Self-assembled calcium pyrophosphate nanostructures for targeted molecular delivery.

Nanostructured, inorganic microspheres have many industrial applications, including catalysis, electronics, and particularly drug delivery, with several advantages over their organic counterparts. However, many current production methods require high energy input, use of harmful chemicals, and extensive processing. Here, the self-assembly of calcium pyrophosphate into nanofibre microspheres is reported. This process takes place at ambient temperature, with no energy input, and only salt water as a by-product. The formation of these materials is examined, as is the formation of nanotubes when the system is agitated, from initial precipitate to crystallisation. A mechanism of formation is proposed, whereby the nanofibre intermediates are formed as the system moves from kinetically favoured spheres to thermodynamically stable plates, with a corresponding increase in aspect ratio. The functionality of the nanofibre microspheres as targeted enteric drug delivery vehicles is then demonstrated in vitro and in vivo, showing that the microspheres can pass through the stomach while protecting the activity of a model protein, then release their payload in intestinal conditions.

Modelling the central nervous system: tissue engineering of the cellular microenvironment.

With the increasing prevalence of neurodegenerative diseases, improved models of the central nervous system (CNS) will improve our understanding of neurophysiology and pathogenesis, whilst enabling exploration of novel therapeutics. Studies of brain physiology have largely been carried out using in vivo models, ex vivo brain slices or primary cell culture from rodents. Whilst these models have provided great insight into complex interactions between brain cell types, key differences remain between human and rodent brains, such as degree of cortical complexity. Unfortunately, comparative models of human brain tissue are lacking. The development of induced Pluripotent Stem Cells (iPSCs) has accelerated advancement within the field of in vitro tissue modelling. However, despite generating accurate cellular representations of cortical development and disease, two-dimensional (2D) iPSC-derived cultures lack an entire dimension of environmental information on structure, migration, polarity, neuronal circuitry and spatiotemporal organisation of cells. As such, researchers look to tissue engineering in order to develop advanced biomaterials and culture systems capable of providing necessary cues for guiding cell fates, to construct in vitro model systems with increased biological relevance. This review highlights experimental methods for engineering of in vitro culture systems to recapitulate the complexity of the CNS with consideration given to previously unexploited biophysical cues within the cellular microenvironment.

Local Structure of Ca2+ Alginate Hydrogels Gelled via Competitive Ligand Exchange and Measured by Small Angle X-Ray Scattering.

Alginates, being linear anionic co-polymers of 1,4-linked residues ß-d-ManA (M) and α-l-GulA (G), are widely applied as hydrogel biomaterials due to their favourable in vivo biocompatibility and convenient ionic crosslinking. The "egg-box" model is the prevailing description of the local structure of junction zones that form between the alginate chains and divalent cations, such as Ca2+, when ionic gelation occurs. In the present study we address to what extent signatures of lateral dimerization and further lateral association of junction zones also represent a valid model for the gelation of alginate using the recently reported method of competitive ligand exchange of chelated Ca2+ ions as a method for introducing gelling ions at constant pH. Small angle X-ray scattering with a q range from 0.1 to 3.3 nm-1 was employed to determine local structure in the hydrogel, using a custom-made fluid sample cell inserted in the X-ray beam. The scattering volume was intended to be localized to the contact zone between the two injected aqueous alginate solutions, and data was captured to resolve the kinetics of the structure formation at three different conditions of pH. The data show evolution of the local structure for the Ca2+ induced formation of junction zones in an alginate with 68% G residues, characterized by cross-sectional radii that could be accounted for by a two-component, broken rod like model. The evolution of the two component weight fractions apparently underpinned the connectivity, as reflected in the rheological data.

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.

Versatile, cell and chip friendly method to gel alginate in microfluidic devices.

Alginate is used extensively in microfluidic devices to produce discrete beads or fibres at the microscale. Such structures may be used to encapsulate sensitive cargoes such as cells and biomolecules. On chip gelation of alginate represents a significant challenge since gelling kinetics or physicochemical conditions are not biocompatible. Here we present a new method that offers a hitherto unprecedented level of control over the gelling kinetics and pH applied to the encapsulation of a variety of cells in both bead and fibre geometries. This versatile approach proved straightforward to adjust to achieve appropriate solution conditions required for implementation in microfluidic devices and resulted in highly reliable device operation and very high viability of several different encapsulated cell types for prolonged periods. We believe this method offers a paradigm shift in alginate gelling technology for application in microfluidics.

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.

Diagenesis-inspired reaction of magnesium ions with surface enamel mineral modifies properties of human teeth.

