaked in a periodically changed phosphate solution to promote the phase transformation of CaCO3 to HA. In this study, however, those procedures have been modified and simplified to just one step. The phosphate was introduced into the collagen suspension immediately after the synthesis of the CaCO3 microparticles, and then the fibrillogenesis of the collagen macromolecules was triggered to construct a composite collagen hydrogel upon elevation of the system temperature to physiological temperature. Thus, the conversion of the CaCO3 particles and the self-assembly of collagen molecules occurred simultaneously in situ. Since the suspension of the collagen-based mixture is injectable, the resultant composite gel is shapeability, and also the multifunctional gel can be constructed by facilely introducing external ingredients such as cells, bioactive factors and drug molecules into the system before the activation of gelation.
Figure 7. TGA curves of (a) pure collagen, (b) collagen/CaCO3microsphere composites and (c) pure CaCO3 microparticles. Collagen/HA-microsphere composites derived in situ from collagen/ CaCO3-microspheres in phosphate ambience for (d) 48, (e) 72 and (f) 96 h.
not fully crystallized hydroxyapatite, but is factually a highly substituted microcrystalline HA containing carbonate and other minerals [36]. Accordingly, collagen and HA are the most necessary components in the synthesized biomimetic scaffold materials for bone tissue engineering and regenerative medicine [7–9, 37]. For utilization of the unique pore sizes and the porous structure of corals, Della M. and his coworkers first prepared HA from coral skeletal carbonate by using hydrothermal chemical exchange with phosphate, and the relevant mechanism is based on the fact that HA is much more thermodynamically stable than CaCO3 [17]. Since then, CHA which is derived from coral skeletal calcium carbonate, has been extensively studied in experiments for development as bone graft substitutes [38–41]. Commercially available forms of CHA are used clinically as a bone substitute in various situations [42–44]. However, this kind of scaffold material contains no collagen composition, and furthermore, harsh reaction conditions of high temperatures and high
5. Conclusions An approach for HA microsphere synthesis in situ from the templates of CaCO3 spherulites under mild conditions has been successfully achieved within a collagen matrix. Asobtained hybrids exhibit multiscale microstructures consisting of nanoscale collagen fibrils and microscale HA particles, which are further composed of carbonated apatite nanocrystals with low crystallinity. This facile strategy for preparing collagen/HA-microsphere hybrid gels may be a promising lead to further develop a novel multifunctional hydrogel 6
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material with shapeability for tissue engineering, and also could find potentials in designing the advanced supporting scaffolds for surgical repair and medical reconstruction.
[15] Ma M G and Zhu J F 2009 Solvothermal synthesis and characterization of hierarchically nanostructured hydroxyapatite hollow spheres Eur. J. Inorg. Chem. 36 5522–6 [16] Zhang C M, Yang J, Quan Z W, Yang P P, Li C X, Hou Z Y and Lin J 2009 Hydroxyapatite nano- and microcrystals with multiform morphologies: controllable synthesis and luminescence properties Cryst. Growth Des. 9 2725–33 [17] Roy D M and Linnehan S K 1974 Hydroxyapatite formed from coral skeletal carbonate by hydrothermal exchange Nature 247 220–2 [18] Fu K, Xu Q G, Czernuszka J, Triffitt J T and Xia Z D 2013 Characterization of a biodegradable coralline hydroxyapatite/calcium carbonate composite and its clinical implementation Biomed. Mater. 8 065007 [19] Ripamonti U et al 2009 The induction of bone formation by coral-derived calcium carbonate/hydroxyapatite constructs Biomaterials 30 1428–39 [20] Wei Q R, Lu J, Ai H and Jiang B 2012 Novel method for the fabrication of multiscale structure collagen/hydroxyapatitemicrosphere composites based on CaCO3 microparticle templates Mater. Lett. 80 91–4 [21] Miller E J 1972 Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin Biochemistry 11 4903-9 [22] Fowler B O 1974 Infrared studies of apatites. I. Vibrational assignments for calcium, strontium, and barium hydroxyapatites utilizing isotopic substitution Inorg. Chem. 13 194–207 [23] Payne K J and Veis A 1988 Fourier transform ir spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies Biopolymers 27 1749–60 [24] Susi H, Ard J S and Carroll R J 1971 The infrared spectrum and water binding of collagen as a function of relative humidity Biopolymers 10 1597–604 [25] Loty C, Sautier J M, Boulekbache H, Kokubo T, Kim H M and Forest N 2000 In vitro bone formation on a bone-like apatite layer prepared by a biomimetic process on a bioactive glassceramic J. Biomed. Mater. Res. 49 423–34 [26] Xiao J, Wang Z, Tang M Y and Yang S 2010 Biomime-tic mineralization of CaCO3 on a phospholipid monolay-er: from an amorphous calcium carbonate precursor tocalcite via vaterite Langmuir 26 4977–83 [27] Tracy S L, Francois C J P and Jennings H M 1998 The growth of calcite spherulites from solution: I. Experimental design techniques J. Cryst. Growth 193 374–81 [28] Zhu H, Stein E W, Lu Z, Yuri Y M and McShane M J 2005 Synthesis of size-controlled monodisperse manganese carbonate microparticles as templates for uniform polyelectrolyte microcapsule formation Chem. Mater. 