Effect of calcium carbonate on hardening, physicochemical properties, and in vitro degradation of injectable calcium phosphate cements

The main disadvantage of apatitic calcium phosphate cements (CPCs) is their slow degradation rate, which limits complete bone regeneration. Carbonate (CO?2?) is the common constituent of bone and it can be used to improve the degradability of the apatitic calcium phosphate ceramics. This study aimed to examine the effect of calcite (CaCO?) incorporation into CPCs. To this end, the CaCO? amount (0-4-8-12 wt %) and its particle size (12.0-μm-coarse or 2.5-μm-fine) were systematically investigated. In comparison to calcite-free CPC, the setting time of the bone substitute was delayed with increasing CaCO? incorporation. Reduction of the CaCO? particle size in the initial powder increased the injectability time of the paste. During hardening of the cements, the increase in calcium release was inversely proportional to the extent of CO?2? incorporation into apatites. The morphology of the carbonate-free product consisted of large needle-like crystals, whereas small plate-like crystals were observed for carbonated apatites. Compressive strength decreased with increasing CaCO? content. In vitro accelerated degradation tests demonstrated that calcium release and dissolution rate from the set cements increased with increasing the incorporation of CO?2?, whereas differences in CaCO? particle size did not affect the in vitro degradation rate under accelerated conditions.     

Conversion of octacalcium phosphate in calcium phosphate cements

This study investigated the in vitro conversion reaction in calcium phosphate cements (CPCs) containing octacalcium phosphate (OCP) as one of the reagents. OCP is known to be a precursor for apatite formation in vivo. The reaction products were characterized using infrared spectroscopy and X-ray diffraction. Although the conversion of OCP into hydroxyapatite is thermodynamically favorable, OCP only yields apatite formation in CPC provided it is combined with a highly soluble Ca(2+) and OH(-) releasing reaction partner. In this respect, tetracalcium phosphate is a promising compound. Adding small amounts of monocalcium phosphate monohydrate can stimulate the setting through intermediate brushite formation. The preparation method of OCP might drastically affect the performance of the cement. The reaction path of the setting of these CPC probably does not conform to the singular point principle described in the literature, and an in situ hydrolysis of OCP to apatite is conceivable. Simulation of apatite formation using OCP as the precursor and/or seed in CPC might be beneficial for some biomedical applications.     

Characterization of calcium phosphate cements modified by addition of amorphous calcium phosphate

In this study the influence of amorphous calcium phosphate (ACP) on the setting of, and the formed apatite crystallite size in, a calcium phosphate cement (CPC) based on α-tricalcium phosphate (α-TCP) or tetracalcium phosphate (TTCP)/monocalcium phosphate monohydrate (MCPM) was investigated. Setting times at 22 °C were measured in air atmosphere; those at 37 °C were measured at 100% relative humidity. The phase composition of the set cements was investigated after 1 week using X-ray diffractometry and infrared spectroscopy and the morphology was investigated using scanning electron microscopy. The compressive strength (CS) of the set CPCs was measured after 1 day. Viability of MC3T3-E1 cells on the CPCs was analyzed after 7, 14 and 21 days of incubation using the CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation Assay. The α-TCP-based cement exhibited long setting times, a high CS and was converted to a calcium-deficient hydroxyapatite (CDHAp). The TTCP/MCPM-based CPC was only partly converted to CDHAp, produced acceptable setting times and had a low CS. Addition of ACP to these two CPCs resulted in cements that exhibited good setting times, CS suitable for non-load-bearing applications and a full conversion to nanocrystalline CDHAp. Moreover, the ACP containing CPCs demonstrated good cell viability, making them suitable candidates for bone substitute materials.     

Frozen delivery of brushite calcium phosphate cements

Calcium phosphate cements typically harden following the combination of a calcium phosphate powder component with an aqueous solution to form a matrix consisting of hydroxyapatite or brushite. The mixing process can be very important to the mechanical properties exhibited by cement materials and consequently when used clinically, since they are usually hand-mixed their mechanical properties are prone to operator-induced variability. It is possible to reduce this variability by pre-mixing the cement, e.g. by replacing the aqueous liquid component with non-reactive glycerol. Here, for the first time, we report the formation of three different pre-mixed brushite cement formulations formed by freezing the cement pastes following combination of the powder and liquid components. When frozen and stored at -80 degrees C or less, significant degradation in compression strength did not occur for the duration of the study (28 days). Interestingly, in the case of the brushite cement formed from the combination of beta-tricalcium phosphate with 2 M orthophosphoric acid solution, freezing the cement paste had the effect of increasing mean compressive strength fivefold (from 4 to 20 MPa). The increase in compression strength was accompanied by a reduction in the setting rate of the cement. As no differences in porosity or degree of reaction were observed, strength improvement was attributed to a modification of crystal morphology and a reduction in damage caused to the cement matrix during manipulation.     

