Stabilisation of amorphous calcium phosphate in polyethylene glycol hydrogels.

Acellular polymer-calcium phosphate composites are promising bone graft materials. Hydrogels are suitable for providing a temporary matrix, while calcium phosphate minerals serve as ion depots for calcium and phosphate required for de novo bone formation. Crystalline calcium phosphates are stable under biological conditions and are commonly used in such scaffolds. However, the low solubility of these phases reduces the availability of free ions and potentially obstructs the remodelling necessary for the formation of mineralised tissue. Here, we investigate two different strategies to stabilise amorphous calcium phosphates in a synthetic polyethylene glycol-based hydrogel matrix. In vitro experiments mimicking an injectable application showed that amorphous calcium phosphate (ACP) of variable stability was formed in the hydrogel matrices. In additive-free composites, ACP transformed into brushite within minutes. Citrate or zinc additives were found to stabilise the formed ACP phase to different degrees. In the presence of citrate, ACP was stable for at least 2 h before it transformed into hydroxyapatite within 3-20 days. Partial calcium substitution with zinc (Zn/Ca = 10%) produced zinc-doped ACP of high stability that did not show signs of crystallisation for at least 20 days. The presented methods and findings open new possibilities for the design of novel injectable synthetic bone graft materials. The possibility to produce ACP with tailorable stability promises great potential for creating temporary scaffolds with good osteogenic properties. STATEMENT OF SIGNIFICANCE: Synthetic hydrogel-calcium phosphate (CaP) composites are promising biomaterials to replace human- and animal-derived bone scaffolds. Most reported hydrogel-CaP composite materials employ crystalline CaP phases that lack the osteoinductive properties of autograft. Stabilising amorphous calcium phosphates (ACP) could overcome this limitation, readily delivering calcium and phosphate ions and facilitating remodelling into new bone tissue. The design of synthetic hydrogel-ACP scaffolds, however, requires more understanding of the mineralisation processes in such matrices. This study presents a model system to characterise the complex mineral formation and transformation processes within a hydrogel matrix. We demonstrate a facile route to produce self-mineralising injectable synthetic hydrogels and prove two different strategies to stabilise ACP for different periods within the formed composites.

Gelling kinetics and in situ mineralization of alginate hydrogels: A correlative spatiotemporal characterization toolbox.

UNLABELLED: Due to their large water content and structural similarities to the extracellular matrix, hydrogels are an attractive class of material in the tissue engineering field. Polymers capable of ionotropic gelation are of special interest due to their ability to form gels at mild conditions. In this study we have developed an experimental toolbox to measure the gelling kinetics of alginate upon crosslinking with calcium ions. A reaction-diffusion model for gelation has been used to describe the diffusion of calcium within the hydrogel and was shown to match experimental observations well. In particular, a single set of parameters was able to predict gelation kinetics over a wide range of gelling ion concentrations. The developed model was used to predict the gelling time for a number of geometries, including microspheres typically used for cell encapsulation. We also demonstrate that this toolbox can be used to spatiotemporally investigate the formation and evolution of mineral within the hydrogel network via correlative Raman microspectroscopy, confocal laser scanning microscopy and electron microscopy. STATEMENT OF SIGNIFICANCE: Hydrogels show great promise in cell-based tissue engineering, however new fabrication and modification methods are needed to realize the full potential of hydrogel based materials. The inclusion of an inorganic phase is one such approach and is known to affect both cell-material interactions and mechanical properties. This article describes the development of a correlative experimental approach where gel formation and mineralization has been investigated with spatial and temporal resolution by applying Raman microspectroscopy, optical and electron microscopy and a reaction-diffusion modeling scheme. Modeling allows us to predict gelling kinetics for other geometries and sizes than those investigated experimentally. Our experimental system enables non-destructive study of composite hydrogel systems relevant for, but not limited to, applications within bone tissue engineering.

A correlative spatiotemporal microscale study of calcium phosphate formation and transformation within an alginate hydrogel matrix.