UNLABELLED: Mineralized tissues such as teeth and bones consist primarily of highly organized apatitic calcium-phosphate crystallites within a complex organic matrix. The dimensions and organization of these apatite crystallites at the nanoscale level determine in part the physical properties of mineralized tissues. After death, geological processes such as diagenesis and dolomitization can alter the crystallographic properties of mineralized tissues through cycles of dissolution and re-precipitation occurring in highly saline environments. Inspired by these natural exchange phenomena, we investigated the effect of hypersalinity on tooth enamel. We discovered that magnesium ions reacted with human tooth enamel through a process of dissolution and re-precipitation, reducing enamel crystal size at the surface of the tooth. This change in crystallographic structure made the teeth harder and whiter. Salt-water rinses have been used for centuries to ameliorate oral infections; however, our discovery suggests that this ancient practice could have additional unexpected benefits. STATEMENT OF SIGNIFICANCE: Here we describe an approach inspired by natural geological processes to modify the properties of a biomineral - human tooth enamel. In this study we showed that treatment of human tooth enamel with solutions saturated with magnesium induced changes in the nanocrystals at the outer surface of the protective enamel layer. As a consequence, the physical properties of the tooth were modified; tooth microhardness increased and the color shade became whiter, thus suggesting that this method could be used as a clinical treatment to improve dental mechanical properties and esthetics. Such an approach is simple and straightforward, and could also be used to develop new strategies to synthesize and modify biominerals for biomedical and industrial applications.

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.

Competitive ligand exchange of crosslinking ions for ionotropic hydrogel formation.

Currently there are limitations to gelation strategies to form ionically crosslinked hydrogels, derived in particular from a lack of control over the release kinetics of crosslinking ions, which severely restrict applications. To address this challenge, we describe a new approach to form hydrogels of ionotropic polymers using competitive displacement of chelated ions, thus making specific ions available to induce interactions between polymer chains and form a hydrogel. This strategy enables control of ion release kinetics within an aqueous polymer solution and thus control over gelation kinetics across a wide range of pH. The described technique simplifies or facilitates the use of ionotropic hydrogels in a range of applications, such as 3D printing, microfluidic-based cell encapsulation, injectable preparations and large scale bubble and solid free mouldable gels. We investigate a range of chelator-ion combinations and demonstrate this powerful method to form hydrogels across a wide range of pH and µm-cm length scales. We highlight our findings by applying this gelation strategy to some of the more challenging hydrogel application areas using alginate and polygalacturonate as model polymer systems.

Elucidating the individual effects of calcium and phosphate ions on hMSCs by using composite materials.

The biological performance of bone graft substitutes based on calcium phosphate bioceramics is dependent on a number of properties including chemical composition, porosity and surface micro- and nanoscale structure. However, in contemporary bioceramics these properties are interlinked, therefore making it difficult to investigate the individual effects of each property on cell behavior. In this study we have attempted to investigate the effects of calcium and inorganic phosphate ions independent from one another by preparing composite materials with polylactic acid (PLA) as a polymeric matrix and calcium carbonate or sodium phosphate salts as fillers. Clinically relevant bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on these composites and proliferation, osteogenic differentiation and ECM mineralization were investigated with time and were compared to plain PLA control particles. In parallel, cells were also cultured on conventional cell culture plates in media supplemented with calcium or inorganic phosphate to study the effect of these ions independent of the 3D environment created by the particles. Calcium was shown to increase proliferation of cells, whereas both calcium and phosphate positively affected alkaline phosphatase enzyme production. QPCR analysis revealed positive effects of calcium and of inorganic phosphate on the expression of osteogenic markers, in particular bone morphogenetic protein-2 and osteopontin. Higher levels of mineralization were also observed upon exposure to either ion. Effects were similar for cells cultured on composite materials and those cultured in supplemented media, although ion concentrations in the composite cultures were lower. The approach presented here may be a valuable tool for studying the individual effects of a variety of soluble compounds, including bioinorganics, without interference from other material properties.

Dissolution of copper mineral phases in biological fluids and the controlled release of copper ions from mineralized alginate hydrogels.

