Development of injectable organic/inorganic colloidal composite gels made of self-assembling gelatin nanospheres and calcium phosphate nanocrystals
Graphical abstract
Introduction
The complexity of biomaterials has increased tremendously over the past decades in terms of both structure and composition by implementing regenerative cues that stimulate regeneration of surrounding tissues [1], [2], [3], [4]. Incorporation of therapeutic biomolecules using traditional carrier materials is still associated with major drawbacks due to the poor control over the release of growth factors at the target sites. A recent study on the clinical efficacy of the well-known osteogenic growth factor bone morphogenetic protein-2 (BMP-2) has confirmed that growth factor delivery from conventional collagen sponges results in serious clinical complications [5], emphasizing the need for novel carrier materials capable of delivering growth factors in a controlled and sustained manner. In that respect, colloidal gels are a particularly attractive class of hydrogels since these materials allow for “bottom-up” synthesis of functional, self-healing materials by employing micro- or nanoscale particles as building blocks to assemble into shape-specific bulk materials [6], [7], [8], [9], [10], [11], [12], [13]. For applications in regenerative medicine, charged micro- or nanospheres made of biocompatible polymers are the most obvious candidates to serve as building blocks since the physicochemical properties of polymeric particles can be tailored to desire in terms of size, charge and chemical derivatization. Therefore, colloidal gels have been formed by self-assembly of oppositely charged microspheres (e.g. chemically functionalized dextran [14]) or nanospheres (e.g. chemically functionalized poly(lactic-co-glycolic acid (PLGA) nanospheres [10], [12], [13]) [8], [10], [12], [13], [14], [15], [16], [17]. Recently, our group has developed a novel class of colloidal gels made of oppositely charged gelatin nanospheres which displayed superior mechanical and biological properties over microstructured colloidal gelatin gels [15], [18]. On the other hand, although the general mechanism of organic/inorganic heteroaggregation has been studied recently [19], [20], this knowledge has not been translated yet towards the development of colloidal composite gels made of biocompatible organic and inorganic nanoparticles for biomedical applications. For regeneration of hard tissues, the introduction of inorganic building blocks into colloidal gels could offer considerable advantages over purely organic colloidal gels by (i) increasing stiffness and mechanical strength of the resulting composite due to the reinforcement effect of ceramic phase, and (ii) improving the biological tissue response owing to the osteoconductive properties of inorganic nanoparticles made of, for example, calcium phosphate (CaP) [21], [22], [23]. Moreover, the addition of a ceramic phase to polymeric gels could lead to improved control over sustained delivery of growth factors, since CaP ceramics have a strong affinity to proteins [24]. This protein-binding capacity of CaP nanoparticles could directly affect the degradation of gelatin-based carrier materials and the release profile of growth factors [25], [26], [27], [28], [29].
Nevertheless, it is not yet known if interparticle forces between organic and inorganic nanoparticles can be sufficiently strong to allow for the formation of cohesive colloidal composite gels for application in regenerative medicine. Therefore, we have studied the feasibility of forming colloidal composite gels by introducing neutral or charged CaP nanoparticles into colloidal gelatin gels. CaP nanoparticles were selected in view of their osteoconductivity [21] as well as inherent affinity to proteins such as collagen, gelatin and growth factors [25], [26], [27], [28], [29], and charged CaP nanoparticles can be easily obtained by decorating CaP nanoparticle surfaces with, for example, negatively charged citrate anions that have a strong affinity for CaP surfaces [30]. Gelatin (Gel) was selected as a source for organic nanoparticles since both positively (type A) and negatively charged (type B) gelatins are commercially available. As such, self-assembly between oppositely charged organic and inorganic nanoparticles was studied without the need for additional chemical functionalizations which might compromise the biocompatibility of the final constructs, thereby opening a simple and promising route for the fabrication of nanostructured composite biomaterials of improved functionality. Compared to conventional organic/inorganic composite hydrogels composed of continuous, monolithic matrices, nanostructured colloidal composite gels can possess superior properties over bulk composites in view of their (i) enhanced control over the properties of macroscopic scaffolds by fine-tuning the characteristics of sub-populations of particulate building blocks [6], [11], [31], [32]; (ii) injectability/moldability allowing for optimal filling of irregularly shaped defects using minimally invasive approaches [10], [14], [15]; (iii) in situ gel formation without using the potentially cytotoxic gelling/cross-linking agents to trigger gelation [6], [11], [31], [32]; and (iv) ease of incorporation of therapeutic agents [18], [33], [34].