17 2323–8 [29] Tampieri A, Celotti G, Landi E, Sandri M, Roveri N and Falini G 2003 Biologically inspired synthesis of bone-like composite: self-assembled collagen fibers/hydroxyapatite nanocrystals J. Biomed. Mater. Res. A 67 618–25 [30] Kim H W, Kim H and Salih V 2005 Stimulation of osteoblast responses to biomimetic nanocomposites of gelatinhydroxyapatite for tissue engineering scaffolds Biomaterials 26 5221–30 [31] Schneiders W et al 2007 Effect of modification of hydroxyapatite/collagen composites with sodium citrate, phosphoserine, phosphoserine/RGD-peptide and calcium carbonate on bone remodelling Bone 40 1048–59 [32] Chen Q Z and Boccaccini A R 2006 Poly(D,L-lactic acid) coated 45S5 Bioglass-based scaffolds for bone engineering J. Biomed. Mater. Res. A 77 445–57 [33] Douglas Timothy E L et al 2014 Injectable self-gelling composites for bone tissue engineering based on gellan gum
Acknowledgments This work was supported by National Natural Science Foundation of China (51203103, 51373105) and National Basic Research Program of China (National 973 program, No. 2011CB606201).
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Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatite-microsphere originating from conversion of CaCO3 templates
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 115605 (http://iopscience.iop.org/0957-4484/26/11/115605) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 169.230.243.252 This content was downloaded on 22/04/2015 at 14:49
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 26 (2015) 115605 (8pp)
doi:10.1088/0957-4484/26/11/115605
Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatitemicrosphere originating from conversion of CaCO3 templates Qingrong Wei1,3, Jian Lu1, Qiaoying Wang2, Hongsong Fan1 and Xingdong Zhang1 1
National Engineering Research Centre for Biomaterials, Sichuan University, Chengdu 610064, People’s Republic of China 2 School of Engineering & Materials Science, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK E-mail: [email protected] Received 28 November 2014, revised 28 January 2015 Accepted for publication 29 January 2015 Published 26 February 2015 Abstract
Inspired by coralline-derived hydroxyapatite, we designed a methodological route to synthesize carbonated-hydroxyapatite microspheres from the conversion of CaCO3 spherulite templates within a collagen matrix under mild conditions and thus constructed the composite hydrogel of collagen/hydroxyapatite-microspheres. Fourier transform infrared spectroscopy (FTIR) and xray diffraction (XRD) were employed to confirm the successful generation of the carbonated hydroxyapatite phase originating from CaCO3, and the ratios of calcium to phosphate were tracked over time. Variations in the weight portion of the components in the hybrid gels before and after the phase transformation of the CaCO3 templates were identified via thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) shows these composite hydrogels have a unique multiscale microstructure consisting of a collagen nanofibril network and hydroxyapatite microspheres. The relationship between the hydroxyapatite nanocrystals and the collagen fibrils was revealed by transmission electron microscopy (TEM) in detail, and the selected area electron diffraction (SAED) pattern further confirmed the results of the XRD analyses which show the typical low crystallinity of the generated hydroxyapatite. This smart synthesis strategy achieved the simultaneous construction of microscale hydroxyapatite particles and collagen fibrillar hydrogel, and appears to provide a novel route to explore an advanced functional hydrogel materials with promising potentials for applications in bone tissue engineering and reconstruction medicine. Keywords: hydroxyapatite microsphere, CaCO3 template, collagen fibrillogenesis, in situ conversion, composite hydrogel, multiscale structure (Some figures may appear in colour only in the online journal) 1. Introduction
which scaffolds play an essential role as a support matrix for three-dimensional tissue reconstruction. Among the various scaffolding systems, hydrogel, especially composed of natural-based polymers, are the most extensively explored categories due to their compositional and structural similarities to the natural extracellular matrices (ECM) in addition
Over recent decades, intense interest has been focused on tissue engineering and regenerative medicine, in field of 3
Author to whom all correspondence should be addressed.
