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Advances in Hydroxyapatites


Dr Phil Jackson and Ben McCarthy of Ceram report on new technology that makes it possible to offer hydroxyapatite-based biomaterials with different surface properties 
and hence different biological properties.

Given that natural bone comprises hydroxyapatite (HA) as a major component, it is unsurprising that so much research effort has been turned to developing synthetic HA versions as bone replacement materials. Although allograft and autograft solutions (in which sections of bone from the iliac crest of a donor or the patient, respectively, are re-sited) are deemed "gold standard,” they offer significant problems in terms of risk of rejection and pain for the donor/patient.
A 3-D porous synthetic HA structure that provides a perfect fit into defect voids represents an attractive alternative. Essential demands for this type of bone replacement are as follows:
  • Bioactivity, whereby attraction of stem and/or osteoblast cells to synthetic HA surfaces is followed by cell proliferation/differentiation and new cell-induced bone growth
  • Antibacterial properties and suppression of hostile immune system responses
  • Superior mechanical properties that avoid both failure and 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
  • Designed-in porosity that allows vessel and new bone impregnation
  • An ability to create bespoke shaped 3-D sections of bone.

An interesting aside, which is not covered in this article, is the use of a sacrificial skeletal structure that slowly dissolves at a rate commensurate with new bone growth within the original porosity. This approach opens up the range of candidate bone replacement materials to include metals such as soluble magnesium alloys, polymers and perhaps glass.

The use of HA powders as 2-D coatings for metal implants has rendered the latter more compatible with the body and led, for example in the case of femoral hip replacements, to implant longevity via stronger bone-to-implant bonding. Despite this, the ultimate goal remains the development of implants comprised of HA alone. 

As a first step along this route to 3-D HA offerings, solutions already exist in the form of porous HA granulates. These materials can be used to fill bone voids arising from disease or trauma, providing plates and mesh are used to contain the free flowing granulate material. However, problems associated with migration or exfoliation of granulate can arise.

In terms of enablers for true 3-D HA structural solutions, combinations of the following aspects are needed:
  • Adaptations on pure HA chemistry to stimulate both bioactivity and enhanced mechanical properties
  • Shaping techniques that offer (directly or indirectly) scope for creating bespoke shapes
  • Nano-manipulation of surfaces to encourage orientation of proteins so that desirable cell and immune system responses are ensured.
Each of these topics will now be discussed in more detail with reference to work at Ceram.

Adaptation of HA chemistry
Various ions can be introduced into the HA crystal lattice. For example cations such as Mg2+ and Ag2+ can partially replace Ca2+. Anions such as F- and SiO44- can partially replace OH- and PO43- respectively. Indeed, the CO32- ion is present in natural bone as a partial replacement for both PO43- and OH-. Depending on the ion chosen (and more specifically the ionic radius and charge), the HA lattice will become distorted to a greater or lesser extent. This distortion of the lattice has notable effects on the surface charge and dissolution rates of the crystals. Changes arise from alterations in the width of ion channels (and the associated difficulty of Ca2+ substitutions leaving via them) and the strength of the phase left behind after Ca2+ leaching.

It is understood that the typical method of HA dissolution (largely under acidic conditions,) is a multi-stage process that can lead to the formation of several different surface "coatings” of a calcium-deficient HA analogue, for example, octacalcium phosphate.1 The route by which these surfaces are formed and removed consists of three general steps as follows.

The HA crystal (when present in the common hexagonal form,) consists of two distinct ion channels running down the c-axis of the lattice. One channel is filled by OH- and lined by Ca2+ in a helix, the other is formed by Ca2+ surrounded by PO43-  groups. The primary ion channel is the OH- with a diameter of approximately 3Å. Dissolution occurs first by 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 restricting the primary ion channel (F-), replacing the Ca2+ with a "harder to remove” cation (Sr2+), or by providing a stronger surface coating (SiO44-).

To illustrate the importance of HA surface charge, Ceram has liaised with Queen Mary University2 (QMU) in London, UK, on preliminary zeta potential analysis of HA powders suspended first in water and then aqueous media containing inorganic electrolytes and organics typically present in body fluid. What can be seen in Figure 1 is that the zeta potential (a measure of powder surface charge) versus the pH profile of HA changes considerably as body fluid species are added. For example it has been seen that
  • Ca2+ ions adsorb on to HA surfaces to create a more positive charge at all pH values
  • mixed cation additions present in simulated body fluid (SBF), raise zeta potential into an even greater positive mV range
  • organics such as Tris-hydroxymethyl aminomethane (TRIS) also seem to adsorb on HA surfaces as evidenced by rises in zeta potential under acidic conditions; this is perhaps achieved by amino groups gaining a proton to form NH3+.
These surface effects will affect the extent to which first proteins (growth factors) and cells orientate towards HA surfaces.

Figure 1: Zeta potential versus pH profiles for sintered HA in a variety of aqueous media
Key: DIW= De-ionised water
MEM= Eagle's minimum essential medium, a cell culture medium with salts, amino acids and vitamins
SBF = Simulated body fluid, inorganic apart from the component Tris-hydroxymethyl aminoethane

Ceram is currently investigating multi-substituted (2- or 3-ion additions) HAs. Studies are focusing initially on how mean deviations from the charge and ionic radius in pure HA impact on (a) the ability to retain the HA structure and (b) how much the HA lattice distorts. Initial evidence is showing that the durability of the system is much more closely linked to maintaining an electronic balance. The charge shift associated with an individual substitution needs to be balanced out by the inclusion of a suitable opposite charge. For example, SO42- to replace PO43- (reduced negative charge) needs to be offset by Na+ replacing Ca2+ (reduced positive charge), thereby allowing much increased loadings.

