Perfluorodecalin and bone regeneration.

Perfluorodecalin (PFD) is a chemically and biologically inert biomaterial and, as many perfluorocarbons, is also hydrophobic, radiopaque and has a high solute capacity for gases such as oxygen. In this article we have demonstrated, both in vitro and in vivo, that PFD may significantly enhance bone regeneration. Firstly, the potential benefit of PFD was demonstrated by prolonging the survival of bone marrow cells cultured in anaerobic conditions. These findings translated in vivo, where PFD incorporated into bone-marrow-loaded 3D-printed scaffolds substantially improved their capacity to regenerate bone. Secondly, in addition to biological applications, we have also shown that PFD improves the radiopacity of bone regeneration biomaterials, a key feature required for the visualisation of biomaterials during and after surgical implantation. Finally, we have shown how the extreme hydrophobicity of PFD enables the fabrication of highly cohesive self-setting injectable biomaterials for bone regeneration. In conclusion, perfluorocarbons would appear to be highly beneficial additives to a number of regenerative biomaterials, especially those for bone regeneration.

Bioinorganics and biomaterials: bone repair.

The field of bioinorganics is well established in the development of a variety of therapies. However, their application to bone regeneration, specifically by way of localized delivery from functional implants, is in its infancy and is the topic of this review. The toxicity of inorganics is species, dose and duration specific. Little is known about how inorganic ions are effective therapeutically since their use is often the result of serendipity, observations from nutritional deficiency or excess and genetic disorders. Many researchers point to early work demonstrating a role for their element of interest as a micronutrient critical to or able to alter bone growth, often during skeletal development, as a basis for localized delivery. While one can appreciate how a deficiency can cause disruption of healing, it is difficult to explain how a locally delivered excess in a preclinical model or patient, which is presumably of normal nutritional status, can evoke more bone or faster healing. The review illustrates that inorganics can positively affect bone healing but various factors make literature comparisons difficult. Bioinorganics have the potential to have just as big an impact on bone regeneration as recombinant proteins without some of the safety concerns and high costs.

In vitro antibacterial efficacy of tetracycline hydrochloride adsorbed onto Bio-Oss bone graft.

Local delivery of antibiotics may provide the advantage of reducing the potential side effects associated with their systemic administration. This study assessed, in vitro, the antimicrobial efficacy of tetracycline hydrochloride (TCH) adsorbed onto Bio-Oss bone grafts against a range of pathogenic bacteria. Various levels of TCH were adsorbed onto Bio-Oss granules by immersing in TCH aqueous solutions of different initial concentrations for 48 h at room temperature. TCH release was assessed in phosphate buffered saline at 37 degrees C, and its antimicrobial efficacy, up to 96 h, was tested against two Gram-negative bacteria associated with periodontal diseases: Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, and Porphyromonas gingivalis, and one Gram-positive bacterium associated with soft-tissue and bone infections: Staphylococcus aureus. The range of TCH concentrations studied was also assessed for cytotoxicity against osteoblast-like human osteosarcoma cell lines. The amount of TCH adsorbed and released from Bio-Oss was concentration dependent. All TCH adsorbed Bio-Oss resulted in a reduction of A. actinomycetemcomitans, P. gingivalis, and S. aureus and higher concentrations were generally more effective in reducing or eliminating bacterial growth. The proliferation of HOS cells was not substantially reduced except for the maximum concentration of TCH. In addition to its osteoconductive role, TCH adsorbed Bio-Oss could also be functional in negating systemically antibiotic prophylactic treatment in the prevention of implant or biomaterial related infections.

Ion adsorption behaviour of hydroxyapatite with different crystallinities.

This study aimed to correlate crystallinity of hydroxyapatite (HA) with the ion adsorption behaviour of the material. Hydroxyapatite powders of various crystallinities (X(c)) and specific surface area (SSA) were prepared by precipitation following heat treatment. Adsorption experiments were carried out by using (i) multi-component ion solutions containing a broad range of light and heavy ions to study competitive adsorption and (ii) lead and zinc solutions with concentrations up to 250 ppm to determine the adsorption isotherms of the material. While as-prepared HA powders of low crystallinity (X(c)=0%) and a high SSA of 170 m(2)/g showed quantitative removal for divalent Pb, Zn, Be, U, Bi, V, Al, Cu and Ga ions, calcined powders with higher crystallinity (X(c)=65-95%) and lower SSA between 5 and 30 m(2)/g led to a quantitative removal only for a few elements (Pb, Bi, Ga). The time and concentration dependant ion removal capacity for Pb(2+) and Zn(2+) single element solutions showed quantitative removal even after short immersion times of less than 10 min for as-prepared HA powders. XRD analysis of the powders after ion adsorption revealed the presence of pyromorphite (Pb(5)(PO(4))(3)OH) and hopeite (Zn(3)(PO(4))(2)) phases, respectively.