Here we investigate the dissolution behaviour of copper minerals contained within biocompatible alginate hydrogels. Copper has a number of biological effects and has most recently been evaluated as an alternative to expensive and controversial growth factors for applications in tissue engineering. Precise control and sustained release of copper ions are important due to a narrow therapeutic window of this potentially toxic ion, and alginate would appear to be a good material of choice for this purpose. We found that aqueously insoluble copper minerals could be precipitated during gelling within or mixed into alginate hydrogels in the form of microbeads prior to gelling to serve as depots of copper. These minerals were found to be soluble in a variety of biological fluids relevant to in vitro and in vivo investigations, and the alginate carrier served as a barrier to diffusion of these ions and therefore offered control over the rate and duration of release (Cu(2+) release rates observed between 10-750 µMol g(-1) h(-1) and duration for up to 32 d). Copper mineral and copper mineralized alginate microbeads were characterized using powder x-ray diffraction, FTIR, thermogravimetric analysis and scanning electron microscopy. Dissolution kinetics were studied based on measurements of copper ion concentrations using colourimetric methods. In addition we characterized the complexes formed between released copper ions and biological fluids by electron paramagnetic spectroscopy which offers an insight into the behaviour of these materials in the body.

Osseointegration of dental implants in 3D-printed synthetic onlay grafts customized according to bone metabolic activity in recipient site.

Onlay grafts made of monolithic microporous monetite bioresorbable bioceramics have the capacity to conduct bone augmentation. However, there is heterogeneity in the graft behaviour in vivo that seems to correlate with the host anatomy. In this study, we sought to investigate the metabolic activity of the regenerated bone in monolithic monetite onlays by using positron emission tomography-computed tomography (PET-CT) in rats. This information was used to optimize the design of monetite onlays with different macroporous architecture that were then fabricated using a 3D-printing technique. In vivo, bone augmentation was attempted with these customized onlays in rabbits. PET-CT findings demonstrated that bone metabolism in the calvarial bone showed higher activity in the inferior and lateral areas of the onlays. Histological observations revealed higher bone volume (up to 47%), less heterogeneity and more implant osseointegration (up to 38%) in the augmented bone with the customized monetite onlays. Our results demonstrated for the first time that it is possible to achieve osseointegration of dental implants in bone augmented with 3D-printed synthetic onlays. It was also observed that designing the macropore geometry according to the bone metabolic activity was a key parameter in increasing the volume of bone augmented within monetite onlays.

A new class of bioactive glasses: calcium-magnesium sulfophosphates.

Low-melting ionic sulfophosphate glasses from the system P2O5-SO4-MO-Na2O (M = Zn(2+), Ca(2+) or Mg(2+)) have been previously shown by us to allow tuneable aqueous dissolution and also enable processing temperatures well below 400°C. Sulfate ions are extremely safe for use in the body as decades of use of calcium sulfate bone grafts testifies and there is no known limit on their adult oral toxicity. This glass system therefore offers great potential for use as biomaterials, especially in organic-inorganic hybrid systems such as glass-polymer composites for tissue engineering or drug encapsulation and delivery applications. A compositional region was identified where stable sulfophosphates of the type P2O5-SO4-(Ca, Mg, Zn)O-Na2O can be fabricated. For these glasses, the viscosity-temperature-dependence, glass transformation temperatures (Tg ) and the onset of crystallization were evaluated as the primary processing parameters. As a first step in exploring their potential as a biomaterial, in this study we examine the bioactivity of several compositions of these glasses using fibroblast, monocyte, and osteoclast cell culture models to determine cellular responses in terms of attachment, proliferation, differentiation, and toxicity.

Ultrasonic phosphate bonding of nanoparticles.

Low intensity ultrasound-induced radicals interact with surface adsorbed orthophosphate to bond nanoparticles with high mechanical strength and surface area. Dissimilar materials could be bonded to form robust metallic, ceramic, and organic composite microparticles. 3D nanostructures of a hydrated and amorphous electrocatalyst with carbon nanotubes were also constructed which exceeded the resistance-limited efficiency of 2D electrodes.

Biocompatibility of magnesium phosphate minerals and their stability under physiological conditions.

Magnesium phosphates such as newberyite (MgHPO(4)·3H(2)O) are formed in vivo and are known to be biodegradable and nontoxic after implantation. Indeed, magnesium apatites have been shown to support osteoblast differentiation and function, and bone formation can occur around metallic magnesium implants. However, very little is known regarding the precipitation and stability of magnesium phosphates in physiological environments. In order to address this, the aqueous formation of magnesium phosphate as a function of pH, temperature and ion concentration is reported. Physicochemical characterization of the precipitates was carried out; additionally, biocompatibility and gene expression of osteoblast differentiation markers for bone formation via an in vitro cell culture assay were determined. Precipitation conditions for newberyite, tribasic magnesium phosphate pentahydrate, holtedahlite, bobierrite and cattiite were determined. Under physiological conditions of pH, temperature and magnesium phosphate concentration, no precipitates were formed. However, at concentrations 10-100 times higher than physiological, magnesium phosphate precipitates of cattiite and newberyite were formed. These two minerals demonstrated biocompatibility with osteoblast cultures and induced osteoblast adhesion and differentiation. The pattern of expression of OCN and CollA1 genes in the presence of newberyite crystals was comparable to that of calcium phosphate bioceramics. In our experiments, we have shown that certain magnesium phosphate phases such as newberyite and cattiite are able to promote in vivo osteogenic activity in a similar way to calcium phosphates such as hydroxyapatite and brushite. This confirms the great potential of magnesium phosphate ceramics in the development of new biomaterials for bone regeneration.

