The pathway to intelligent implants: osteoblast response to nano silicon-doped hydroxyapatite patterning.

Bioactive hydroxyapatite (HA) with addition of silicon (Si) in the crystal structure (silicon-doped hydroxyapatite (SiHA)) has become a highly attractive alternative to conventional HA in bone replacement owing to the significant improvement in the in vivo bioactivity and osteoconductivity. Nanometre-scaled SiHA (nanoSiHA), which closely resembles the size of bone mineral, has been synthesized in this study. Thus, the silicon addition provides an extra chemical cue to stimulate and enhance bone formation for new generation coatings, and the next stage in metallic implantation design is to further improve cellular adhesion and proliferation by control of cell alignment. Topography has been found to provide a powerful set of signals for cells and form contact guidance. Using the recently developed novel technique of template-assisted electrohydrodynamic atomization (TAEA), patterns of pillars and tracks of various dimensions of nanoSiHA were achieved. Modifying the parameters of TAEA, the resolution of pattern structures was controlled, enabling the topography of a substrate to be modified accordingly. Spray time, flow rate and distance between the needle and substrate were varied to improve the pattern formation of pillars and tracks. The 15 min deposition time provided the most consistent patterned topography with a distance of 50 mm and flow rate of 4 µl min(-1). A titanium substrate was patterned with pillars and tracks of varying widths, line lengths and distances under the optimized TAEA processing condition. A fast bone-like apatite formation rate was found on nanoSiHA after immersion in simulated body fluid, thus demonstrating its high in vitro bioactivity. Primary human osteoblast (HOB) cells responded to SiHA patterns by stretching of the filopodia between track and pillar, attaching to the apex of the pillar pattern and stretching between two. HOB cells responded to the track pattern by elongating along and between the track, and the length of HOB cells was proportional to the gaps between track patterns, but this relationship was not observed on the pillar patterns. The study has therefore provided an insight for future design of next generation implant surfaces to control and guide cellular responses, while TAEA patterning provides a controllable technique to provide topography to medical implants.

The role of surface wettability and surface charge of electrosprayed nanoapatites on the behaviour of osteoblasts.

A new deposition method is presented, based on electrospraying, that can build bioceramic structures with desirable surface properties. This technology allows nanoapatite crystals, including hydroxyapatite (nHA), carbonate-substituted HA (nCHA) and silicon-substituted HA (nSiHA), to be electrosprayed on glass substrates. Human osteoblast cells cultured on nSiHA showed enhanced cell attachment, proliferation and protein expression, namely alkaline phosphatase, type 1 collagen and osteocalcin, as compared to nHA and nCHA. The modification of nanoapatite by the addition of silicon into the HA lattice structure renders the electrosprayed surface more hydrophilic and electronegatively charged.

Development and characterization of titanium-containing hydroxyapatite for medical applications.

Hydroxyapatite containing levels of titanium (TiHA) of up to 1.6 wt.% has been produced via a chemical co-precipitation route. The distribution of Ti was seen by transmission electron microscopy/energy-dispersive X-ray analysis to be uniform throughout as-prepared nanosized TiHA particles (20 nm x 100 nm). The incorporation of Ti into the HA structure was found to influence the ceramic microstructure on sintering and the grain size was found to decrease from 0.89 microm with HA to 0.63 microm with 0.8 wt.% TiHA (0.8 TiHA) and 0.45 microm with 1.6 wt.% TiHA (1.6 TiHA). Rietveld refinement analysis showed that there was a proportional increase in both the a and c axis with incorporation of Ti into the HA lattice structure, leading to an increase in the cell volume with the addition of Ti. Fourier transform-Raman analysis showed a slight increase in the ratio of O-H/P-O peaks on TiHA, in comparison with HA. A bone-like apatite layer was formed on the surface of TiHA after immersion in simulated body fluid for 3 days, which demonstrated the high in vitro bioactivity of TiHA. In vitro culture with primary human osteoblast (HOB) cells revealed that TiHA was able to support the growth and proliferation of HOB cells in vitro, with a significantly higher cell activity being observed on 0.8 TiHA after 7 days of culture in comparison with that on HA. Well-organized actin cytoskeletal protein was developed after 1 day of culture, and an increase in cell filopodia (attachment) was observed on TiHA sample surfaces. The results indicate that TiHA has great potential for biomedical applications.

Deposition of nano-hydroxyapatite particles utilising direct and transitional electrohydrodynamic processes.