High-strength resorbable brushite bone cement with controlled drug-releasing capabilities.

Brushite cements differ from apatite-forming compositions by consuming a lot of water in their setting reaction whereas apatite-forming cements consume little or no water at all. Only such cement systems that consume water during setting can theoretically produce near-zero porosity ceramics. This study aimed to produce such a brushite ceramic and investigated whether near elimination of porosity would prevent a burst release profile of incorporated antibiotics that is common to prior calcium phosphate cement delivery matrices. Through adjustment of the powder technological properties of the powder reactants, that is particle size and particle size distribution, and by adjusting citric acid concentration of the liquid phase to 800mM, a relative porosity of as low as 11% of the brushite cement matrix could be achieved (a 60% reduction compared to previous studies), resulting in a wet unprecompacted compressive strength of 52MPa (representing a more than 100% increase to previously reported results) with a workable setting time of 4.5min of the cement paste. Up to 2wt.% of vancomycin and ciprofloxacin could be incorporated into the cement system without loss of wet compressive strength. It was found that drug release rates could be controlled by the adjustable relative porosity of the cement system and burst release could be minimized and an almost linear release achieved, but the solubility of the antibiotic (vancomycin>ciprofloxacin) appeared also to be a crucial factor.

Carvable calcium phosphate bone substitute material.

This study investigated the use of partially set hydroxyapatite forming calcium phosphate cement as a carvable and mechanically stable bone substitute material. Hydroxyapatite-forming cements were made of either mechanically activated alpha-tricalcium phosphate or a mixture of tetracalcium phosphate and dicalcium phosphate anhydrous and setting was arrested up to 4 h post setting. The study showed that these partially set rigid samples of defined geometry could be carved into a desired shape when the degree of reaction was 30-40% and the relative porosity between 40 and 50%; samples are then expected to set completely after implantation in the presence of water or serum, having the same compressive strength as a continuously set calcium phosphate cement (up to 36 MPa). The development of compressive strength, phase composition, and crystallinity when varying production parameters of these partially "preset" bone substitute materials are presented for both cement systems.

Real-time monitoring of the setting reaction of brushite-forming cement using isothermal differential scanning calorimetry.

The setting behavior of a brushite-forming cement (beta-tricalcium phosphate/mono calcium monophosphate) was investigated using an indentation technique (the Gillmore needles method) and isothermal differential scanning calorimetry (DSC). The two objectives of the study were to investigate whether DSC could be used to real-time monitor a fast-setting calcium phosphate cement (CPC) and to determine if it is possible to correlate DSC results directly with conventional setting-time measurements. Best-fit linear correlation analysis revealed that both the initial and final setting time (T(i) and T(f)) measured by indentation were strongly correlated to the maximum heat flow measured with DSC. It seems therefore possible to predict the setting times, usually achieved with user dependent indentation methods, of this specific fast setting CPC on the basis of objective DSC measurements. The drawbacks of DSC, however, are its overall complexity and expense and the fact that only exothermal reactions can be investigated in comparison to the Gillmore needles method, furthermore, it is not possible to monitor the complete reaction as the first 2 or 3 min are lost due to sample preparation and apparatus set up.

Effects of fibre reinforcement on the mechanical properties of brushite cement.

In this study the effect of structure and amount of polyglactin fibre incorporation into a brushite forming calcium phosphate cement system and the effect of mechanical compaction on the fibre modified system were investigated. In comparison the effect of resorbable polycaprolactone surface coating of cement specimens was investigated. The results showed that, apart from the mechanical properties of the reinforcing material, the structure of the incorporated fibres, regular or random, is crucial for the resulting flexural strength and modulus of elasticity. Fibre reinforcement could also be combined with mechanical compaction of the cement/fibre composite paste leading to a possible 7-fold increase in flexural strength or an almost 5-fold increase in modulus of elasticity. Reinforcement of the tensile surface of cement grafts may ultimately improve strength where required, especially in conjunction with bone fixation devices.

Bone marrow cell gene expression and tissue construct assembly using octacalcium phosphate microscaffolds.

