Monetite, an important calcium phosphate compound-Its synthesis, properties and applications in orthopedics.

This review recognizes a unique calcium phosphate (CaP) phase known as monetite or dicalcium phosphate anhydrous (DCPA, CaHPO4), and presents an overview of its properties, processing, and applications in orthopedics. The motivation for the present effort is to highlight the state-of-the-art research and development of monetite and propel the research community to explore more of its potentials in orthopedics. After a brief introduction of monetite, we provide a summary of its various synthesis routes like dehydration, solvent-based, energy-assisted processes and also discuss the formation of different crystal structures with respect to the synthesis conditions. Subsequently, we discuss the material's noteworthy physico-chemical properties including the crystal structure, vibrational spectra, solubility, thermal decomposition, and conversion to other phases. Of note, we focus on the biological (in vitro and in vivo) properties of monetite, given its ever-increasing popularity as a biomaterial for medical implants. Appropriately, we discuss various orthopedic applications of monetite as bone cement, implant coatings, granules for defect fillers, and scaffolds. Many in vitro and in vivo studies confirmed the favorable osteointegration and osteoconduction properties of monetite products, along with a better balance between implant resorption and new bone formation as compared to other CaP phases. The review ends with translational aspects of monetite and presents thoughts about its possible future research directions. Further research may explore but not limited to improvements in mechanical strength of monetite-based scaffolds, using monetite particles as a therapeutic agent delivery, and tissue engineering strategies where monetite serves as the biomaterial. STATEMENT OF SIGNIFICANCE: This is the first review that focusses on the favorable potential of monetite for hard tissue repair and regeneration. The article accurately covers the "Synthesis-Structure-Property-Applications" correlations elaborating on monetite's diverse material properties. Special focus is put on the in vitro and in vivo properties of the material highlighting monetite as an orthopedic material-of-choice. The synthesis techniques are discussed which provide important information about the different fabrication routes for monetite. Most importantly, the review provides comprehensive knowledge about the diverse biomedical applications of monetite as granules, defect--specific scaffolds, bone cements and implant coatings. This review will help to highlight monetite's potential as an effective regenerative medicine and catalyze the continuing translation of this bioceramic from the laboratory to clinics.

Multifunctional calcium phosphate based coatings on titanium implants with integrated trace elements.

For decades, the main focus of titanium implants developed to restore bone functionality was on improved osseointegration. Additional antimicrobial properties have now become desirable, due to the risk that rising antibiotic resistance poses for implant-associated infections. To this end, the trace elements of copper and zinc were integrated into calcium phosphate based coatings by electrochemically assisted deposition. In addition to their antimicrobial activity, zinc is reported to attract bone progenitor cells through chemotaxis and thus increase osteogenic differentiation, and copper to stimulate angiogenesis. Quantities of up to 68.9 ± 0.1 µg cm- 2 of copper and 56.6 ± 0.4 µg cm- 2 of zinc were deposited; co-deposition of both ions did not influence the amount of zinc but slightly increased the amount of copper in the coatings. The release of deposited copper and zinc species was negligible in serum-free simulated body fluid. In protein-containing solutions, a burst release of up to 10 µg ml-1 was observed for copper, while zinc was released continuously for up to 14 days. The presence of zinc was beneficial for adhesion and growth of human mesenchymal stromal cells in a concentration-dependent manner, but cytotoxic effects were already visible for coatings with an intermediate copper content. However, co-deposited zinc could somewhat alleviate the adverse effects of copper. Antimicrobial tests with E. coli revealed a decrease in adherent bacteria on brushite without copper or zinc of 60%, but if the coating contained both ions there was almost no bacterial adhesion after 12 h. Coatings with high zinc content and intermediate copper content had the overall best multifunctional properties.

Silver and copper addition enhances the antimicrobial activity of calcium hydroxide coatings on titanium.

Electrochemically assisted deposition of Ca(OH)2 (Portlandite) coatings on titanium surfaces has been proven as a promising method to provide the substrate with a most desirable combination of significant bacterial growth reduction on one hand and good biocompatibility on the other. Due to the rapid in vivo transformation of Ca(OH)2 to hydroxyapatite, the antimicrobial activity will be an ephemeral property of the coating when implanted into the human body. In this study, the ability to reduce bacterial growth of such portlandite coatings was significantly enhanced by an ionic modification with copper and silver ions. Antibacterial tests revealed a noticeably elevated reduction of bacterial growth, especially for copper and even at a relatively low copper content of about 0.3 wt.%. In addition, the cytocompatibility, a crucial prerequisite for potential in vivo biocompatibility, of the copper-modified coating was comparable to pure calcium hydroxide coatings.

Calcium phosphate bone cement/mesoporous bioactive glass composites for controlled growth factor delivery.