Electrohydrodynamic spraying is a well established process used to deposit, coat, analyse and synthesise materials within the biomedical remit. Recently, electrohydrodynamic printing has been developed to afford structures for potential applications in the biomedical and medical engineering fields. Both of these processes rely on the formation of an electrically-induced jet, however the resulting products can be made strikingly different and offer potential in broader applications. Here we show how spraying and printing are linked by elucidating the ease of transition between the processes. Changes in the deposition distance can result in either spray (>10 mm) or print formation (<3 mm), with an overlap of the two in between this range. For the optimal printing distance of 0.5 mm, gradual changes in the applied voltage (0-4.5 kV) encounters transitional printing modes (dripping, micro-dripping, rapid micro-dripping, unstable and stable jetting) which can be utilised for patterning. The results indicate the robustness of the electrohydrodynamic route in the nano-materials processing arena, with emphasis on biomedical materials.

In vitro evaluation of nanosized carbonate-substituted hydroxyapatite and its polyhydroxyethylmethacrylate nanocomposite.

Nanometer scale carbonate-substituted hydroxyapatite (nanoCHA) particles were prepared and examined using transmission electron microscopy, which revealed their polycrystalline nature with a rod-like morphology (20-30 nm in width and 50-80 nm in length). In vitro cytotoxicity study showed that there was some evidence of lactate dehydrogenase (LDH) release when macrophages were in contact with high concentrations of nanoCHA particles. The levels of LDH release decreased significantly with a reduction in nanoCHA concentration. A similar trend was observed for the inflammatory cytokine TNF-alpha. nanoCHA particles with high carbonate content induced a high level of TNF-alpha release. Biological testing using a human osteoblast (HOB) cell model found that HOB cells were able to grow and proliferate on a nanoCHA deposited surface. Well organized actin fibers were observed for HOB cells in contact with nanoCHA particles with low carbonate content and the cell proliferation rate was higher on these particles in comparison with those of high carbonate nanoCHA particles. Therefore, low carbonate nanoCHA particles were incorporated into poly-(2-hydroxyethylmethacrylate) matrix to make a nanocomposite. It was found that the nanoCHA composite was hydrophilic and became rubber-like after hydration. Both 20 wt % and 40 wt % composites were able to induce the formation of bone-like apatite after immersion in simulated body fluid. A high bioactivity of the composite was obtained with high loading of the nanoCHA filler. These results demonstrate the potential of formulating nanocomposites for biomedical applications.

Influence of nanohydroxyapatite patterns deposited by electrohydrodynamic spraying on osteoblast response.

Electrohydrodynamic spraying has been used to produce patterns of line width up to 100 microm in size on glass discs, using nanohydroxyapatite (nHA). A human osteoblast (HOB)-like cell model was then used to study the interaction between the HOB cells and nHA patterns in vitro. Growth of the cells was significantly increased (p < 0.05) on the nHA surfaces. In addition, HOBs attached and spread well, secreting extracellular matrix. It was found that a confluent, aligned cell layer was achieved on nHA patterns by day 9. Immunofluorescent staining indicated that these cells showed elongated nuclei, enhanced adhesion (vinculin adhesion plaques) and a well-aligned cytoskeleton (actin stress fibres). This work suggests that this type of spraying may provide a route for the production of nanoscale features on implants for biomedical applications.

In vitro testing of Nd:YAG laser processed calcium phosphate coatings.

Nd:YAG laser cladding is a new method for deposition of a calcium phosphate onto metallic surfaces of interest in implantology. The aim of this study was to compare the biologic response of MG-63 human osteoblast-like cells grown on Ti-6Al-4V substrates coated with a calcium phosphate layer applied using different methods: plasma spraying as reference material and Nd:YAG laser cladding as test material. Tissue culture polystyrene was used as negative control. The Nd:YAG laser clad material showed a behaviour similar to the reference material, plasma spray, respective to cell morphology (SEM observations), cell proliferation (AlamarBlue assay) and cytotoxicity of extracts (MTT assay). Proliferation, as measured by the AlamarBlue assay, showed little difference in the metabolic activity of the cells on the materials over an 18 day culture period. There were no significant differences in the cellular growth response on the test material when compared to the ones exhibited by the reference material. In the solvent extraction test all the extracts had some detrimental effect on cellular activity at 100% concentration, although cells incubated in the test material extract showed a proliferation rate similar to that of the reference material. To better understand the scope of these results it should be taken into account that the Nd:YAG clad coating has recently been developed. The fact that its in vitro performance is comparable to that produced by plasma spray, a material commercially available for more than ten years, indicates that this new laser based method could be of commercial interest in the near future.

