Multi-Substituted Hydroxyapatites and the Role They Can Play in Enhanced Bone Replacement Solutions

Author: Ben McCarthy & Dr. Phil Jackson

This work by Ceram is licensed under a Creative Commons AttributionNonCommercial-ShareAlike 3.0 Unported License

Much research has been done into developing synthetic Hydroxyapatite (HA) as materials for bone replacement, due to the fact that natural bone comprises HA. In addition, HA powders have been used as coatings on metal implants in a bid to make them more compatible with the body and to promote stronger bone-to-implant bonding and hence increased longevity of the implant (for example, in the case of femoral hip implants). In the quest to develop implants that comprise HA alone, porous HA granulates have already been developed. Used to fill bone voids originating from disease or trauma (as long as plates and mesh are used to contain the free flowing granulate material) they do, however, also have their problems, namely migration or exfoliation of the granulate. What potential alternatives exist and what key requirements should such they fulfil? It is our belief that a 3-D porous synthetic HA structure, which would provide a perfect fit into defect voids, would provide an answer. Ideally it would fulfil the following needs: • Bioactivity – i.e. stem and/or osteoblast cells are attracted to synthetic HA surfaces - this is followed by cell proliferation/differentiation and new cell-induced bone growth Anti-bacterial properties Suppression of hostile immune system responses Superior mechanical properties to minimise (i) failure and (ii) stress shielding (the latter has potential to alter the stresses experienced in areas away from any insert and so induce distorted bone growth or high osteoclast activity) Porosity that is designed to allow the impregnation of vascular and new bonemineral / collagen structures • An ability to create bespoke shaped 3-D sections of bone, ideally via some form of Additive Layer Manufacturing, to cut down on waste created by machining. Extending the concept a stage further, one can envisage the aforementioned properties in a sacrificial skeletal structure that provides mechanical integrity initially but then slowly dissolves at a rate commensurate with new bone growth. Such an approach opens up the range of candidate materials to include metal alloys (e.g. soluble magnesium alloys) and inorganic powder/soluble polymer composites. The basis of the goals defined above however is an optimised primary HA powder and ideally a range of HA powders with bioactivity behaviour tuned to the needs of different patients is required. This paper explores the role of (multi-element) substitution in HA and how this can impact on behaviour of HA in aqueous physiological environments.

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Adaptation Of HA Chemistry
The HA lattice can be altered by the specific inclusion of target ions. For example cations like Sr2+ and Zn2+ can partially replace Ca2+ whilst anions such as Cl- and SO43- can partially replace OH- and PO43- respectively. Some ions can exist in multiple
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sites, for example CO32- is present in natural bone in both the PO43- and OH- site. The HA lattice can be distorted to a greater or lesser extent based on the properties of the ion substituting into the lattice (ionic radius and charge simply, however access to different electron orbits may be important). Subtle distortions to the lattice can have large consequences to the final properties, for instance surface charge and dissolution rates. In the case of dissolution rates the difficulty of Ca2+ and its substitutions leaving via the main ion channel and the strength of the anion rich phase left behind after cation leaching can also be a consequence. It is understood that the typical method of HA dissolution (under acidic conditions) is a multi-stage process that can lead to the formation of several different surface morphologies of a calcium deficient HA analogue (for example, octacalcium phosphate (OCP))[1]. The method by which the dissolution method works consists of 3 general steps; the HA crystal (commonly found in hexagonal form, although monoclinic does exist – i.e. tooth enamel) consists of 2 distinct ion channels running down the c-axis of the lattice, one channel being filled by OH- and lined by Ca2+ in a helix, the other being formed by Ca2+ surrounded by PO43- groups. The primary ion channel is that of the OH- with a diameter of approximately 3 Å. Dissolution occurs by first acidic neutralisation of the OH- leading to removal of H2O and the opening up of the channel. This is followed by the migration of Ca2+ out of this channel. The removal of Ca2+ leads to the formation of the PO43- rich layer, which then dissolves due to a lack of cross linking between PO43- tetrahedra. Substitutions can alter these processes by either restricting the primary ion channel (F-), replacing the Ca with an easier to remove cation (Mg2+), or by providing a salting out ion (SO43-). Current work at Ceram involves investigations into, and developments of, multisubstituted (2- or 3- ion additions) HAs. Initial studies aim to establish how mean (average) deviations from the charge and ionic radius in pure HA impact on both the ability to retain the HA structure and the distortion of the HA lattice. Initial findings have shown areas and potential trends where increased loadings are possible.