Calcium phosphates have been widely used in bone and soft tissue applications and are of considerable interest as scaffold materials due to properties of osteoconduction, resorbability and in some cases osteoinduction. These materials are microcrystalline and as such are processed using sintering, surface coating or cement technologies. However calcium phosphates containing HPO(4)(2-) ions often have layered crystal structures and can form macrocrystals in an aqueous environment at room temperature and pressure. This study aimed to investigate the potential of octacalcium phosphate (OCP) crystals for the attachment, proliferation and differentiation of bone marrow stromal cells and the potential of these cell seeded crystals as 'building blocks' for manufacture of self-supporting macroscale tissue constructs. An inverse relationship between cell number and crystal surface area was found and marrow cells grown on OCP crystals expressed osteocalcin and osteopontin mRNA, markers of osteoblastic differentiation, even in the absence of inductive media additives. Self-supporting crystal tissue macroscale constructs could be fabricated by culturing cell loaded crystals in moulds of the desired shape. Due to the low packing efficiency as a consequence of the high aspect ratio of OCP crystals, this microscaffold approach may offer the potential for ex vivo construction of large volumes of tissue which forms as a physiologically vascularised tissue.

Cement from nanocrystalline hydroxyapatite: effect of calcium phosphate ratio.

Nanocrystalline hydroxyapatite (nHA) can be mixed with phosphoric acid to form a brushite cement; a degradable inorganic bone filling material. nHA was precipitated from reactants of calcium to phosphate (Ca/P) ratio 0.8 to 2.0 and mixed with phosphoric acid, which resulted in the formation of a brushite cement. Cement was also formed by mixing microcrystalline calcium phosphates, beta-tricalcium phosphate, hydroxyapatite and tetracalcium phosphate with phosphoric acid solution. Cement produced with nHA was stronger in compression than that formed with crystalline calcium phosphate phases. Setting time, strength and composition of cement produced with nHA was dependant on both the Ca/P ratio of nHA and the concentration of phosphoric acid in cement slurry. Increasing phosphoric acid concentration increased compressive strength whilst reducing the initial setting time of cement. Reducing the Ca/P ratio of nHA precipitation reactants retarded the setting and increased the extent of reaction of cements. This finding was unexpected and suggests that Ca/P ratio may strongly affect dissolution behaviour and this parameter is more important than stoichiometry in determining extent of reaction in this system. This study demonstrated that the wide variation in stoichiometry that may be attained in nanocrystalline apatite may be utilised to change cement performance and setting behaviour.

Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels.

Calcium cross-linked sodium alginate hydrogels have several advantageous features making them potentially suitable as tissue engineering scaffolds and this material has been previously used in many biomedical applications. 3D cell culture systems are often very different from 2D petri dish type cultures. in this study the effect of alginate hydrogel architecture was investigated by comparing rat bone marrow cell proliferation and differentiation on calcium cross linked sodium alginate discs and 1mm internal diameter tubes. It was found that bone marrow cell proliferation was diminished as the concentration of alginate in the 2D hydrogel substrates increased, yet proliferation was extensive on tubular alginate constructs with high alginate contents. Alginate gel thickness was found to be an important parameter in determining cell behaviour and the different geometries did not generate significant alterations in BMC differentiation profiles.

Technological issues for the development of more efficient calcium phosphate bone cements: a critical assessment.

The first calcium phosphate cements (CPCs) were discovered in the 1980s. Two decades later, the interest for these materials is still rising. The goal of the present document is to review the most recent achievements in the field and to analyze future directions in research and development.

Alkali ion substituted calcium phosphate cement formation from mechanically activated reactants.

Potassium and sodium containing nanoapatite cements were produced from Ca2KNa(PO4)2 by prolonged high energy ball milling of the compound for up to 24 h. This mechanical treatment resulted in the decrease of the crystal size and a partial amorphisation of the cement reactant as shown by X-ray diffraction analysis and the appearance of strong exothermic peaks in differential scanning calorimetry measurements. The pH of water saturated with Ca2KNa(PO4)2 was 12.5 when the material was mechanically activated but was only 9.5 for the untreated compound suggesting an increase in solubility following milling. The cements set following mixing with a 2.5% Na2HPO4 solution in clinically acceptable times between 5-12 min and showed compressive strengths of up to 11 MPa after 24 h setting. The strong alkaline pH value of the cements may provide antimicrobial potential for an application in dentistry as pulp capping agents or cavity liners or for the treatment of infected bone sites.