Human osteoblast response to silicon-substituted hydroxyapatite.

Human osteoblasts were cultured on hydroxyapatite (HA), 0.8 wt % silicon substituted hydroxyapatite (Si-HA) and 1.5 wt % Si-HA discs. The influence of these substrates on cell behaviour in vitro was assessed by measuring total protein in the cell lysate and the production of several phenotypic markers: collagen type I (COL I), alkaline phosphatase (ALP), osteocalcin (OC), and the formation of bone mineral. After 7 days, beta-glycerophosphate and physiological levels of hydrocortisone were added to the culture medium to stimulate cell differentiation and mineral production. There was a significantly higher production of ALP on 1.5 wt % Si-HA at day 7 following which, the addition of hydrocortisone promoted the differentiation of cells on the other two substrates. Hydrocortisone addition also decreased the production of OC. During the period, when hydrocortisone was present, no significant difference in behavior was seen between cells on Si-HA and HA; however, following removal of hydrocortisone, cells responded to 0.8 wt % Si-HA with a significant increase in protein production. Using fluorescence microscopy, nodular structures labeled with tetracycline were observed on the surface of all substrates after 21 days. These structures were deposited on areas of high cell density but were not related to the presence or level of silicon in the substrate. These results indicate that human osteoblasts are affected by the presence of silicon in the HA substrate and that the timing of these effects may be dependent upon the level of silicon substitution.

Differentiation of mononuclear precursors into osteoclasts on the surface of Si-substituted hydroxyapatite.

In healthy bone, resorption and synthesis are in perfect coordination. In previous studies we demonstrated that the incorporation of silicon into the hydroxyapatite (HA) lattice enhances the proliferation and differentiation of human osteoblasts. Therefore, the aim of this study was to demonstrate the effect of silicon-substituted HA (0.8 and 1.5 wt % Si-HA) on the differentiation of mononuclear cells into osteoclasts, using two different starting cultures, peripheral blood mononuclear cells (PBMC) and monocytes expressing the CD14 antigen (CD14+). Through this study, it was possible to demonstrate that Si-HA allows the differentiation of mononuclear cells into mature osteoclasts, independent of the starting culture, PBMC or CD14+. Most of the cells on the surface of the materials expressed osteoclastic markers: actin rings, several nuclei, positivity for tartrate-resistant acid phosphatase (TRAP), and vitronectin receptor. In the presence of osteoclasts, a higher release of calcium and phosphate into the medium from the 1.5 wt % Si-HA substrate was detected when compared to the HA substrate; therefore, these results indicate higher osteoclastic resorptive activity on the 1.5 wt % Si-HA surface. Si-HA can be resorbed by cellular mechanisms and have a stimulatory effect on osteoclasts, although the underlying mechanism is still poorly understood.

Silicon-substituted hydroxyapatite thin films: effect of annealing temperature on coating stability and bioactivity.

The effect of annealing temperature on the physicochemical and biological characteristics of magnetron cosputtered silicon-substituted hydroxyapatite (SiHA) thin coatings was studied. Annealing is required to transform as-sputtered amorphous films into crystalline coatings. A nanocrystalline, single-phase apatite structure was achieved for coatings heated to 600 or 700 degrees C and, with increasing annealing temperature, the crystallite size increased. Small crystallites were found to be more soluble in the physiological environment but, at the same time, were able to induce early formation of a new apatite layer. A human osteoblast-like (HOB) cell model was used to evaluate the performance of these annealed SiHA coatings. HOB cells attached and grew well on coatings and, after 42 days in culture, a mineralization process was observed to be taking place, with evidence of calcium phosphate minerals throughout the extracellular matrix. Our findings indicated that an annealing temperature of 600 degrees C is sufficient to achieve crystalline SiHA coatings and exhibiting good chemical stability and bioactivity.

Novel silicon-doped hydroxyapatite (Si-HA) for biomedical coatings: an in vitro study using acellular simulated body fluid.