Graph 1. Volume Deviation Vs HA Yield

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Graph 1 plots the mean deviation in ionic volume (due to substitution) from the original lattice against the HA yield. Data was generated by modifying the data set from a FED (Factorial Experimental Design) experimental group to include ‘true’ values for the substitutions (values gathered by X-Ray Fluorescence – XRF). This initial graph heavily suggests that the HA lattice will more readily form via wet precipitation when the substituents it is attempting to incorporate lead to a positive volume deviation rather than a negative deviation. This may be due to the nature of the synthesis meaning that the energy of formation is related to the energy of dissolution – a value that decreases with smaller ions and increases with larger ions. In addition to looking at ionic volume changes, Ceram has also investigated the impact of mean ionic charge variations arising from substitutions. This work is led by the belief that a combination of charge stability and ionic volume distortion is acceptable within the lattice and that by finding these ‘sweet spots’ it is possible to increase the amount of substituted element incorporated. The initial findings for charge balance are included below.

Graph 2. Charge Deviation Vs HA Yield

Whilst further work is needed to fully analyse the data presented in Graph 2, there is already some information that can be gathered: Firstly, there seems to be a definite preference for the HA lattice to support an increase in negative charge. We theorise that this is due to an associated loss of OH- whereas an increase in positive charge cannot be supported in a similar fashion. The amount of data sitting outside of the stylised ‘curve’ could be due to several issues, such as volume deviations, and work is going on to build sufficient data for a comprehensive 3 dimensional contour system. Once studies into the impact of ion substitution on retention of an HA crystalline structure (all be it distorted as quantified by 2 theta values shifts in XRD traces) is completed, Ceram will turn its attention to how this data correlates to bioactivity. A simple test that can be deployed is the SBF immersion trial as defined in ISO 23317.
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Initial data has been very promising with significant increases in HA deposited as shown in Graph 3. Ceram hopes to enhance this test by using low angle XRD analysis to better quantify rate of fresh HA deposition vs time. By optimising the incident angle and the 2theta data gathered, it should be possible to pick up signals for both the substituted HA substrate disc AND freshly deposited pure HA. With time, the ratio of these two signals should change allowing calculation of a rate of fresh HA desposition. Ceram also proposes to monitor the concentration of key soluble ions over time to establish the extent to which HA dissolution (as well as retention of initial crystalline HA phase) has a role to play in bioactivity. Once the ISO23317 test has established whether multi-substitutions create enhanced bioactivity, work will turn to selected in-vitro testing. Whilst SBF tests mimic the chemical environment of the implant, they poorly mimic the deposition method of fresh HA growth. For instance, in preliminary work carried out at Ceram it has been noted that cell-deposited HA has a very different morphology to crystals grown in SBF when viewed via Scanning Electron Microscopy (SEM). What the SBF test can establish is that the surface characteristics of the material present lend themselves to the formation of additional HA using dissolved mineral supplies – be this by having a crystal face with the correct growing front to initialise precipitation or a correct distribution of charge to initiate Ca2+ and PO43orientation in a manner conducive to crystal formation. By introducing cell activity, a whole range of measurements open up to help characterise and understand the HA re-precipitation method. Whilst Ceram has no in-house cell testing capabilities, strong links have been established to organisations that do. For example, Ceram is already sub-contracting experts at the Guy Hilton Research Centre, Keele University to investigate cell response to zirconia implants as part of a TSB project led by Ceram on the development of nano-structured hip replacements. One cell test that will be imployed in zirconia and HA work is the so-called “MTT” test for cytotoxicity. The test essentially evaluates how healthy the cell is when subjected to powdered zirconia, HA. When healthy, an enzyme known as SDH will be present in the cell to carry out oxidation reactions. By adding a chemical to the mixture that changes to a blue colour when oxidised, it is possible to use UV-Vis spectroscopy to measure cell health (the more intense the absorbance at 540 and 655nm, the higher the number of healthy cells). Whilst MTT gives a measure of cell proliferation across a surface, tests such as Alkaline Phosphatase (ALP) assays measure the amount of an enzyme present and compare it to a DNA count to give an enzyme/cell count. ALP is measured because whilst in ‘normal’ cells ALP is used to dephosphorylate (remove PO43- groups) organic molecules, in Human Osteoblast-like Cells (HOB), ALP-2 (a specific ALP enzyme found in bone mineralizing cells) can be linked to bone turnover and therefore cell activity levels. Therefore an ALP assay with HOB can give a much stronger indication as to how conductive a material surface is to osteoblast activity. The stage on from ALP assays is to look at differentiation of stem cells to measure how conductive a material is to the generation of bone forming cells in preference to anything else. Ceram also intends to build on their links with CRANN in Dublin by using high resolution SEM analysis to gain micrographs of cells adhered to different HA surfaces. The way cells adhere and the shapes they assume can be indicative of bioactivity.