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surfactants [15, 16], most of which are hazardous to human health and environment. Hydrothermic chemical reaction is the most utilized approach for producing coralline HA (CHA) scaffold from calcium carbonate exoskeleton of sea coral calcite [17–19]. This strategy always includes harsh conditions such as high temperature and high pressure which undoubtedly can damage the bioactivity of the biomacromolecules which comprise the hydrogel matrix. In the present study, inspired by the smart route of CHA formation from coral skeletal carbonate, we have developed a CaCO3 template-based method for preparation of carbonated HA spherulites within a collagen matrix and then obtained a composite hydrogel of collagen/HA-microspheres under mild conditions. Based on the previous work [20], this study mainly focuses on the in situ derivation of HA microspheres from CaCO3 templates within a collagen hydrogel matrix by only one procedure. As illustrated in scheme 1, the phase transformation of the inorganic particles from CaCO3 to HA proceeds along with the self-assembly of collagen molecules. After this process, HA microspheres can be obtained in situ from the conversion of the CaCO3 templates within a simultaneously constructed collagen hydrogel. As fabricated, the collagen/HA-microparticle hybrid gels exhibit multiscale structures including nanofibrils of collagen, microscale spherulites of HA and macroscale blocks of the composites. Despite apparent structural differences between these hybrids and nature bone tissue, the significant concept of a multiscale structure for hydrogel materials has been introduced and achieved here. Meanwhile, a novel route to prepare carbonated HA microspheres from the conversion of CaCO3 templates within a bio-macromolecule matrix under mild conditions has been successfully established in this study and would pave the way for designing further smart hydrogel scaffolds for tissue engineering and regenerative medicine.
Scheme 1. Schematic illustration of collagen/HA-microsphere composite scaffold fabrication in situ: (a) Formation of CaCO3 microspheres in an aqueous phase in the presence of collagen molecules. (b) Addition of phosphate into the suspension of collagen molecules and CaCO3 microspheres. (c) Upon elevating the system temperature to 35 °C, fibrillogenesis of collagen molecules into a fibrillar network occurs along with conversion of CaCO3 particles into HA microspheres.
