Karlis Agris Gross

Micro-Raman spectroscopy shows how the coating process affects the characteristics of hydroxylapatite

The diversity in the structural and chemical state of apatites allows implant manufacturers to fine-tune implant properties. This requires suitable manufacturing processes and characterization tools to adjust the amorphous phase and hydroxyl content from the source hydroxylapatite. Hydroxylapatite was processed by high-velocity oxy-fuel spraying, plasma spraying and flame spraying, and primarily analyzed by Raman spectroscopy. Investigation of rounded splats, the building blocks of thermal spray coatings, allowed correlation between the visual identity of the splat surface and the Raman spectra. Splats were heat-treated to crystallize any remaining amorphous phase. The ν1 PO4 stretching peak at 950-970 cm(-1) displayed the crystalline order, but the hydroxyl peak at 3572 cm(-1) followed the degree of dehydroxylation. Hydroxyl loss was greatest for flame-sprayed particles, which create the longest residence time for the melted particle. Higher-frequency hydroxyl peaks in flame- and plasma-sprayed splats indicated a lower structural order for the recrystallized hydroxylapatite within the splats. Crystallization at 700 °C has shown potential for revealing hydroxyl ions previously trapped in amorphous calcium phosphate. This work compares Fourier transform infrared and Raman spectroscopy to measure the hydroxyl content in rapidly solidified apatites and shows that Raman spectroscopy is more suitable.

Effect of processing conditions on the crystallinity and structure of carbonated calcium hydroxyapatite (CHAp)

Previous synthesis routes created apatites in low crystallinity and high crystallinity states, but a wider range will extend the design capabilities of apatites for hard tissue replacements. While high crystallinity apatites are more conventional, this work investigated lower crystallinity variations from an amorphous state to low crystallinity apatite. Carbonated hydroxyapatite was prepared by precipitating an amorphous phase followed by crystallization at 650 °C at slow (5 °C min−1) and fast heating rates (60 °C min−1). The effect of processing conditions on crystallinity and structural changes was evaluated by thermal analysis, X-ray diffraction, transmission electron microscopy, Fourier transform infrared and Raman spectroscopy. Furthermore, peak deconvolution of IR and Raman spectra resolved the carbonate and phosphate bands and revealed the carbonate and crystalline phase content in CHAp. Similar to precipitation of crystalline apatites, the crystallization at elevated temperature led to carbonate in both the phosphate and hydroxyl positions. Heating at 650 °C provided a nanosized spherical hydroxyapatite containing carbonate controlled by the heating rate. This creates a mechanism for creating a large range in crystallinity with a greater resorption capability for regenerative medicine.

Amorphous calcium phosphate offers improved crack resistance: a design feature from nature?

Amorphous calcium phosphate (ACP) is found in biological organisms and coated implants, used in calcium phosphate cements, and has been recently confirmed as a precursor in mineralized tissue; however, nothing is known about crack initiation in ACP or its fracture toughness. The objective of this study was to determine the crack resistance of ACP to help understand its role in biological organisms and assist in the design of calcium-phosphate-based implants. ACP was manufactured by quenching droplets to form a bulk sample and individual splats. Testing of Berkovich and cube-corner indenter types revealed that the Berkovich indenter was more suitable, providing ease of crack detection. Nanoindentation was performed on polished ACP and hydroxyapatite (HAp), and cracks were identified with scanning electron microscopy. Additional nanoindentation was done on splats to assess the suitability for testing microarrays used in high throughput discovery of new bioceramics. ACP required about three times more force to initiate a crack compared to sintered HAp, but about nine times more than a single crystal. Crack initiation resistance decreased with increasing grain size. The fracture toughness of ACP was comparable to a single crystal, but higher for nanograined HAp. The crack initiation load can be potentially used for evaluating microsized samples. ACP prevents crack formation, but requires the presence of nanograins to provide a greater toughness. The implications of the higher crack initiation load in ACP are discussed for biological organisms and thermally processed biomaterials such as thermally sprayed and sintered HAp.