Calcium phosphate (CaP) bone cements are widely used for the treatment of bone defects and have been proposed to serve as a delivery platform for therapeutic drugs, proteins and growth factors into the defect region. However, they lack sufficient porosity to allow immediate bone ingrowth and thus foster rapid integration into the bone tissue. In this study we investigated a composite prepared from a hydroxyapatite forming bone cement and mesoporous bioactive glass (MBG) granules as a potential carrier for biologically active proteins. The mechanical properties of the composite were not compromised by up to 10 wt% MBG granule addition, which can be attributed to the strong interface between the cement matrix and MBG particles, however this modification induced a significant increase in porosity within 3 weeks ageing in an aqueous liquid. The release profiles of two proteins, lysozyme and the vascular endothelial growth factor (VEGF), could be controlled when they were loaded onto MBG granules that were subsequently embedded into the cement when compared to direct loading into the cement precursor. Both proteins were also demonstrated to maintain their biologic activity during embedding and release from the composite. These findings suggest the CaP bone cement/MBG composite developed in this study as a potential delivery platform for growth factors or other bioactive substances.

The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model.

OBJECTIVE: To investigate the effect of decellularized cartilage-derived matrix (CDM) scaffolds, by itself and as a composite scaffold with a calcium phosphate (CaP) base, for the repair of osteochondral defects. It was hypothesized that the chondral defects would heal with fibrocartilaginous tissue and that the composite scaffold would result in better bone formation. METHODS: After an 8-week pilot experiment in a single horse, scaffolds were implanted in eight healthy horses in osteochondral defects on the medial trochlear ridge of the femur. In one joint a composite CDM-CaP scaffold was implanted (+P), in the contralateral joint a CDM only (-P) scaffold. After euthanasia at 6 months, tissues were analysed by histology, immunohistochemistry, micro-CT, biochemistry and biomechanical evaluation. RESULTS: The 8-week pilot showed encouraging formation of bone and cartilage, but incomplete defect filling. At 6 months, micro-CT and histology showed much more limited filling of the defect, but the CaP component of the +P scaffolds was well integrated with the surrounding bone. The repair tissue was fibrotic with high collagen type I and low type II content and with no differences between the groups. There were also no biochemical differences between the groups and repair tissue was much less stiff than normal tissue (P < 0.0001). CONCLUSIONS: The implants failed to produce reasonable repair tissue in this osteochondral defect model, although the CaP base in the -P group integrated well with the recipient bone. The study stresses the importance of long-term in vivo studies to assess the efficacy of cartilage repair techniques.

Electrochemically assisted deposition of strontium modified magnesium phosphate on titanium surfaces.

Electrochemically assisted deposition was utilized to produce ceramic coatings on the basis of magnesium ammonium phosphate (struvite) on corundum-blasted titanium surfaces. By the addition of defined concentrations of strontium nitrate to the coating electrolyte Sr(2+) ions were successfully incorporated into the struvite matrix. By variation of deposition parameters it was possible to fabricate coatings with different kinetics of Sr(2+) into physiological media, whereas the release of therapeutically relevant strontium doses could be sustained over several weeks. Morphological and crystallographic examinations of the immersed coatings revealed that the degradation of struvite and the release of Sr(2+) ions were accompanied by a transformation of the coating to a calcium phosphate based phase similar to low-crystalline hydroxyapatite. These findings showed that strontium doped struvite coatings may provide a promising degradable coating system for the local application of strontium or other biologically active metal ions in the implant-bone interface.

Real-time measurement of protein adsorption on electrophoretically deposited hydroxyapatite coatings and magnetron sputtered metallic films using the surface acoustic wave technique.

Surface acoustic wave (SAW) biosensors are highly sensitive for mass binding and are therefore used to detect protein-protein and protein-antibody interactions. Whilst the standard surface of the chips is a thin gold film, measurements on implant- or bone-like surfaces could significantly enhance the range of possible applications for this technique. The aim of this study was to establish methods to coat biosensor chips with Ti, TiN, and silver-doped TiN using physical vapor deposition as well as with hydroxyapatite by electrophoresis. To demonstrate that protein adsorption can be detected on these surfaces, binding experiments with fibronectin and fibronectin-specific antibodies have been performed with the coatings, which successfully proved the applicability of PVD and EPD for SAW biosensor functionalization.

Silicon calcium phosphate ceramic as novel biomaterial to simulate the bone regenerative properties of autologous bone.