to their desirable framework for cellular proliferation and survival. A natural bone matrix is an organic/inorganic hybrid mainly consisting of hydroxyapatite (HA) and collagen, and it can be characterized by a multiscale distribution of nano-, micro- and macroscale spatial structures [1–3], which have been found to contribute to the physical and biological properties of natural or engineered tissues [4–6]. Accordingly, hydrogel-based scaffolds with structures and compositions similar to those of natural tissues are highly desirable for building appropriate microenvironments for cellular behaviours. Following this principle, extensive efforts have been exerted to develop biomimetic matrices using collagen/HA composite hydrogels or their blends with other inorganic and organic elements, such as magnetite nano-particles, synthetic polymers and biomacromolecules [7–9]. Most of the available composite hydrogels usually comprise single-scale building blocks including nanofibers and nanoparticles. Some biomimetic scaffolds with hierarchical porosity have been developed [10, 11] from the aspect of the pore architecture of the scaffolds. From another angle, however, the bulk material itself comprising the hydrogel-scaffold can be designed as a multiscale distribution in space from the nanoscale to the micro- and macroscales. On the other hand, as the most important inorganic component for bone tissue engineering, HA crystals are generally obtained at the nanoscale rather than at the microscale by using conventional methods such as in situ deposition or mineralization within a biomacromolecular matrix [10–14], because it is comparatively difficult to synthesize microscale HA particles under mild conditions. Traditional methods for fabricating HA microspheres mainly rely on the approaches involving organic solvents and
2. Materials and methods 2.1. Materials
Highly purified collagen (type I) was extracted from calf skin in our laboratory following the procedures described by Miller, with some modifications [21]. Anhydrous Na2CO3, anhydrous CaCl2, Na2HPO4·12H2O and other chemicals (Kermel Ltd, Chengdu, China) were of analytical grade and used as received. The water used in all experiments was super-purified using a Milli-Q system. 2.2. Synthesis of collagen/hydroxyapatite-microsphere hybrid composites
CaCl2 was added dropwise to a 20 ml acidic collagen solution (4 mg ml−1, pH2.5) with gentle stirring to obtain a final concentration of 0.22 M Ca2+. Approximately 21 ml of a 0.22 M Na2CO3 aqueous solution was quickly added into the dispersion of the collagen/Ca2+ under rapid stirring for two minutes. Then, a concentrated aqueous phosphate solution was introduced slowly into the system with stirring. The 2
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amount of phosphate added was calculated from the ratio of calcium to phosphorus (Ca/P ratio) in stoichiometric HA based on the existing amount of Ca2+ in the synthesis system. The suspension of products was then degassed via centrifugation and transferred into beakers, which were then placed in a water bath and incubated at 35 °C for 48-120 h. All experimental procedures above that involved collagen were performed at 4 °C. Along with the occurrence of fibrillogenesis and the gradual construction of a collagen gel initiated by elevating the temperature, the CaCO3 microparticles distributing throughout the fibrillar network of the gel were gradually converted to calcium phosphate (CaP) particles in the presence of ambient phosphate. As obtained, the collagen/HAmicrosphere hybrid gels were thoroughly rinsed in deionized water to remove salt residues and then lyophilized for subsequent uses or analyses.
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Figure 1. FTIR spectra of (a) pure collagen, (b) collagen/CaCO3microsphere composites, and collagen/HA-microsphere composites derived in situ from collagen/CaCO3-microspheres in phosphate ambience for (c) 72 and (d) 48 h.
2.3. Characterization 2.3.1. Scanning electron microscopy. The microstructure of
Germany) at a heating rate of 10 °C min−1 from 30 °C to 1200 °C under a nitrogen atmosphere.
the collagen/HA-microsphere composites was analysed using a field-emission scanning electron microscope (FESEM, S4800, Hitachi, Japan) with energy dispersive spectroscopy (EDS, Inca Energy 250 with liquid nitrogen cooling, Oxford Instruments, UK). All of the specimens for SEM were sputtercoated with Au, and the specimens for the EDS analysis were applied to a conductive adhesive directly without Au coating.
3. Results 3.1. Functional groups by FTIR
As shown in figure 1, the FTIR spectra show the typical apatite signals together with the signals of CaCO3 and pure collagen.The absorption bands at 713 (v4), 876 (v2) and 1437 cm−1 (v3) were derived from the characteristic vibrations of carbonate in the collagen/CaCO3-microparticle composites (figure 1(b)). After more than 48 h of conversion of CaCO3 to CaP inside the hybrid gels, the bands at 1062 (v3), 604 (v4) and 563 cm−1 (v4) attributed to the PO3− 4 groups of apatite were legibly identified (figures 1(c)–(d)) [22]. The absorption peak at 872 cm−1 (v2) and the bimodal bands at 1421 (v3) and 1469 cm−1 (v3) are evidences for the carbonate substitution in the phosphate B-site of the HA lattice. Compared with the amide vibrational band groups (1654, 1550 and 1239 cm−1 for amide I, amide II and amide III bands, respectively [23, 24]) derived from pure collagen (figure 1(a)), the amide I bands are obviously present in the hybrid gels, with the peak at 1650 cm−1 and 1651 cm−1 for the composite hydrogel of collagen/CaCO3-microparticles and the collagen/HA-microparticles, respectively (figures 1(b)–(d)). The weaker band complexes of the amide II and amide III are absent in the hybrids.
