Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers.

The study of environmental DNA (eDNA) released by aquatic organisms in their habitat offers a fast, noninvasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as water filtration and DNA precipitation are time-consuming, require difficult-to-handle equipment and partially integrate eDNA signals. To overcome these limitations, we created the first proof of concept of a passive, 3D-printed and easy-to-use eDNA sampler. We designed the samplers from hydroxyapatite (HAp samplers), a natural mineral with a high DNA adsorption capacity. The porous structure and shape of the samplers were designed to optimize DNA adsorption and facilitate their handling in the laboratory and in the field. Here we show that HAp samplers can efficiently collect genomic DNA in controlled set-ups, but can also collect animal eDNA under controlled and natural conditions with yields similar to conventional methods. However, we also observed large variations in the amount of DNA collected even under controlled conditions. A better understanding of the DNA-hydroxyapatite interactions on the surface of the samplers is now necessary to optimize eDNA adsorption and to allow the development of a reliable, easy-to-use and reusable eDNA sampling tool.

Electrospinning of in situ synthesized silica-based and calcium phosphate bioceramics for applications in bone tissue engineering: A review.

The field of bone tissue engineering (BTE) focuses on the repair of bone defects that are too large to be restored by the natural healing process. To that purpose, synthetic materials mimicking the natural bone extracellular matrix (ECM) are widely studied and many combinations of compositions and architectures are possible. In particular, the electrospinning process can reproduce the fibrillar structure of bone ECM by stretching a viscoelastic solution under an electrical field. With this method, nano/micrometer-sized fibres can be produced, with an adjustable chemical composition. Therefore, by shaping bioactive ceramics such as silica, bioactive glasses and calcium phosphates through electrospinning, promising properties for their use in BTE can be obtained. This review focuses on the in situ synthesis and simultaneous electrospinning of bioceramic-based fibres while the reasons for using each material are correlated with its bioactivity. Theoretical and practical considerations for the synthesis and electrospinning of these materials are developed. Finally, investigations into the in vitro and in vivo bioactivity of different systems using such inorganic fibres are exposed.

Effect of grain orientation and magnesium doping on ß-tricalcium phosphate resorption behavior.

The efficiency of calcium phosphate (CaP) bone substitutes can be improved by tuning their resorption rate. The influence of both crystal orientation and ion doping on resorption is here investigated for beta-tricalcium phosphate (ß-TCP). Non-doped and Mg-doped (1 and 6 mol%) sintered ß-TCP samples were immersed in acidic solution (pH 4.4) to mimic the environmental conditions found underneath active osteoclasts. The surfaces of ß-TCP samples were observed after acid-etching and compared to surfaces after osteoclastic resorption assays. ß-TCP grains exhibited similar patterns with characteristic intra-crystalline pillars after acid-etching and after cell-mediated resorption. Electron BackScatter Diffraction analyses, coupled with Scanning Electron Microscopy, Inductively Coupled Plasma-Mass Spectrometry and X-Ray Diffraction, demonstrated the influence of both grain orientation and doping on the process and kinetics of resorption. Grains with c-axis nearly perpendicular to the surface were preferentially etched in non-doped ß-TCP samples, whereas all grains with simple axis (a, b or c) nearly normal to the surface were etched in 6 mol% Mg-doped samples. In addition, both the dissolution rate and the percentage of etched surface were lower in Mg-doped specimens. Finally, the alignment direction of the intra-crystalline pillars was correlated with the preferential direction for dissolution. STATEMENT OF SIGNIFICANCE: The present work focuses on the resorption behavior of calcium phosphate bioceramics. A simple and cost-effective alternative to osteoclast culture was implemented to identify which material features drive resorption. For the first time, it was demonstrated that crystal orientation, measured by Electron Backscatter Diffraction, is the discriminating factor between grains, which resorbed first, and grains, which resorbed slower. It also elucidated how resorption kinetics can be tuned by doping ß-tricalcium phosphate with ions of interest. Doping with magnesium impacted lattice parameters. Therefore, the crystal orientations, which preferentially resorbed, changed, explaining the solubility decrease. These important findings pave the way for the design of optimized bone graft substitutes with tailored resorption kinetics.

Novel calcium phosphate/PCL graded samples: Design and development in view of biomedical applications.

Scaffolds for bone tissue engineering require a combination of bioactivity and bioresorption at the sample surface and high mechanical properties in the bulk. This work presents a novel calcium phosphate (CaP)/polycaprolactone (PCL) scaffold with graded composition and porosity fraction. The scaffold is made of (i) a dense hydroxyapatite (HA)/ß-tricalcium phosphate (ß-TCP) core, (ii) a macroporous HA/ß-TCP transition layer and (iii) a macroporous PCL/(HA/ß-TCP) external layer. The ceramic layers were fabricated by gel-casting whereas the outer composite layer was obtained by a solvent casting/particle leaching process. The microstructure, phase composition and biodegradation of the scaffolds were characterized. The gradient of porosity was clearly obtained whereas the gradation of phase composition was less pronounced. An in vitro dissolution test was performed by immersing the scaffolds in a TRIS solution. The results showed a dissolution phenomenon with possible differentiated mechanisms in the different layers, in relation with the targeted multi-functionality.

