David C. Bassett

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.

Resorption of monetite granules in alveolar bone defects in human patients

Bone grafting is often required to restore mandibular or maxillary bone volume prior to prosthetic tooth root implantation. Preclinical animal models are often used to study the in vivo properties of new bone graft products designed for human use. Although animal studies may offer valuable data regarding bioperformance, materials do not necessarily perform the same in human patients. In this study we implanted bovine hydroxyapatite (BH), a widely used porous apatite granule, and dicalcium phosphate anhydrous (monetite) granules, bilaterally in human patients post extraction alveolar sockets. After six months, histomorphometrical analysis of the biopsies revealed that the amount of bone regenerated with monetite (59.5 +/- 13%) was significantly higher than that obtained with BH (33.1% +/- 4.9), while the amount of unresorbed graft was higher in the sockets treated with BH (37.8 +/- 6.1) than in those implanted with monetite (25.8 +/- 14.3). Resorption of calcium phosphate ceramics is discussed by applying the Hixon-Crowell dissolution model.

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.

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.

Collagen Biomineralization In Vivo by Sustained Release of Inorganic Phosphate Ions

A new strategy for mineralized tissue formation in vivo is presented based on localized sustained delivery of inorganic orthophosphate (Pi) sufficient to supersaturate tissue surrounding an implant and induce mineralization of collagen. After 15 days implantation mineral formation around the implants was detected. Histology and electron microscopy show two populations of apatite; inter-fibrillar microcrystals and nanocrystals associated with collagen.

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.

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.

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.

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.

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.