2.3.2. X-ray diffraction. The phase identification of the
composites was performed on an x-ray diffractometer (XRD, X'Pert Pro, PHILIPS) using a Cu-Kα radiation (λ = 1.540 56 Å, 40 kV and 35 mA). Samples were scanned in 0.03° steps from 20° to 70° (2θ) with 0.2 s dwell time per step. 2.3.3. Fourier transform infrared spectroscopy.
For precise chemical composition and structure investigations, Fourier transform infrared spectroscopy (FTIR) was employed running on a NEXUS 670 instrument (Thermo Electron, USA) in the range of 4000 to 400 cm−1 with a resolution of 1.0 cm−1 and 20 scan accumulations. Transmission electron microscopy. The spatial relationship between the collagen fibrils and mineral nanoparticles was revealed using transmission electron microscopy (TEM, FEI Tecnai F20). The samples were crushed into a nanoscale powder in liquid nitrogen with a mortar and pestle. A few small drops of ethanol were then applied to the powder, followed by transferring the slurry to carbon coated copper grids and air drying. Selected area electron diffraction (SAED) was also conducted on the crystals to identify their phase.
2.3.4.
3.2. Phase identification by XRD spectrometry
The evolvement of the mineral phase transformation inside the composite gel was demonstrated by XRD patterns (figure 2). It is shown that the inorganic component of the collagen/CaCO3-microparticle composite hydrogel comprises CaCO3 vaterite and calcite (figure 2(a)). The characteristic
Thermogravimetric analysis. Thermogravimetric analyses (TGA) of the lyophilized hybrids were carried out using a Thermal Analysis Instrument (STA 49C, Netzsch,
2.3.5.
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microspheres (figure 2(b)). This information suggests the successful conversion of CaCO3 to a crystalline HA phase. The broaden peaks of the XRD patterns imply weak crystallinity in the apatite.
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3.3. Analysis of the Ca/P ratio evolving process
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As shown in the graph of the EDS analyses (figure 3), the final Ca/P ratio of the collagen/CaP-microsphere composites is 2.08 ± 0.14 at 25 °C and 1.53 ± 0.06 at 37 °C. This difference suggests that the process of the conversion of CaCO3 to CaP is mainly affected by temperature rather than by reaction time and that higher temperatures are favourable for achieving the phase transformation. Additionally, the Ca/P ratio shows small fluctuations after 72 h at 37 °C, and finally, it settles approximately 1.5, indicating that the phase transformation reaction is almost complete after 72 h. The final Ca/P ratio of the inorganic component of the collagen/HA-microsphere composites is slightly lower than that of stoichiometric HA and is similar to biologically inspired HA. Such carbonated apatite possesses the bioactivity of providing a ‘bone bond’ capability in vivo [25].
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3.4. Microstructures and morphologies of hybrid gels containing inorganic microspheres
Figure 2. XRD patterns of (a) collagen/CaCO3-microsphere composites and (b) collagen/HA-microsphere composites derived in situ from collagen/CaCO3-microspheres in phosphate ambience for (a) 48 and (b) 72 h.