Magnetron co-sputtering was used to produce silicon-doped hydroxyapatite (Si-HA) as coatings intended for potential applications such as orthopedic and dental implants. It was found that the crystallinity of the as-sputtered coatings increased after annealing, resulting in a nanocrystalline apatite structure. Subsequently, the bioactivity of the coatings was evaluated in an acellular simulated body fluid (SBF). Physicochemical evaluation demonstrated that a carbonate-containing apatite layer, which is essential for bonding at the bone/implant interface, was formed on the coating surfaces after immersion in SBF between 4 and 7 days. The annealed coatings exhibited enhanced bioactivity and chemical stability under physiological conditions, as compared with the as-sputtered coatings. It is proposed that the rate at which the carbonate-containing apatite layer forms is dependent on the scale factor of the structure. A nanocrystalline structure can provide a higher number of nucleation sites for the formation of apatite crystallites, leading to a more rapid precipitation of carbonate-containing apatite layer. This work shows that Si-HA coatings offer considerable potential for applications in hard tissue replacement, owing to their ability to form a carbonate-containing apatite layer rapidly.

Novel deposition of nano-sized silicon substituted hydroxyapatite by electrostatic spraying.

Suspensions containing nano-sized silicon substituted hydroxyaptite (nSiHA) particles were produced and processed for electrostatic spray deposition. No secondary phases were detected by X-ray diffraction, which indicated that the nSiHA was phase pure. Electrostatic spraying of nSiHA in cone-jet mode was achieved at flow rate of 10(-9) m3s(-1) with an applied voltage between the needle and the ring-shaped ground electrode set at 6 to 8 kV. Micrometer- and submicrometer-scaled islands of nSiHA have been deposited on glass and titanium substrates. The surface roughness of such nHA and nSiHA islands was in the range 60 to 80 nm, as measured from atomic force microscopy in tapping mode. The growth of primary human osteoblast (HOB) cells on the nSiHA deposited substrates increased with time during the 4 days of culture, and the increase was related with the Si content in substituted HA, indicating that nSiHA was able to promote and support the growth of HOB cells. Scanning electron microscopy (SEM) revealed that extracellular matrix (ECM) produced by the HOB cells on these nSiHA deposits was well organized. In addition, the presence of Ca and P containing nodules in the ECM were also confirmed by Energy Dispersive X-ray (EDX) analysis, indicating early signs of calcification fronts. The results showed that nSiHA produced by electrostatic spray deposition was able to promote the attachment and the growth of HOB cells. Therefore, electrostatic spray deposition offers great potential for the creation of bioactive surfaces to provide improved interfacial bonding with host tissues.

Effect of carbonate substitution on the ultrastructural characteristics of hydroxyapatite implants.

Carbonate ion substitution has been shown to be beneficial for increasing the amount of in vivo osseointegration to hydroxyapatite (HA). Nevertheless, mechanisms by which carbonate ions increase in vivo bioactivity are not fully understood. Sintered granules of HA and carbonate-substituted hydroxyapatite (CHA) were implanted for 6 and 12 weeks in an ovine model. Samples containing the bone-implant interface were prepared for transmission electron microscopy (TEM) and TEM was used to compare the in vivo reactivity of sintered granules of HA and CHA. The current findings demonstrated that CHA (1.2 and 2.05 wt.%) is more soluble than pure HA in vivo. More dissolution was observed from the CHA, at the bone-implant interface and within the implant, when compared to pure HA. A less crystalline phase was formed between the 2.05 wt.% CHA and bone at 12 weeks in vivo. Bone surrounding both the pure HA and 1.2 wt.% CHA was relatively disorganised at 12 weeks. In comparison, bone surrounding the 2.05 wt.% CHA was considerably more organised and in many regions collagen fibrils were present. Despite increased quality of bone surrounding 2.05 wt.% CHA, compared to 1.2 wt.% CHA, the amount of dissolution from both materials was similar.

Composition-controlled nanocomposites of apatite and collagen incorporating silicon as an osseopromotive agent.

The development of a novel biocomposite of apatite (Ap) and collagen incorporating low-level additions of silicon (Si) as an osseopromotive agent is detailed. Designed to mimic the structural and compositional characteristics of developing bone, this composite is produced via a coprecipitation method, through which the weight percentage of Ap (i.e., the Ap/collagen ratio) can be varied. Coprecipitates produced at Ap contents of 80 wt % (Ap/collagen=4:1), 60 wt % (Ap/collagen=3:2), and 40 wt % (Ap/collagen=2:3) Ap showed markedly different morphologies, ranging from ceramic-like particulates to rope-like macro-fibrils; at all three Ap contents, however, the nanostructural features of the composites remained qualitatively indistinguishable, with equiaxed Ap nanocrystals distributed randomly throughout a matrix of amorphous collagen. Si incorporation was observed to occur preferentially in the collagenous phase-a result with potential impact on local controlled release of Si.