Physico-chemical-mechanical and in vitro biological properties of calcium phosphate cements with doped amorphous calcium phosphates

Calcium phosphate cements (CPCs) are successfully used as bone substitutes in dentistry and orthopaedic applications. This study investigated the physico-chemical-mechanical properties of and in vitro biological properties (cell response) of CPCs prepared with amorphous calcium carbonate phosphate (ACCP) doped with magnesium (ACCP-Mg), zinc (ACCp-Zn) or fluoride (ACCP-F) ions. The experimental CPC consisted of alpha-TCP, doped ACCP, and MPCM powders as matrix and biphasic calcium phosphate (BCP) granules. X-ray diffraction analysis showed that the matrix converted to apatite with poor crystallinity (reflecting small crystal size) after setting for 24 h, while BCP remained apparently unchanged. Cements with ACCP-F (F-CPC) had shorter setting times and greater compressive strength compared to cements with ACCP-Mg (Mg-CPC) or ACCP-Zn (Zn-CPC). Scanning electron microscopy (SEM) showed that crystals set on Mg-CPC and Zn-CPC were smaller compared to those on F-CPC. The total porosity of Mg-CPC was greater compared to Zn-CPC or F-CPC. Osteoblast-like cells, MC3T3-E1, remained viable and maintained their ability to express alkaline phosphatase in contact with the CPCs with doped ACCPs.     

Fibre-reinforced calcium phosphate cements: a review

Calcium phosphate cements (CPC) consist of one or more calcium orthophosphate powders, which upon mixing with water or an aqueous solution, form a paste that is able to set and harden after being implanted within the body. Different issues remain still to be improved in CPC, such as their mechanical properties to more closely mimic those of natural bone, or their macroporosity to favour osteointegration of the artificial grafts. To this end, blends of CPC with polymer and ceramic fibres in different forms have been investigated. The present work aims at providing an overview of the different approaches taken and identifying the most significant achievements in the field of fibre-reinforced calcium phosphate cements for clinical applications, with special focus on their mechanical properties.     

Soft-tissue response to injectable calcium phosphate cements

In this study, the soft tissue reaction to two newly developed injectable calcium phosphate bone cements (cement D and W) was evaluated after implantation in the back of goats. For one of the cements (cement D) the tissue reaction was also investigated after varying the concentration of accelerator Na(2)HPO(4) in the cement liquid (resulting in cement D1 and D2). Eight healthy mature female Saanen goats were used. The cement was applied 10min after mixing while it was still moldable and plastic. The material was given a standardized cylindrical shape. Thirty-two implants of each cement formulation were inserted and left in place for 1, 2, 4, and 8weeks. At the end of the study, eight specimens of each material and healing period were available for further analysis. Two specimens were used for X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) and six specimens were used for light microscopical evaluation. XRD and FTIR showed that the cements did set as microcrystalline carbonate apatite with the disappearance of monetite from the cements during implantation. Histological analysis showed that after 8weeks of implantation around all materials a thin soft-tissue capsule was formed (thickness ranging from 5 to 15 cell layers) with almost complete absence of inflammatory cells. Only in some specimens a slightly higher inflammatory reaction was observed. This was due to cement surface defects and a zone of dispersed particles near the cement-soft tissue interface. There was almost no resorption of the material after 8 weeks of implantation. In a few 4 and 8weeks samples, small areas of calcification were found in the fibrous capsule surrounding the implants. On the basis of our observations, we conclude that the tested cements were biocompatible and can be used next to soft tissue.     