This study was conducted to develop novel ceramic bone substitute that resembles the autologous bone behavior when used as graft material. Solid-state reaction at 1100°C was performed to synthesize ß-tricalcium phosphate (ß-TCP) and biphasic calcium phosphate (BCP). The ceramics were further analyzed to characterize phase composition, microstructural properties, cytocompatability and then challenged to regenerate critical bone defects in the parietal bone of rabbits. X-ray diffraction analysis confirmed the production of ß-TCP and indicated the synthesis of novel BCP composed of ß-TCP and silicocarnotite (calcium phosphate silicate mineral). The cytocompatibility test with human osteoblast cell line revealed enhanced cell proliferation on the BCP ceramic. The novel BCP induced the filling of about 73% of the bone defect with a newly formed bone tissue and an almost complete degradation after 12 weeks of healing. This novel ceramic resembles the autologous bone properties of complete degradation and efficient enhancement of bone formation, making it promising as bone graft material.

Structural characterisation of oxygen diffusion hardened alpha-tantalum PVD-coatings on titanium.

Titanium substrates were coated with tantalum layers of 5 µm thickness using physical vapour deposition (PVD). The tantalum layers showed a (110)-preferred orientation. The coated samples were hardened by oxygen diffusion. Using X-ray diffraction the crystallographic structure of the tantalum coatings was characterised, comparing untreated and diffusion hardened specimen conditions. Oxygen depth profiles were determined by glow discharge spectrometry. The hardening effect of the heat treatment was examined by Vickers microhardness testing. The increase of surface hardness caused by oxygen diffusion was at least 50%.

Ready-to-use injectable calcium phosphate bone cement paste as drug carrier.

Current developments in calcium phosphate cement (CPC) technology concern the use of ready-to-use injectable cement pastes by dispersing the cement powder in a water-miscible solvent, such that, after injection into the physiological environment, setting of cements occurs by diffusion of water into the cement paste. It has also been demonstrated recently that the combination of a water-immiscible carrier liquid combined with suitable surfactants facilitates a discontinuous liquid exchange in CPC, enabling the cement setting reaction to take place. This paper reports on the use of these novel cement paste formulations as a controlled release system of antibiotics (gentamicin, vancomycin). Cement pastes were applied either as a one-component material, in which the solid drugs were physically dispersed, or as a two-component system, where the drugs were dissolved in an aqueous phase that was homogeneously mixed with the cement paste using a static mixing device during injection. Drug release profiles of both antibiotics from pre-mixed one- and two-component cements were characterized by an initial burst release of ∼7-28%, followed by a typical square root of time release kinetic for vancomycin. Gentamicin release rates also decreased during the first days of the release study, but after ∼1 week, the release rates were more or less constant over a period of several weeks. This anomalous release kinetic was attributed to participation of the sulfate counter ion in the cement setting reaction altering the drug solubility. The drug-loaded cement pastes showed high antimicrobial potency against Staphylococcus aureus in an agar diffusion test regime, while other cement properties such as mechanical performance or phase composition after setting were only marginally affected.

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.

Dual setting α-tricalcium phosphate cements.

An extension of the application of calcium phosphate cements (CPC) to load-bearing defects, e.g. in vertebroplasty, would require less brittle cements with an increased fracture toughness. Here we report the modification of CPC made of alpha-tricalcium phosphate (α-TCP) with 2-hydroxyethylmethacrylate (HEMA), which is polymerised during setting to obtain a mechanically stable polymer-ceramic composite with interpenetrating organic and inorganic networks. The cement liquid was modified by the addition of 30-70 % HEMA and ammoniumpersulfate/tetramethylethylendiamine as initiator. Modification of α-TCP cement paste with HEMA decreased the setting time from 14 min to 3-8 min depending on the initiator concentration. The 4-point bending strength was increased from 9 MPa to more than 14 MPa when using 50 % HEMA, while the bending modulus decreased from 18 GPa to approx. 4 GPa. The addition of ≥50 % HEMA reduced the brittle fracture behaviour of the cements and resulted in an increase of the work of fracture by more than an order of magnitude. X-ray diffraction analyses revealed that the degree of transformation of α-TCP to calcium deficient hydroxyapatite was lower for polymer modified cements (82 % for polymer free cement and 55 % for 70 % HEMA) after 24 h setting, while the polymerisation of HEMA in the cement liquid was quantitative according to FT-IR spectroscopy. This work demonstrated the feasibility of producing fracture resistant dual-setting calcium phosphate cements by adding water soluble polymerisable monomers to the liquid cement phase, which may be suitable for an application in load-bearing bone defects.

Oxygen diffusion hardening of cp-titanium for biomedical applications.

Common methods to increase the wear resistance of titanium by surface hardening in biomedical applications, such as chemical/physical vapour deposition techniques or thermal/electrochemical oxidation, result in a layer of titanium dioxide or titanium nitride on the metal surface with a sharp interface between the hard and brittle coating and the ductile metallic substrate. A major disadvantage of these methods is that the sharp transition in material properties may cause exfoliation of these coatings. In this work, a two-step heat treatment was used to investigate oxygen diffusion hardening and its capability to produce hard surfaces with a transition zone between the coating and the ductile substrate. During the first step, the native oxide layer was strengthened. In the second step, oxygen diffusion was activated and a transition zone was formed. Different methods of analysis confirmed the success of the thermal treatment, as well as the change of the mechanical properties.