Contact nanofatigue shows crack growth in amorphous calcium phosphate on Ti, Co-Cr and Stainless steel.

Fatigue testing of load-bearing coated implants is usually very time-consuming and so a new contact nanofatigue test using a nanoindenter has been evaluated. A cube corner indenter provided the fastest indication of failure, through crack formation, compared to a spherical indenter. Contact nanofatigue was performed on a sintered hydroxyapatite and then on amorphous calcium phosphate splats produced on titanium, stainless steel and Co-Cr surfaces, made either at room temperature or on 250°C preheated surfaces. Sintered hydroxyapatite showed continual plastic deformation, but this is not that apparent for splats on metal surfaces. Substrate preheating was found to induce cracking in splats, explained by greater thermal residual stresses. Endurance during contact nanofatigue, measured as time to crack formation, was the lowest for splats on titanium followed by Co-Cr and stainless steel. The splat on titanium showed both cracking and plastic deformation during testing. Good agreement has been reached with previous studies with cracking directed to the substrate without splat delamination. Contact nanofatigue with the nanoindenter easily and quickly identifies cracking events that previously required detection with acoustic emission, and shows good feasibility for mechanical testing of discs and splats produced by thermal spraying, spray forming, laser-ablation, aerosol jet and ink jet printing.

Nano-indentation on amorphous calcium phosphate splats: Effect of droplet size on mechanical properties

Droplet processing technologies and many biological processes use disk-like or hemispherical shapes for construction or the design of surfaces. The ability to tune the characteristics and properties of a surface is important at the micro- and nano-scale. The influence of size on the mechanical properties is presently unknown. This work set out to produce splats from different droplet sizes (20-40 μm, 40-60 μm and 60-80 μm), and then determine the deposit characteristics and mechanical properties. All splats produced by melting particles in a flame and depositing onto a polished titanium surface were amorphous, as determined by Raman micro-spectrometry. The topography shown in an optical and scanning electron microscope and topographically mapped using the scanning mode of the nano-indenter revealed a flattened hemispherical deposit. The critical nano-indentation load for determining the true hardness decreased with increasing splat size; for 20-40 μm, 40-60 μm and 60-80 μm splats the critical load was 19, 16, 11 mN respectively compared to 30 mN for sintered hydroxyapatite. Higher loads are required to cause cracking and delamination in smaller splats. A load between 40 and 60 mN was required for delamination of the splat. Delamination of the splats could offer a new means to determine the adhesion of splats on low roughness surfaces.

Characterization and dissolution of functionalized amorphous calcium phosphate biolayers using single-splat technology.

New processing routes and characterization techniques underpin further growth of biomaterials for improved performance and multifunctionality. This study investigates the characteristics and solubility of amorphous calcium phosphate (ACP) printed splats. Splats made from 20 to 60 μm molten hydroxyapatite particles were classified for shape (rounded/splashed) and cracking. Recoil of the spread droplet created a bowl-shaped splat. This has previously not been observed and could be related to the longer solidification time associated with solidification to an ACP. A central depression was created from 20 μm particles, but a bowl-shaped splat from 60 μm particles. Cracking was more prevalent for splats that solidified with an edge discontinuity. Splats immersed in pH 7.3 tris buffer displayed dissolution followed by cracking. Cracking continued over a period of 15 min as dissolution induced more cracks. Further degradation occurred by delamination of splat segments. Delamination accelerated the process of splat removal. Applied to thermal spray coatings, this highlights topography and dissolution at the splat level. The use of separate splats can potentially be used as a biolayer where splats are separate, in a line or on top of each other.