Craniofacial vertical bone augmentation: a comparison between 3D printed monolithic monetite blocks and autologous onlay grafts in the rabbit.

Onlay autografting is amongst the most predictable techniques for craniofacial vertical bone augmentation, however, complications related to donor site surgery are common and synthetic alternatives to onlay autografts are desirable. Recent studies have shown that the acidic calcium phosphates, brushite and monetite, are osteoconductive, osteoinductive and resorb faster in vivo than hydroxyapatite. Moreover, they can be 3D printed allowing precise host bone-implant conformation. The objectives of this study were to confirm that craniofacial screw fixation of 3D printed monetite blocks was possible and to compare the resulting vertical bone augmentation with autograft. 3D printed monolithic monetite onlay implants were fixed with osteosynthesis screws on the calvarial bone surface of New Zealand rabbits. After 8 weeks, integration between the implant and the calvarial bone surface was observed in all cases. Histomorphometry revealed that 42% of the monetite was resorbed and that the new bone formed within the implant occupied 43% of its volume, sufficient for immediate dental implant placement. Bone tissue within the autologous onlay occupied 60% of the volume. We observed that patterns of regeneration within the implants differed throughout the material and propose that this was due to the anatomy and blood supply pattern in the region. Rapid prototyped monetite being resorbable osteoconductive and osteoinductive would appear to be a promising biomaterial for many bone regeneration strategies.

Minimally invasive maxillofacial vertical bone augmentation using brushite based cements.

An ideal material for maxillofacial vertical bone augmentation procedures should not only be osteoconductive, biocompatible and mechanically strong, but should also be applied using minimally invasive procedures and remain stable with respect to the original bone surfaces. This way, implant exposure and infection might be reduced and good mechanical stability may be achieved. Calcium phosphate cements are proven biocompatible and osteoconductive materials that can be injected using minimally invasive procedures. Among these cements, brushite based cements have the added advantage of being biodegradable in vivo. Therefore, this material has the potential for use in the aforementioned procedures. An in vivo study was performed in rabbits to evaluate the potential use of brushite cements in minimally invasive maxillofacial vertical bone augmentation procedures. In this study, we injected self-setting brushite cements on the subperiosteal bone surface using a minimally invasive tunnelling technique. The cement pastes were stable on the bone surface and hardened soon after they were injected thereby negating the need for additional supports such as membranes or meshes. The animals were sacrificed 8 weeks after the intervention and histological observations revealed signs of successful vertical bone augmentation. Therefore, we have demonstrated a minimally invasive vertical bone augmentation procedure that is an attractive alternative to current surgical procedures in terms of increased simplicity, reduced trauma, and lower cost of surgery.

The importance of particle size and DNA condensation salt for calcium phosphate nanoparticle transfection.

Calcium phosphate has been used for over 30 years to deliver genetic material to mammalian cells. This vector has proven advantages over other transfection species such as viruses and dendrimers in terms of superior biocompatibility and reduced immune response. However, clinical application of calcium phosphate based transfection techniques is hampered by poor understanding of the key factors underlying its action. Despite widespread in vitro use, little attention has been given to the physico-chemical characteristics of the calcium phosphate particles mediating transfection. In this study parameters were optimised to produce calcium phosphate nanoparticles onto which plasmid DNA (pDNA) was adsorbed that were more effective than a commercial dendrimer vector in delivering pDNA to an osteoblastic cell line and compared favourably in a fibroblastic cell line without the need for special culture conditions such as cell cycle synchronization or glycerol shock treatment. Addition of the pDNA after nanoparticle synthesis allowed for characterisation of particle morphology, size, surface charge and composition. We found that the key parameters for effective calcium phosphate nanoparticle transfection were an optimal concentration of calcium and chloride ions and a nanosized non-agglomerated precipitate.