3-D printing of chitosan-calcium phosphate inks: rheology, interactions and characterization.

Bone substitute fabrication is of interest to meet the worldwide incidence of bone disorders. Physical chitosan hydrogels with intertwined apatite particles were chosen to meet the bio-physical and mechanical properties required by a potential bone substitute. A set up for 3-D printing by robocasting was found adequate to fabricate scaffolds. Inks consisted of suspensions of calcium phosphate particles in chitosan acidic aqueous solution. The inks are shear-thinning and consist of a suspension of dispersed platelet aggregates of dicalcium phosphate dihydrate in a continuous chitosan phase. The rheological properties of the inks were studied, including their shear-thinning characteristics and yield stress. Scaffolds were printed in basic water/ethanol baths to induce transformation of chitosan-calcium phosphates suspension into physical hydrogel of chitosan mineralized with apatite. Scaffolds consisted of a chitosan polymeric matrix intertwined with poorly crystalline apatite particles. Results indicate that ink rheological properties could be tuned by controlling ink composition: in particular, more printable inks are obtained with higher chitosan concentration (0.19 mol·L-1).

The in vitro evolution of resorbable brushite cements: A physico-chemical, micro-structural and mechanical study.

The mechanisms by which calcium phosphate bone substitutes evolve and are resorbed in vivo are not yet fully known. In particular, the formation of intermediate phases during resorption and evolution of the mechanical properties may be of crucial interest for their clinical efficiency. The in vitro tests proposed here are the first steps toward understanding these phenomena. Microporous Dicalcium Phosphate Dihydrate (DCPD) samples were immersed in tris(hydroxymethyl)aminomethane (TRIS) and Phosphate Buffered Saline (PBS) solutions, with or without daily refresh of the medium, for time-points up to 14days. Before and after immersion, samples were extensively characterised in terms of morphology, chemistry (XRD coupled with Rietveld analysis), microstructure (X-ray tomography, SEM observations) and local mechanical properties (instrumented micro-indentation). The composition of the immersion solutions was monitored in parallel (pH, elemental analysis). The results show the influence and importance of the experimental set-up and protocol on the formation of apatite and octacalcium phosphate concurrently to DCPD dissolution; moreover, strong inter-correlations between physico-chemistry, microstructure and mechanics are demonstrated. STATEMENT OF SIGNIFICANCE: Ideally, the resorption kinetics of biodegradable bone substitutes should be controlled to favor the healing processes of bone. Although biodegradable bone grafts are already used in surgeries, their resorption process is still partially unknown. The present work studies these resorption phenomena, their kinetics and mechanisms and their consequences on the properties of a calcium phosphate resorbable material. The original in vitro approach developed in this work couples for the first time physico-chemical, micro-structural and mechanical assessments. The dissolution of the CaP phase in body fluids and the reprecipitation of more stable phases are studied on a local scale, which has permitted to evidence and monitor the development of a gradient of properties between the surface and the core of the samples.

Microstructural control of modular peptide release from microporous biphasic calcium phosphate.

Drug release from tissue scaffolds is commonly controlled by using coatings and carriers, as well as by varying the binding affinity of molecules being released. This paper considers modulating synthetic peptide incorporation and release through the use of interconnected microporosity in biphasic calcium phosphate (BCP) and identifies the microstructural characteristics important to the release using experiments and a model of relative diffusivity. First, the release of three modular peptides designed to include an osteocalcin-inspired binding sequence based on bone morphogenic protein-2 (BMP-2) was compared and one was selected for further study. Next, the incorporation and release of the peptide from four types of substrates were compared: non-microporous (NMP) substrates had no microporosity; microporous (MP) substrates were either 50% microporous with 5µm pores (50/5), 60% microporous with 5µm pores (60/5), or 50% microporous with 50µm pores (50/50). Results showed that MP substrates incorporated significantly more peptide than NMP ones, but that the three different microporous substrates all incorporated the same total amount of peptide. NMP had a markedly lower release rate compared to each of three of the MP samples, though the initial burst release was the highest. The initial release and the release rate for the 60/5 samples were different from the 50/50, though they were not statistically different from the 50/5. The model indicated that the pore interconnection to pore size ratio, affecting the constriction between pores, had the greatest influence on the calculated relative diffusivity. While the model was consistent with the trends observed experimentally, the quantitative experimental results suggested that to attain an appreciable difference in release characteristics, both pore size and pore fraction should be changed for this system. These results contribute to rational scaffold design by showing that microstructure, specifically microporosity, can be used to modulate drug release.

Hydrogen-substituted ß-tricalcium phosphate synthesized in organic media.