Importance of FTIR Spectra Deconvolution for the Analysis of Amorphous Calcium Phosphates

This work will consider Fourier transform infrared spectroscopy – diffuse reflectance infrared reflection (FTIR-DRIFT) for collecting the spectra and deconvolution to identify changes in bonding as a means of more powerful detection. Spectra were recorded from amorphous calcium phosphate synthesized by wet precipitation, and from bone. FTIR-DRIFT was used to study the chemical environments of PO4, CO3 and amide. Deconvolution of spectra separated overlapping bands in the ʋ4PO4, ʋ2CO3, ʋ3CO3 and amide region allowing a more detailed analysis of changes at the atomic level. Amorphous calcium phosphate dried at 80 oC, despite showing an X-ray diffraction amorphous structure, displayed carbonate in positions resembling a carbonated hydroxyapatite. Additional peaks were designated as A1 type, A2 type or B type. Deconvolution allowed the separation of CO3 positions in bone from amide peaks. FTIR-DRIFT spectrometry in combination with deconvolution offers an advanced tool for qualitative and quantitative determination of CO3, PO4 and HPO4 and shows promise to measure the degree of order.

Synthesis of Tetracalcium Phosphate at Reduced Temperatures

Tetracalcium phosphate (TTCP) requires the highest synthesis temperatures of all the calcium phosphates, but now a new process is available at 400 °C lower than previously, at 900 °C. Instead of ball-milling reactants for a homogeneous mix, the reactants were included in an amorphous phase. Heating produced hydroxyapatite, oxyapatite and then TTCP. Amorphous nanoparticles were synthesized and heated in air or in vacuum. The sequence of solid-state reactions were tracked with X-ray diffraction and Fourier transform infra-red spectroscopy. Heating in air stabilized the carbonate containing apatite, thereby requiring higher temperatures for decomposition, as per previous studies. Heating in vacuum promoted oxyapatite; a critical step for reaction with calcium oxide to generate TTCP. This faster process enables production at a lower temperature and reduces the use of ball milling for producing fine TTCP powders.

Traversing Phase Fields towards Nanosized Beta Tricalcium Phosphate

The difficulty of beta tricalcium phosphate (β-TCP) crystallization in aqueous media opens the question whether β-TCP can be produced using an alternative pathway. Amorphous calcium phosphate (ACP) is metastable in an aqueous environment and prefers a more stable apatite phase. Others have transformed a crystallized calcium deficient hydroxyapatite (Ca-def HAp) into β-TCP, but automatic transformation from ACP to Ca-def HAp followed by transformation to β-TCP has not been addressed. This work shows the formation of Ca-def HAp after different aging times of ACP and the subsequent transition to β-TCP. An amorphous phase with a Ca/P ratio of 1.5 was synthesized, rinsed, filtered and excess fluid removed for maturation. The resulting apatite was monitored with X-ray diffraction at different temperatures. Heating at 700 °C then investigated the transition to β-TCP. It was found that Ca-def HAp formed at short aging times produced a combination of alpha and beta phases, but a longer aging time led to pure β-TCP.

Crystallized nano-sized alpha-tricalcium phosphate from amorphous calcium phosphate: microstructure, cementation and cell response

New insight on the conversion of amorphous calcium phosphate (ACP) to nano-sized alpha tricalcium phosphate (α-TCP) provides a faster pathway to calcium phosphate bone cements. In this work, synthesized ACP powders were treated with either water or ethanol, dried, crystallized between 700 and 800 °C, and then cooled at different cooling rates. Particle size was measured in a scanning electron microscope, but crystallite size calculated by Rietveld analysis. Phase composition and bonding in the crystallized powder was assessed by x-ray diffraction and Fourier-transform infrared spectroscopy. Results showed that 50 nm sized α-TCP formed after crystallization of lyophilized powders. Water treated ACP retained an unstable state that may allow ordering to nanoapatite, and further transition to β-TCP after crystallization and subsequent decomposition. Powders treated with ethanol, favoured the formation of pure α-TCP. Faster cooling limited the growth of β-TCP. Both the initial contact with water and the cooling rate after crystallization dictated β-TCP formation. Nano-sized α-TCP reacted faster with water to an apatite bone cement than conventionally prepared α-TCP. Water treated and freeze-dried powders showed faster apatite cement formation compared to ethanol treated powders. Good biocompatibility was found in pure α-TCP nanoparticles made from ethanol treatment and with a larger crystallite size. This is the first report of pure α-TCP nanoparticles with a reactivity that has not required additional milling to cause cementation.