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Graph 3. Demonstration of bioactivity by measurement of wt% HA detectable by XRD in the top ~30 microns. HA* and HA** are novel multi-substituted HA materials currently under investigation. The substrate is a bio-inert polymer.

To illustrate the importance of HA surface charge (which can be significantly altered via substitution of calcium and phosphate ions with alternative elements), Ceram has liaised with Queen Mary University (QMU) in London[2] on preliminary zeta potential analysis of HA powders suspended in (a) water and (b) aqueous media containing inorganic electrolytes and organics typically present in body fluid. What can be seen from figure 1 is that the zeta potential (a measure of powder surface charge) vs pH profile of HA changes considerably as body fluid species are added. For example it has been shown that (i) Ca2+ ions adsorb on to HA surfaces to create a more positive charge at all pH values, (ii) mixed cation additions (as present in SBF) raise zeta potential into an even greater positive mV range, and (iii) organics such as TRIS etc. also seem to adsorb on HA surfaces as evidenced by rises in zeta potential under acidic conditions (achieved by perhaps amino groups gaining a proton to form NH3+). These surface effects will impact on the extent to which first proteins (growth factors) and cells orientate towards HA surfaces.

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0 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5

) V m ( l i n o P a t e Z


-20 DIW -30 DIW with Ca ions MEM MEM with 10% FBS S BF SBF with NO TRIS



Graph 4 - Zeta Potential vs pH profiles for sintered HA in a variety of aqueous media (DIW = De-ionized water; MEM = Minimum Essential Eagles Medium (a cell culture medium with salts, amino acids and vitamins); SBF = Simulated Body Fluid (inorganic apart from the component TRIS – Tris-hydroxymethyl aminomethane)

Successfully preparing multi-substituted HAs represents just the first step in generating synthetic bone solutions. Much work is the needed to (a) establish correlations to bioactivity and propose mechanisms behind enhanced bioactivity and (b) convert primary HA powders into shaped porous structures that surgeons can employ. Central to making progress is the need for a multi-disciplinary approach to R&D. Managing such teams and encouraging individual teams to ‘go the extra mile’ and bridge gaps in knowledge to allow meaningful dialogue represents perhaps the greatest challenge.


K. Matsunga, J. Am. Ceram. Soc., 2010, 93 (1), 1-14


Work undertaken by Krystelle Mafina as part of a 6-month QMU IMPACT scholarship, supervised by Phil Jackson at Ceram and Karin Hing at QMU

Article in ASM International “Medical Materials e-news” posted 12/08/2011

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About Ceram
Ceram is an independent expert in innovation, sustainability and quality assurance of materials. With a long history in the ceramics industry, Ceram has diversified into other materials and other markets including aerospace and defence, medical and healthcare, minerals, electronics and energy and environment. Partnership is central to how we do business; we work with our clients to understand their needs so that we can help them overcome materials challenges, develop new products, processes and technologies and gain real, tangible results. Headquartered in Staffordshire, UK, Ceram has approved laboratories around the world.

About the Author
Ben McCarthy Technical Consultant
Ben has a BSc in Chemistry from the University of Manchester. His third year project involved synthesising block co-polymers for use as a drug delivery capsule via a controlled radical polymerisation. At Ceram, Ben is currently working in the area of biomedical devices, with projects including novel HA formulations and controlled release work from bioglasses/solgels. He is also working with polymer emulsions and using zeta to track and characterise different systems to build a better understanding of what's occurring in more complex formulations (ranging from shampoos to anaesthetics/non-water soluble drug injections).

Dr. Phil Jackson Business Development Manager, Healthcare
Phil Jackson holds a BSc Hons in Chemistry and a PhD in Thermodynamics, both gained at Leicester University. Owing to over twenty years of experience, Phil’s areas of expertise lie in glass chemistry, novel shaping processes and in the practical control of powder suspensions. Moving from a focus on glaze and ceramic powder processing for the traditional ceramic industry in the early part of his career, Phil transferred his expertise to advanced ceramic process issues, including control of nano-suspensions and precipitation processes for electronics applications. More recently Phil has focused on applications in the medical devices sector and in particular to the development of implanted structures, surgical instrumentation and implant/anti-bacterial coatings. Phil is also a Technology Translator for the Materials KTN, identifying powder needs in various industries and helping to connect companies and universities with complementary skills.
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