Tetracalcium phosphate: Synthesis, properties and biomedical applications.

Monoclinic tetracalcium phosphate (TTCP, Ca(4)(PO(4))(2)O), also known by the mineral name hilgenstockite, is formed in the (CaO-P(2)O(5)) system at temperatures>1300 degrees C. TTCP is the only calcium phosphate with a Ca/P ratio greater than hydroxyapatite (HA). It appears as a by-product in plasma-sprayed HA coatings and shows moderate reactivity and concurrent solubility when combined with acidic calcium phosphates such as dicalcium phosphate anhydrous (DCPA, monetite) or dicalcium phosphate dihydrate (DCPD, brushite). Therefore it is widely used in self-setting calcium phosphate bone cements, which form HA under physiological conditions. This paper aims to review the synthesis and properties of TTCP in biomaterials applications such as cements, sintered ceramics and coatings on implant metals.

Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation.

Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 x 10(5) cells ml(-1) in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.

Loading and release of doxycycline hyclate from strontium-substituted calcium phosphate cement.

Novel Sr-substituted calcium phosphate cement (CPC) loaded with doxycycline hyclate (DOXY-h) was employed to elucidate the effect of strontium substitution on antibiotic delivery. The cement was prepared using as reactants Sr-substituted beta-tricalcium phosphate (Sr-beta-TCP) and acidic monocalcium phosphate monohydrate. Two different methods were used to load DOXY-h: (i) the adsorption on CPC by incubating the set cement in drug-containing solutions; and (ii) the use of antibiotic solution as the cement liquid phase. The results revealed that the Sr-substituted cement efficiently adsorbs the antibiotic, which is attributed to an enhanced accessibility to the drug-binding sites within this CPC. DOXY-h desorption is influenced by the initial adsorbed amount and the cement matrix type. Furthermore, the fraction of drug released from CPCs set with DOXY-h solution was higher, and the release rate was faster for the CPC prepared with 26.7% Sr-beta-TCP. The analysis of releasing profiles points to Fickian diffusion as the mechanism responsible for antibiotic delivery. We can conclude that Sr substitution in secondary calcium phosphate cements improves their efficiency for DOXY-h adsorption and release. The antibiotic loading method provides a way to switch from rapid and complete to slower and prolonged drug release.

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.

The effect of hyaluronic acid on brushite cement cohesion.

The improvement of calcium phosphate cement (CPC) cohesion is essential for its application in highly blood perfused regions. This study reports the effectiveness of hyaluronic acids of different molecular weights in the enhancement of brushite cement cohesion. The cement was prepared using a powder phase composed of a mixture of beta-tricalcium phosphate and monocalcium phosphate monohydrate, whereas the liquid phase was formed by 0.5M citric acid solution modified by the addition of hyaluronic acid of different molecular weights. It was found that medium and high molecular weight hyaluronic acid enhances the cement cohesion and scarcely affects the cement mechanical properties. However, concentrations >0.5% (w/v) were less efficient to prevent the cement disintegration. It is concluded that hyaluronic acid could be applied efficiently to reduce brushite cement disintegration.

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.

Cytocompatibility of brushite and monetite cell culture scaffolds made by three-dimensional powder printing.

This study investigated the cytocompatibility of low-temperature direct 3-D printed calcium phosphate scaffolds in vitro. The fabrication of the scaffolds was performed with a commercial 3-D powder printing system. Diluted phosphoric acid was printed into tricalcium phosphate powder, leading to the formation of dicalcium phosphate dihydrate (brushite). Hydrothermal conversion of the brushite matrices led to the formation of dicalcium phosphate anhydrous (monetite). The biocompatibility was investigated using the osteoblastic cell line MC3T3-E1. Cell viability and the expression of alkaline phosphatase served as parameters. The culture medium was analyzed for pH value, concentration of free calcium and phosphate ions and osteocalcin. Both types of scaffolds showed a considerable increase of cell proliferation and viability; the monetite matrices were a little inferior compared with the brushite ones. The activity of alkaline phosphatase showed a similar pattern. Optical and electron microscopy revealed an obvious cell growth on the surface of both materials. Analysis of the culture medium showed minor alterations of pH value within the physiological range. The concentrations of free calcium and phosphate ions were obviously different among brushite and monetite cultures, due to their different solubility. The content of osteocalcin of the culture medium was reduced by the printed scaffolds due to adsorption. We conclude that the powder printed brushite and monetite matrices have a suitable biocompatibility for their use as cell culture scaffolds. Both materials enable osteoblastic cells in vitro to proliferate and differentiate due to the expression of typical osteoblastic markers.