Addition of cohesion promotors to calcium phosphate cements

Many calcium phosphate cements (CPC) pastes tend to disintegrate upon early contact with blood or other aqueous (body) fluids, which inhibits the use of these materials for clinical use as for bone repair, reconstruction and augmentation. In studies on CPCs based on tetracalcium phosphate and dicalcium hydrogen phosphate others have suggested to use sodium alginate, cellulose derivatives or chitosan derivatives dissolved in the cement liquid for improving the cohesion of CPC pastes. In this study 10 other organic compounds were shown to act as cohesion promotors in the case of CPCs based on alpha-tertiary calcium phosphate as the main active ingredient.     

Injectable PLGA microsphere/calcium phosphate cements: physical properties and degradation characteristics

Calcium phosphate (CaP) cements show an excellent biocompatibility and often have a high mechanical strength, but in general degrade relatively slow. To increase degradation rates, macropores can be introduced into the cement, e.g., by the inclusion of biodegradable microspheres into the cement. The aim of this research is to develop an injectable PLGA microsphere/CaP cement with sufficient setting/cohesive properties and good mechanical and physical properties. PLGA microspheres were prepared using a water-in-oil-in-water double-emulsion technique. The CaP-cement used was Calcibon, a commercially available hydroxyapatite-based cement. 10:90 and 20:80 dry wt% PLGA microsphere/CaP cylindrical scaffolds were prepared as well as microporous cement (reference material). Injectability, setting time, cohesive properties and porosity were determined. Also, a 12-week degradation study in PBS (37 degree C) was performed. Results showed that injectability decreased with an increase in PLGA microsphere content. Initial and final setting time of the PLGA/CaP samples was higher than the microporous sample. Porosity of the different formulations was 40.8% (microporous), 60.2% (10:90) and 69.3% (20:80). The degradation study showed distinct mass loss and a pH decrease of the surrounding medium starting from week 6 with the 10:90 and 20:80 formulations, indicating PLGA erosion. Compression strength of the PLGA microsphere/CaP samples decreased siginificantly in time, the microporous sample remained constant. After 12 weeks both PLGA/CaP samples showed a structure of spherical micropores and had a compressive strength of 12.2 MPa (10:90) and 4.3 MPa (20:80). Signs of cement degradation were also found with the 20:80 formulation. In conclusion, all physical parameters were well within workable ranges with both 10:90 and 20:80 PLGA microsphere/CaP cements. After 12 weeks the PLGA was totally degraded and a highly porous, but strong scaffold remained.     

Effect of the calcium to phosphate ratio of tetracalcium phosphate on the properties of calcium phosphate bone cement

Six different tetracalcium phosphate (TTCP) products were synthesized by solid state reaction at high temperature by varying the overall calcium to phosphate ratio of the synthesis mixture. The objective was to evaluate the effect of the calcium to phosphate ratio on a TTCP-dicalcium phosphate dihydrate (DCPD) cement. The resulting six TTCP-DCPD cement mixtures were characterized using X-ray diffraction analysis, scanning electron microscopy, and pH measurements. Setting times and compressive strength (CS) were also measured. Using the TTCP product with a Ca/P ratio of 2.0 resulted in low strength values (25.61 MPa) when distilled water was used as the setting liquid, even though conversion to hydroxyapatite was not prevented, as confirmed by X-ray diffraction. The suspected CaO presence in this TTCP may have affected the cohesiveness of the cement mixture but not the cement setting reaction, however no direct evidence of CaO presence was found. Lower Ca/P ratio products yielded cements with CS values ranging from 46.7 MPa for Ca/P ratio of 1.90 to 38.32 MPa for Ca/P ratio of 1.85. When a dilute sodium phosphate solution was used as the setting liquid, CS values were 15.3% lower than those obtained with water as the setting liquid. Setting times ranged from 18 to 22 min when water was the cement liquid and from 7 to 8 min when sodium phosphate solution was used, and the calcium to phosphate ratio did not have a marked effect on this property.     

Antimicrobial properties of nanocrystalline tetracalcium phosphate cements

The antimicrobial properties of cements prepared from mechanically activated tetracalcium phosphate (maTTCP) were tested with the agar diffusion test using Streptococcus salivarius, Staphylococcus epidermidis, and a clinically isolated plaque mixture. All maTTCP cements showed a significantly higher antimicrobial potency as revealed by inhibition zones of approximately 3-5 mm width, compared with a commercial Ca(OH)(2)//salicylate cement which only produced small inhibition zones around the cement specimens of 1.5 mm or less. This behavior was explained by the formation of amorphous Ca(OH)(2) during setting of maTTCP cements, which is thought to have a higher solubility and may release more OH(-) ions than conventional Ca(OH)(2)//salicylate cements. In fact, the pH value in the agar gel around the specimens was higher in the case of maTTCP cements (7.8-8.7) compared with the Ca(OH)(2)//salicylate control (7.0-8.0). The maTTCP cements did not affect the photoactivation of resin-based composites, and their antimicrobial activity is making them interesting candidates for the use as pulp-capping agents, endodontic sealers, or cavity liners in dentistry.     