The size difference of added substituents appears to have much less effect on the stability of the crystal. However, there is an impact on solubility and therefore potentially bioavailability. During the next year, Ceram will be performing simple simulated body fluid immersion studies to establish whether multi-substitutions suggest enhanced bioactivity. This will involved using ISO 23317, Implants for surgery, In vitro evaluation for apatite-forming ability of implant materials, and low angle X-ray diffraction (XRD) to quantify rate of fresh HA deposition versus time. Initial data has been promising with significant increases in HA deposited as shown in Figure 2.

Figure 2:
Demonstration of bioactivity by measurement of percentage weight of HA detectable by XRD in the top approximately 30 microns. HA* and HA** are novel multi-substituted HA materials currently under investigation; the substrate is a bio-inert polyme

There is also some suggestion that substituting elements for Ca2+ enhances mechanical properties. Recent reported work from Washington State University on additive layer manufacturing has indicated that final structures made from HA containing zinc or silicon have improved strength.3

Novel shaping to create 3-D porous structures
Once the initial primary HA powder particles have been produced there is a need to shape and consolidate (that is, sinter to high temperatures) 3-D structures. Following basic ceramic principles, bulk shapes can be created from
  • fluid slurries (these can be cast in moulds)
  • plastic pastes (the water content is lower to allow extrusion)
  • dry powders (for pressing).
The need to design porosity into structures has led to research into the use of sacrificial organic powder additives (that burn-out upon sintering), foaming from fluid slurries and impregnation of sacrificial polymer foams.

Warwick University has developed some interesting intellectual property in which researchers propose a catalytic convertor "monolith” structure as the basis for bespoke bone grafts. The unidirectional channels (that approximate to the open porosity of cancellous bone) have been shown to have a compressive strength of 250 MPa in line with cortical bone. Warwick University is currently working with Ceram on optimising paste composition and rheology for enhanced extrusion and micro-structural properties, including tailored porosity in the cell walls. The overall concept involves manipulating the initial sintered monolithic structure through computer numerically controlled machining to deliver bespoke bone grafts for large bone defects intrinsic to the stability of the skeleton.

Given the need to recognise that bone shapes, surgery and damage in accidents is unique to each patient, additive layer manufacturing is an exciting proposition. The ability to use computer tomography scans and then create CAD files prior to building up replica structures layer-by-layer is extremely attractive. Washington University reports an approach using ink jet printing in which a binder glue is used to adhere areas of a calcium phosphate powder bed prior to applying a fresh powder layer and repeating the process.3

Alternatively, laser fusion of a resin filled with HA on a layer-by-layer basis could be employed. Direct laser fusion of HA powder particles represents perhaps too great a challenge given the high temperatures and heat-work profiles required for solid-state sintering; however for structures built from metal powder, this may be a viable route.

Surface structure nano-manipulation
It has been reported by the University of Gothenburg4 that deposition of gold nano-particles on surfaces creates a "cobbled stone” structure that encourages preferential protein deposition and thus desirable immune system responses that protect against rejection by the body. Instead of deposition of nano-features, an alternative approach may be to nano-etch surfaces. Ceram has recently signed a memorandum of understanding with the Centre for Adaptive Nanostructures and Nanodevices (CRANN) in Dublin, Ireland, which recognises complementary surface analytical tools at both organisations. Ceram and CRANN hope to build this relationship through staff exchanges and work on targeted medical-related projects through applying for European funding such as the Industry–Academia Partnerships and Pathways programme. One potential project would investigate the use of helium and focused ion beam facilities at CRANN for surface manipulation of HA, bioglass and composite surfaces.

To conclude, it is clear that delivery of 3-D HA bone replacement inserts depends on collaboration between a number of scientific disciplines including inorganic chemistry (primary HA particle optimisation), physical chemistry (particle size/shape measurements; particle surface characterisation) engineering (shaping from primary HA powders, powder suspensions and/or sintered structures) and cell biology.

1. K. Matsunga et al., "Theoretical Defect Energetics in Calcium Phosphate Bioceramics,” J. Am. Ceram. Soc., 93, 1, 1–14, 2010.

2. Work undertaken by Krystelle Mafina as part of a six-month QMU IMPACT scholarship supervised by Dr Phil Jackson at Ceram and Dr Karin Hing at QMU.

3. 3-D Printer Used to Make Bone-Like Material, Science Daily, 29 November 2011, www.sciencedaily.com. This references a report from Washington State University in Dental Materials Journal.

4. "Implants with a Nanostructured Surface Reduce Risk of Rejection,” article in ASM International’s Medical Materials e-news, posted 12 August 2011, www.asminternational.org.

A white paper by Ceram titled "Multi-Substituted Hydroxyapatites & Their Role in Bone Replacement" can be downloaded by clicking here.

Dr Phil Jackson is Business Development Manager, Healthcare

Ben McCarthy is Technical Consultant
both at Ceram, Queens Road, Penkull
Stoke-on-Trent ST 4 7LQ, UK
tel. +44 (0)1782 764 428
e-mail: enquiries@ceram.com 


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