ß-Tricalcium phosphate (ß-TCP) platelets synthesized in ethylene glycol offer interesting geometries for nano-structured composite bone substitutes but were never crystallographically analyzed. In this study, powder X-ray diffraction and Rietveld refinement revealed a discrepancy between the platelet structure and the known ß-TCP crystal model. In contrast, a model featuring partial H for Ca substitution and the inversion of P1O4 tetrahedra, adopted from the whitlockite structure, allowed for a refinement with minimal misfits and was corroborated by HPO42- absorptions in Fourier-transform IR spectra. The Ca/P ratio converged to 1.443 ±â€…0.003 (n = 36), independently of synthesis conditions. As a quantitative verification, the platelets were thermally decomposed into hydrogen-free ß-TCP and ß-calcium pyrophosphate which resulted in a global Ca/P ratio in close agreement with the initial ß-TCP Ca/P ratio (ΔCa/P = 0.003) and with the chemical composition measured by inductively coupled plasma (ΔCa/P = 0.003). These findings thus describe for the first time a hydrogen-substituted ß-TCP structure, i.e. a Mg-free whitlockite, represented by the formula Ca21 - x(HPO4)2x(PO4)14 - 2x, where x = 0.80 ±â€…0.04, and may have implications for resorption properties of bone regenerative materials.

Synthesis of spherical calcium phosphate particles for dental and orthopedic applications.

Calcium phosphate materials have been used increasingly in the past 40 years as bone graft substitutes in the dental and orthopedic fields. Accordingly, numerous fabrication methods have been proposed and used. However, the controlled production of spherical calcium phosphate particles remains a challenge. Since such particles are essential for the synthesis of pastes and cements delivered into the host bone by minimally-invasive approaches, the aim of the present document is to review their synthesis and applications. For that purpose, production methods were classified according to the used reagents (solutions, slurries, pastes, powders), dispersion media (gas, liquid, solid), dispersion tools (nozzle, propeller, sieve, mold), particle diameters of the end product (from 10 nm to 10 mm), and calcium phosphate phases. Low-temperature calcium phosphates such as monetite, brushite or octacalcium phosphate, as well as high-temperature calcium phosphates, such as hydroxyapatite, ß-tricalcium phosphate or tetracalcium phosphate, were considered. More than a dozen production methods and over hundred scientific publications were discussed.

Strontium-loaded mineral bone cements as sustained release systems: Compositions, release properties, and effects on human osteoprogenitor cells.

This study aims to evaluate in vitro the release properties and biological behavior of original compositions of strontium (Sr)-loaded bone mineral cements. Strontium was introduced into vaterite CaCO3 -dicalcium phosphate dihydrate cement via two routes: as SrCO3 in the solid phase (SrS cements), and as SrCl2 dissolved in the liquid phase (SrL cements), leading to different cement compositions after setting. Complementary analytical techniques implemented to thoroughly investigate the release/dissolution mechanism of Sr-loaded cements at pH 7.4 and 37°C during 3 weeks revealed a sustained release of Sr and a centripetal dissolution of the more soluble phase (vaterite) limited by a diffusion process. In all cases, the initial burst of the Ca and Sr release (highest for the SrL cements) that occurred over 48 h did not have a significant effect on the expression of bone markers (alkaline phosphatase, osteocalcin), the levels of which remained overexpressed after 15 days of culture with human osteoprogenitor (HOP) cells. At the same time, proliferation of HOP cells was significantly higher on SrS cements. Interestingly, this study shows that we can optimize the sustained release of Sr(2+) , the cement biodegradation and biological activity by controlling the route of introduction of strontium in the cement paste.

Cogrinding significance for calcium carbonate-calcium phosphate mixed cement. II. Effect on cement properties.

In the present study, we aim to evaluate the contribution of the cogrinding process in controlling calcium carbonate-dicalcium phosphate dihydrate cement properties. We set a method designed to evaluate phase separation, usually occurring during paste extrusion, which is quantitative, reliable, and discriminating and points out the determining role of cogrinding to limit filter-pressing. We show that solid-phase cogrinding leads to synergistic positive effects on cement injectability, mechanical properties, and radio-opacity. It allows maintaining a low (<0.4 kg) and constant load during the extrusion of paste, and the paste's composition remains constant and close to that of the initial paste. Analogous behavior was observed when adding a third component into the solid phase, especially SrCO(3) as a contrasting agent. Moreover, the cement's mechanical properties can be enhanced by lowering the L/S ratio because of the lower plastic limit. Finally, unloaded or Sr-loaded cements show uniform and increased optical density because of the enhanced homogeneity of dry component distribution. Interestingly, this study reveals that cogrinding improves and controls essential cement properties and involves processing parameters that could be easily scaled up. This constitutes a decisive advantage for the development of calcium carbonate-calcium phosphate mixed cements and, more generally, of injectable multicomponent bone cements that meet a surgeon's requirements.