Figure 4 presents the microstructures of the composite hydrogels of collagen/inorganic-microparticles. Morphological features reveal that these hybrids possess multiscale structures comprising collagen nanofibrils and inorganic microspheres and that the collagen fibrils and the inorganic microspheres interpenetrate. The CaCO3 spherulites, as the templates for fabrication of the HA microspheres, are present throughout the collagen fibrillar network and further composed of nanocrystal particles (figure 4(a)). After undergoing the phase transformation in phosphate ambience, the CaCO3 nanoparticles were thoroughly substituted with platy or needle-like HA nanocrystals either forming porous HA microspheres with ‘cauliflower’ structures or aggregating into clusters that enwrap the collagen fibrils (figure 4(b)). TEM analyses were performed to depict the CaP minerals and their distribution relationship to the collagen fibrils in great detail. The inorganic crystals existing in clusters or as individual needle-like particles attach to the surface of the collagen fibrils (figure 5). From the SAED pattern (figure 5 insert), the mineral content has been identified as HA with low crystallinity, which further confirms the results of the XRD analyses. Figure 6 shows the macroscopic morphology of the collagen/HA-microsphere composites. The shape of the hybrid hydrogel (figure 6(a)) is mouldable because the mixture suspension produced from this synthesis strategy are initially injectable. A sponge-like scaffold material (figure 6(b)) can be obtained from the hybrid gel after lyophilisation.
Figure 3. EDS analyses of the evolving Ca/P ratios of the composite gels over the conversion reaction time.
diffraction peaks of apatite became evident when the reaction time reached 48 h, and the positions of these diffuse peaks are located at 2θ = 26.3° (002), 32.3° (211), 40.3° (310), 47.0° (222) and 49.6° (213) for the hybrid gel of collagen/CaP4
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Figure 4. SEM images showing the morphology of the composite hydrogel scaffolds of (a) collagen/CaCO3-microspheres and (b) collagen/ HA-microspheres derived in situ from collagen/CaCO3-microspheres in phosphate ambience for 48 h.
78.8%, 79.3% and 79.9% for composites of collagen/HAmicrospheres-48 h (figure 7(d)), collagen/HA-microspheres72 h (figure 7(e)) and collagen/HA-microspheres-96 h (figure 7(f)), respectively. Very small difference between the weight percentage of the inorganic residues at 48 and 96 h indicates that the conversion of CaCO3 to a crystalline HA phase is almost complete after two days of reaction time in phosphate ambience.
4. Discussion It has been demonstrated that homogeneous isotropic crystal growth on tiny crystal nuclei yields a spherical shape and a uniform distribution of porous CaCO3 spheres under strong stirring [27, 28]. In the present work, this conventional synthesis route for CaCO3 particles has been introduced into an aqueous collagen solution to obtain the templates of CaCO3 microspheres within a biomacromolecular matrix. The inorganic component of the collagen/CaP-particle hybrid gel mainly consisted of carbonate-substituted apatite, which is thought to more closely resemble biologically inspired HA such as bone mineral [29–31]. As most of the calcium phosphate phase in biomimetic scaffolds for bone tissue engineering [32, 33], the apatite microspheres derived from CaCO3 templates show the typical ‘cauliflower-like’ morphology. Such carbonated apatite with poorly crystallized feature is significant for reabsorption and remodelling processes in tissues [34, 35]. From the absence of the absorption peaks of the amide II and amide III for the hybrid gels, it has been hypothesized that there would exist a certain interaction between the inorganic minerals and the collagen molecules, which could exert some constrain on the movements of stretching and bending of the N-H and C-N bonds in the collagen molecules. However, the fibrillogenesis behaviour of the collagen molecules received no any significant effect from this interaction. Nature bone consists of both inorganic and organic ECM. The organic matrix is mainly collagen type I which is an important structural protein in ECM. Collagen macromolecules are characterized by self-assembling into fibrillar network of hydrogel in vitro under suitable conditions. The inorganic ECM, namely natural bone mineral, is not pure and
Figure 5. TEM micrograph showing a typical mineralized collagen
fibril from a collagen/HA-microsphere composites derived in situ from collagen/CaCO3-microspheres in phosphate ambience for 48 h; the inset shows the SAED pattern of this mineralized fibril.