Cement from magnesium substituted hydroxyapatite.

Brushite cement may be used as a bone graft material and is more soluble than apatite in physiological conditions. Consequently it is considerably more resorbable in vivo than apatite forming cements. Brushite cement formation has previously been reported by our group following the mixture of nanocrystalline hydroxyapatite and phosphoric acid. In this study, brushite cement was formed from the reaction of nanocrystalline magnesium-substituted hydroxyapatite with phosphoric acid in an attempt to produce a magnesium substituted brushite cement. The presence of magnesium was shown to have a strong effect on cement composition and strength. Additionally the presence of magnesium in brushite cement was found to reduce the extent of brushite hydrolysis resulting in the formation of HA. By incorporating magnesium ions in the apatite reactant structure the concentration of magnesium ions in the liquid phase of the cement was controlled by the dissolution rate of the apatite. This approach may be used to supply other ions to cement systems during setting as a means to manipulate the clinical performance and characteristics of brushite cements.

Cortical bone screw fixation in ionically modified apatite cements.

Hydroxyapatite cements are used in reconstruction of the face; usually in well-defined cavities where the cement can be stabilized without the need for internal fixation. A hydroxyapatite cement that could enable screw fixation and some loading therefore has considerable potential in maxillofacial reconstruction. It has been demonstrated recently that water demand of calcium phosphate cements can be reduced by ionically modifying the liquid component. This study investigated the capacity of an ionically modified precompacted apatite cement to retain self-tapping cortical bone screws. Screw pullout forces were determined in the direction of the screw long axis and perpendicular to it, using cortical bone and polymethylmethacrylate cement as a control. In bending pullout tests, measured forces to remove screws from ionically modified precompacted cement were insignificantly different from cortical bone. However, pullout forces of bone screws from hydroxyapatite cement decreased with aging time in vitro.

Improving peptide-based assays to differentiate between vaccination and Mycobacterium bovis infection in cattle using nanoparticle carriers for adsorbed antigens.

The development of diagnostic tests to differentiate between vaccinated animals and those infected with Mycobacterium bovis is required so that test and slaughter control strategies can continue alongside vaccination. In this work, the peptide antigen, ESAT-6, p45, derived from the N-terminal sequence of the ESAT-6 protein, was adsorbed onto a range of microparticulate and nanoparticulate substrates to enhance the in vitro immune response of blood lymphocytes previously sensitised to M. bovis. Two types of hydroxyapatite (HA) nanoparticles (both approximately 300 nm in linear dimension), carbonate hydroxyapatite nanospheres (CHA, approximately 50 nm), two sizes of polystyrene nanospheres ( approximately 500 and 40 nm), calcium carbonate microparticles (0.3-1.0 microm) and glass microspheres (1.0-3.0 microm) were incubated in a solution of the peptide in PBS. Peptide adsorption increased on the nanoparticle carriers in the order HA (2.5+/-0.12%w/w), CHA (4.9+/-0.12) polystyrene (500 nm, 6.8+/-0.15%, 40 nm, 9.2+/-0.07) and these systems exhibited fairly low levels of desorption (approximately 10-15% peptide release) over a 24-h incubation period in PBS at 37 degrees C. HA, CHA and polystyrene carriers with adsorbed peptide were subsequently tested in the BOVIGAM assay to investigate the efficiency of the immune response of blood lymphocytes in terms of interferon-gamma (IFN-gamma) production. A general elevation of IFN-gamma production resulted for particle-bound peptide relative to free peptide at high peptide concentrations (>10 microg/ml). Only HA-adsorbed peptide resulted in consistently higher immune responses at low peptide concentration (<0.1 microg/ml) compared with the free peptide, indicating that peptide antigens adsorbed to hydroxyapatite nanoparticles may be useful, in diagnostic assays, for differentiating between tuberculosis (TB)-infected and vaccinated animals.

Rheological enhancement of mechanically activated alpha-tricalcium phosphate cements.