To investigate the interactions between CaP nanoparticles and gelatin nanospheres, the self-assembly process of colloidal mixtures containing organic and inorganic particles at diluted conditions (∼0.02 w/v%) was monitored using dynamic light scattering (DLS) and transmission electron microscopy (TEM) as a function of parameters including particle charge, CaP-to-gelatin ratio (CaP/Gel ratio) and ionic strength, whereas the formation of colloidal composite gels at concentrated conditions was investigated using rheometry as a function of solid content (up to 20 w/v%), CaP/Gel ratio and ionic strength. Moreover, the biological performance of the resulting Gel–CaP colloidal composite gels composed of gelatin and CaP nanoparticles was preliminarily studied in vitro by evaluating (i) gel degradation, (ii) release kinetics of a (radiolabeled) osteogenic protein (BMP-2) and (iii) the cellular response of bone marrow stem cells cultured in contact with the colloidal composite gels.
Section snippets
Materials
Gelatin A (GelA, from porcine skin, 300 Bloom, isoelectric point (IEP) ∼9) and gelatin B (GelB from bovine skin, 225 Bloom, IEP ∼5) were purchased from Sigma–Aldrich. Calcium hydroxide (Ca(OH)2, 98+%, extra pure), sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O), and glutaraldehyde (GA, 25 wt.% solution in water) were purchased from Acros Organics. Phosphoric acid (H3PO4, 85%) and acetone were from J.T. Baker. Recombinant human bone morphogenetic protein-2 (BMP-2, carrier-free, catalog no.
Preparation and characterization of CaP nanoparticles
Formation of CaP nanoparticles using an established wet-chemical precipitation method [35] resulted in the formation of needle-shaped apatitic crystals with an average length of 173 ± 52 nm and a width of 30 ± 8 nm (Table 1). The addition of sodium citrate to suspensions containing these CaP crystals shifted the ζ-potential of CaP nanoparticles from relatively neutral (+3.0 ± 0.2 mV) to highly negative (−20.8 ± 0.7 mV) (Table 1), corresponding to effective adsorption of citrate anions onto CaP
Discussion
Electrostatic interparticle forces are the most widely employed non-covalent interactions between colloidal particles for the formation of colloidal gels [8], [9], [10], [14], [15], [16], [31], [32]. Our group recently discovered that colloidal gels made of oppositely charged gelatin nanospheres were surprisingly cohesive, elastic and self-healing, while their beneficial properties for growth factor release were recently confirmed in vitro and in vivo [18], [34], [45]. Building on this concept,
Conclusions
The current study has provided firm evidence for the facile bottom-up synthesis of organic–inorganic colloidal composite gels using CaP nanoparticles and gelatin nanospheres as building blocks. Depending on the ratio between gelatin and CaP nanoparticles, these novel colloidal Gel–CaP composite gels exhibited a strongly enhanced gel elasticity, self-healing behavior and gel stability at high ionic strengths without the need for chemical – potentially cytotoxic – functionalizations to introduce
Acknowledgements
Cathelijne Frielink of the Nuclear Medicine Department of Radboud University Nijmegen Medical Centre is gratefully acknowledged for assistance with the preparation and measurement of radiolabeled biomolecules. We are grateful for the support from funding KNAW, China-Netherlands Programme Strategic Alliances (PSA) (No. 2008DFB50120).
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