3.5. Depiction of the components of the hybrid gels via TGA
TGA was used to analyse the weight loss of the composites under elevated temperatures (figure 7). The sharpest decreases in weight were extrapolated to be at approximately 283 °C and 752 °C for pure collagen (figure 7(a)) and pure CaCO3 microparticles (calcite, figure 7(c)), respectively, while at approximately 282 °C and 710 °C for the components of collagen and CaCO3, respectively, in the collagen/CaCO3microsphere composites (figure 7(b)). Calcite is more thermodynamically stable than vaterite [26]. And additionally, the presence of collagen macromolecules could result in some defects in the CaCO3 crystal lattice. Consequently, the decomposition temperature of the CaCO3 in the collagen/ CaCO3-microparticle hybrids is slightly lower than that of pure CaCO3. The onset of the decomposition temperature of collagen in the collagen/HA-microsphere composites was recorded near 330 °C. In the range of 670–770 °C, a weight loss was recorded for the composites (figures 7(d)–(f)), and this phenomenon could be contributed to the presence of trace CaCO3 components. The residue degrees at 1000 °C are 5
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Figure 6. Images of macroscale morphology recorded by a camera showing (a) a composite hydrogel of collagen/HA-microspheres in a glass beaker and (b) a sponge-like scaffold block obtained from the same hybrid gel after lyophilisation.
pressures have to be needed in the exchanging process. So, based on the idea of fabricating CHA constructs, we synthesized micro-scale calcium carbonate component within collagen matrix and further obtain HA products from these CaCO3 templates by inorganic phase transformation in situ. In our previously described work [20], the hybrid collagen hydrogels containing CaCO3 microspheres were fabricated at the first step, then the gels were soaked in a periodically changed phosphate solution to promote the phase transformation of CaCO3 to HA. In this study, however, those procedures have been modified and simplified to just one step. The phosphate was introduced into the collagen suspension immediately after the synthesis of the CaCO3 microparticles, and then the fibrillogenesis of the collagen macromolecules was triggered to construct a composite collagen hydrogel upon elevation of the system temperature to physiological temperature. Thus, the conversion of the CaCO3 particles and the self-assembly of collagen molecules occurred simultaneously in situ. Since the suspension of the collagen-based mixture is injectable, the resultant composite gel is shapeability, and also the multifunctional gel can be constructed by facilely introducing external ingredients such as cells, bioactive factors and drug molecules into the system before the activation of gelation.
Figure 7. TGA curves of (a) pure collagen, (b) collagen/CaCO3microsphere composites and (c) pure CaCO3 microparticles. Collagen/HA-microsphere composites derived in situ from collagen/ CaCO3-microspheres in phosphate ambience for (d) 48, (e) 72 and (f) 96 h.
not fully crystallized hydroxyapatite, but is factually a highly substituted microcrystalline HA containing carbonate and other minerals [36]. Accordingly, collagen and HA are the most necessary components in the synthesized biomimetic scaffold materials for bone tissue engineering and regenerative medicine [7–9, 37]. For utilization of the unique pore sizes and the porous structure of corals, Della M. and his coworkers first prepared HA from coral skeletal carbonate by using hydrothermal chemical exchange with phosphate, and the relevant mechanism is based on the fact that HA is much more thermodynamically stable than CaCO3 [17]. Since then, CHA which is derived from coral skeletal calcium carbonate, has been extensively studied in experiments for development as bone graft substitutes [38–41]. Commercially available forms of CHA are used clinically as a bone substitute in various situations [42–44]. However, this kind of scaffold material contains no collagen composition, and furthermore, harsh reaction conditions of high temperatures and high
5. Conclusions An approach for HA microsphere synthesis in situ from the templates of CaCO3 spherulites under mild conditions has been successfully achieved within a collagen matrix. Asobtained hybrids exhibit multiscale microstructures consisting of nanoscale collagen fibrils and microscale HA particles, which are further composed of carbonated apatite nanocrystals with low crystallinity. This facile strategy for preparing collagen/HA-microsphere hybrid gels may be a promising lead to further develop a novel multifunctional hydrogel 6
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material with shapeability for tissue engineering, and also could find potentials in designing the advanced supporting scaffolds for surgical repair and medical reconstruction.
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Acknowledgments This work was supported by National Natural Science Foundation of China (51203103, 51373105) and National Basic Research Program of China (National 973 program, No. 2011CB606201).
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