A new way of incorporating silicon in hydroxyapatite (Si-HA) as thin films.

Bioactive silicon-containing hydroxyapatite (Si-HA) thin films that can be used as coatings for bone tissue replacement have been developed. A magnetron co-sputtering technique was used to deposit Si-HA films up to 700 nm thick on titanium substrates, with a silicon level up to 1.2 wt%. X-ray diffraction demonstrated that annealing transformed the as-deposited Si-HA films which were amorphous, into a crystalline HA structure. A human osteoblast-like (HOB) cell model was used to determine the biocompatibility of these films. HOB cells were seen to attach and grow well on the Si-HA films, and the metabolic activity of HOB cells on these films was observed to increase with culture time. Furthermore, mineralisation of the cell layers was observed after 8 weeks of culture. Based on the present findings, Si-HA of different film compositions demonstrate bioactive properties in-vitro, and indicate the potential as biocoatings for a wide variety of medical implants including load-bearing applications such as the femoral stem of hip replacement implants.

In vivo assessment of hydroxyapatite and silicate-substituted hydroxyapatite granules using an ovine defect model.

Phase pure hydroxyapatite (HA) and two silicate-substituted hydroxyapatites (0.8 and 1.5 wt% Si, or 2.6 and 4.9 wt% SiO4) were prepared by aqueous precipitation methods. The filter-cakes of HA and silicate-substituted hydroxyapatite (SiHA) compositions were processed into granules 1.0-2.0 mm in diameter and sintered at 1200 degrees C for 2 h. The sintered granules underwent full structural characterisation, prior to assessment in an ovine defect model by implantation for a period of 6 and 12 weeks. The results indicate that HA and SiHA implants were well accepted by the host tissue, with no evidence of inflammation. New bone formation was observed directly on the surfaces and in the spaces between the granular implants. Quantitative histomorphometry as determined by the percentage of bone ingrowth and bone coverage for both SiHA implant compositions was significantly greater than that for phase pure HA. These findings indicate that the in vivo bioactivity of hydroxyapatite was significantly improved by the incorporation of silicate ions into the HA structure, making SiHA ceramics attractive alternatives to conventional HA materials for use as bone graft substitute ceramics.

A novel method for the simultaneous, titrant-free control of pH and calcium phosphate mass yield.

Calcium phosphate (CaP) bioceramics have long been of interest for the unique properties that they exhibit as bone substitute materials. By harnessing the unique bone-bonding capacity of CaP's, biomaterials scientists have made great strides over the past 2 decades to produce novel materials to assist in the treatments of defects caused by trauma, disease, or both. In recent years, however, it has become apparent that the traditional set of techniques used to produce calcium phosphates does not satisfy all of the requirements necessary to meet the challenges of emerging applications. In particular, recent interest in (i) the synthesis of coprecipitated CaP/bioorganic composites and (ii) the investigation of the mechanisms of biomineralization has highlighted the need for new methods to control pH and CaP mass yield.

Magnetron co-sputtered silicon-containing hydroxyapatite thin films--an in vitro study.

The use of silicon-substituted hydroxyapatite (Si-HA) as a biomaterial has been reported recently. In vivo testing has shown that Si-HA promotes early bonding of the bone/implant interface. In order to extend its usage to major load-bearing applications such as artificial hip replacement implants, it has been proposed that the material could be used in the form of a coating on implant surfaces. This paper reports a preliminary study of the biocompatibility of magnetron co-sputtered silicon-containing hydroxyapatite (Si-HA) coatings on a metallic substrate. Magnetron co-sputtered Si-HA films of thickness 600 nm with a Si content of approximately 0.8 wt% were produced on titanium substrates. X-ray diffraction analysis showed that the as-deposited Si-HA films were either amorphous or made up of very small crystals. The crystallinity of Si-HA films was increased after post-deposition heat treatment at 700 degrees C for 3 h, and the principal peaks were attributable to HA. The formation of nano-scale silicon-calcium phosphate precipitates was noted on the heat-treated films. In vitro cell culture has demonstrated that human osteoblast-like cells attached and grew well on all films, with the highest cell growth and signs of mineralisation observed on the heat-treated Si-HA films. In addition, many focal contacts were produced on the films and the cells had well-defined actin cytoskeletal organisation. This work shows that as-deposited and heat-treated Si-HA films have excellent bioactivity and are good candidates when rapid bone apposition is required. Furthermore, heat-treated Si-HA films have improved biostability compared to as-deposited films under physiological conditions.