Most biocements are two- or three-component acid-based systems with large differences in the component particle sizes, which occurs by virtue of the differing processing routes. This work aimed to improve injectability and strength of a single reactive component cement, that is, mechanically activated alpha-tricalcium phosphate (TCP)-based cement by adding 13-33 wt % of several fine-particle-sized (d(50) of 0.5-1.1 microm) fillers [dicalcium phosphate anhydrous (DCPA), titanium dioxide (TiO(2)), and calcium carbonate] to the monomodal alpha-TCP matrix (d(50) = 9.8 microm). A high zeta-potential was measured for all particles in trisodium citrate solution. The fraction of alpha-TCP cement "injected" through an 800-microm hypodermic needle was found to be only 35% at a powder-to-liquid ratio of 3.5 g/mL. In contrast, the use of fillers decreased cement viscosity to a point, where complete injectability could be obtained. Mechanistically, these additives disrupted alpha-TCP particle packing yet decreased the interparticle spacing by a factor of approximately 5.5 such that the electrostatic repulsion effect was enhanced. A strength improvement was found when DCPA and TiO(2) were used as fillers despite the lower degree of conversion of these cements. Compressive strengths of precompacted cement samples increased from 70 MPa for unfilled alpha-TCP cement to 140 (110) MPa for 23 wt % DCPA (or TiO(2)) fillers as a result of porosity reduction. Strength improvement for more clinically relevant uncompacted cements was achieved by higher powder-to-liquid ratio mixes for filled cements such that maximum strengths of 90 MPa were obtained for 23 wt % DCPA filler compared with 50 MPa for single-component alpha-TCP cement.

Modified PMMA cements for a hydrolysis resistant metal-polymer interface in orthopaedic applications.

Amongst the many factors influencing the long-term stability of cemented hip prostheses, the interface between the implant and bone cement is considered to be one of the most susceptible to failure. Osteolysis and loosening of the implant can occur by the interaction of mechanically and/or hydrolytically induced bond failure of the metal-cement interface. In this work, an improvement of the hydrolysis resistance of the titanium-bone cement interface was obtained by cement modification with a bifunctional coupling agent combined with a tribochemical TiO2-modification of the metal surface. Methacryloxypropyl-trimethoxysilane was added as coupling agent to the PMMA monomer in concentrations between 5 and 20 wt.% followed by the testing the shear bond strength of PMMA/titanium joints before and after ageing in physiological saline solution. It was found that the hydrolysis resistance of the metal-PMMA interface could be significantly improved by the modification of the cement. At the same time, the mechanical properties (compressive and bending strength) of the modified cement were not altered by the addition of the coupling agent. The advantage of the modification of the cement matrix is an easy clinical applicability of the procedure maintaining the processing and implantation techniques of the cement material.

Adhesion and growth of bone marrow stromal cells on modified alginate hydrogels.

Alginate is a biodegradable, immunocompatible biopolymer that is capable of immobilizing viable cells and bioactive factors. Few investigations have analyzed the efficacy of alginate gels as substrata for cell attachment and proliferation. Here we have compared the adhesion and subsequent growth of human and rat bone marrow stromal fibroblastic cells on unmodified alginate hydrogel surfaces. It was found that, in contrast to rat cells, human cells did not readily attach or proliferate on unmodified alginates. In attempts to enhance these features, or collagen type I was incorporated into the gels, with no significant improvements in prolonged human cell adherence. However, alginate gels containing both collagen type I and beta-tricalcium phosphate were found to enhance human cell adherence and proliferation. Furthermore, interactions between the collagen and beta-tricalcium phosphate prevented loss of the protein from the hydrogels. These results indicate that alginate gels containing collagen have potential uses as vehicles for delivery of adherent cells to a tissue site. In addition, gels containing beta-tricalcium phosphate, with or without collagen type I incorporation, have potential to support cell growth and differentiation in vitro before implantation. This study emphasizes the limitations of the uses of cells derived from experimental animals in certain model studies relating to human tissue engineering.

Surfactant vesicle-mediated delivery of DNA vaccines via the subcutaneous route.

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper we have compared the potency of lipid-based and non-ionic surfactant based vesicle carrier systems for DNA vaccines after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into various vesicle formulations. The DRV method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded high DNA vaccine incorporation values (85-97% of the DNA used) in all formulations. Studies on vesicle size revealed lipid-based systems formed cationic submicron size vesicles whilst constructs containing a non-ionic surfactant had significantly large z-average diameters (>1500 nm). Subcutaneous vesicle-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded nucleoprotein were substantially higher after dosing twice, 28 days apart with 10 microg DRV-entrapped DNA compared to naked DNA. Comparison between the lipid and non-ionic based vesicle formulations revealed no significant difference in stimulated antibody production. These results suggest that, not only can DNA be effectively entrapped within a range of lipid and non-ionic based vesicle formulations using the DRV method but that such DRV vesicles containing DNA may be a useful system for subcutaneous delivery of DNA vaccines.