UNLABELLED: The modification of soft hydrogels with hard inorganic components is a method used to form composite materials with application in non-load-bearing bone tissue engineering. The inclusion of an inorganic component may provide mechanical enhancement, introduce osteoconductive or osteoinductive properties, or change other aspects of interactions between native or implanted cells and the material. A thorough understanding of the interactions between such components is needed to improve the rational design of such biomaterials. To achieve this goal, model systems which could allow study of the formation and transformation of mineral phases within a hydrogel network with a range of experimental methods and high spatial and time resolution are needed. Here, we report a detailed investigation of the formation and transformation process of calcium phosphate mineral within an alginate hydrogel matrix. A combination of optical microscopy, confocal Raman microspectroscopy and electron microscopy was used to investigate the spatial distribution, morphology and crystal phase of the calcium phosphate mineral, as well as to study transformation of the mineral phases during the hydrogel mineralization process and upon incubation in a simulated body fluid. It was found, that under the conditions used in this work, mineral initially formed as a metastable amorphous calcium phosphate phase (ACP). The ACP particles had a distinctive spherical morphology and transformed within minutes into brushite in the presence of brushite seed crystals or into octacalcium phosphate, when no seeds were present in the hydrogel matrix. Incubation of brushite-alginate composites in simulated body fluid resulted in formation of hydroxyapatite. The characterization strategy presented here allows for non-destructive, in situ observation of mineralization processes in optically transparent hydrogels with little to no sample preparation. STATEMENT OF SIGNIFICANCE: The precipitation and transformations of calcium phosphates (CaP) is a complex process, where both formation kinetics and the stability of different mineral phases control the outcome. This situation is even more complex if CaP is precipitated in a hydrogel matrix, where one can expect the organic matrix to modulate crystallization by introducing supersaturation gradients or changing the nucleation and growth kinetics of crystals. In this study we apply a range of characterization techniques to study the mineral formation and transformations of CaP within an alginate matrix with spatiotemporal resolution. It demonstrates how a detailed investigation of the mineral precipitation and transformations can aid in the future rational design of hydrogel-based materials for bone tissue engineering and studies of biomineralization processes.

Controlled mineralisation and recrystallisation of brushite within alginate hydrogels.

Due to high solubility and fast resorption behaviour under physiological conditions, brushite (CaHPO4⋅2H2O, calcium monohydrogen phosphate dihydrate, dicalcium phosphate dihydrate) has great potential in bone regeneration applications, both in combination with scaffolds or as a component of calcium phosphate cements. The use of brushite in combination with hydrogels opens up possibilities for new cell-based tissue engineering applications of this promising material. However, published preparation methods of brushite composites, in which the mineral phase is precipitated within the hydrogel network, fail to offer the necessary degree of control over the mineral phase, content and distribution within the hydrogel matrix. The main focus of this study is to address these shortcomings by determining the precise fabrication parameters needed to prepare composites with controlled composition and properties. Composite alginate microbeads were prepared using a counter-diffusion technique, which allows for the simultaneous crosslinking of the hydrogel and precipitation of an inorganic mineral phase. Reliable nucleation of a desired mineral phase within the alginate network proved more challenging than simple aqueous precipitation. This was largely due to ion transport within the hydrogel producing concentration gradients that modified levels of supersaturation and favoured the nucleation of other phases such as hydroxyapatite and octacalcium phosphate, which would otherwise not form. To overcome this, the incorporation of brushite seed crystals resulted in good control during the mineral phase, and by adjusting the number of seeds and amount of precursor concentration, the amount of mineral could be tuned. The material was characterised with a range of physical techniques, including scanning electron microscopy, powder x-ray diffraction and Rietveld refinement, Fourier transform infrared spectroscopy, and thermogravimetric analysis, in order to assess the mineral morphology, phase and amount within the organic matrix. The mineral content of the composite material converted from brushite into hydroxyapatite when submerged in simulated body fluid, indicating possible bioactivity. Additionally, initial cell culture studies revealed that both the material and the synthesis procedure are compatible with cells relevant to bone tissue engineering.