Antimicrobial potency of alkali ion substituted calcium phosphate cements

Potassium and sodium containing nanoapatite cements were produced by the reaction of mechanically activated CaNaPO(4) (CSP), CaKPO(4) (CPP) and Ca(2)KNa(PO(4))(2) (CPCP) with a 2.5% Na(2)HPO(4) solution. The cements exhibited clinically acceptable setting times of approximately 5 min and compressive strengths of 5-10 MPa. The antimicrobial properties of the cements were tested with the agar diffusion test using Streptococcus salvarius, Staphylococcus epidermis and Candida albicans. All types of alkali ion containing cements showed a significantly higher antimicrobial potency with inhibition zones of approx. 4-11 mm than a commercial calcium hydroxide cement which resulted in small inhibition zones around the cement samples of a maximum of 1.5 mm. The antimicrobial properties of all the cements were not found to diminish even after longer incubation times. This behaviour was attributed to the formation of soluble alkaline metal phosphates during setting which increased the pH value in the agar gel around the alkali containing calcium phosphate cement to 8.5-10.7 compared to 6.5-8.0 for the Ca(OH)(2) product. The high antimicrobial potency of alkali-calcium phosphate cements may find an application in dentistry as pulp capping agents, root fillers or cavity liners.     

The effect of ball milling grinding pathways on the bulk and reactivity properties of calcium phosphate cements

Calcium phosphate cements (CPCs) are significant alternatives to autologous bone grafting. CPCs can be composed of biphasic or multiphase calcium phosphate (CaP) compounds. A common way to process CPCs is by ball milling. Ball milling can be used for grinding or mechanosynthesis. The aim of this study was to determine the effect of well-defined ball milling grinding parameters, applied via different milling pathways, on the properties of CPCs. Starting CaP compounds used included α-tricalcium phosphate, dicalcium phosphate anhydrous and precipitated hydroxyapatite. Scanning electron microscopy showed changes in the powder morphology, which were related to the behavior of the starting CaP materials. Specific surface area (SSA) and particle size (PS) measurements exposed the effect of ball milling on the CaP compounds and CPC powders. X-ray diffraction revealed no effect of ball milling pathways or milling time on the composition of CPCs or the starting materials, but affected their crystallographic properties. No contamination of the milling media or transformation into an amorphous calcium phosphate compound was found. The milling pathways affected setting and cohesion. Fourier transform infrared spectroscopy (FTIR) revealed differences on the CPC v?-PO?3? bands according to the interaction, created between the CaP compounds by the milling pathways. FTIR confirmed that the milling pathways changed the crystallographic properties. This study demonstrates that the pathways used for milling grinding modify the PS, SSA, and crystallographic properties of the powders, without affecting their composition. These modifications affected the bulk and reactivity properties of the CPCs by creating different setting and cohesion behaviors.     

A theranostic agent to enhance osteogenic and magnetic resonance imaging properties of calcium phosphate cements

With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies.     

Development of a new calcium phosphate cement that contains sodium calcium phosphate

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.     

The Ca/P range of nanoapatitic calcium phosphate cements

Nanoapatites are apatites consisting of nanometer size crystals. The commercial calcium phosphate cements set by the precipitation of nanoapatitic calcium phosphates in the range 1.5 < or = Ca/P < 1.8. In this study it is shown that a continuum of nanoapatites can precipitate in the range 0.8 < Ca/P< or = 1.5. In order to be formed these nanoapatites need to incorporate K+ ions. In addition they can incorporate some Na+ ions. Upon immersion in aqueous solutions these nanoapatites loose phosphate, K+ and Na+ so that in an open system they are transformed into calcium deficient hydroxyapatite Ca9(HPO4)(PO4)5OH within about 2 months.     

Heparin modification of calcium phosphate bone cements for VEGF functionalization

A promising strategy to promote angiogenesis within an engineered tissue is the local and sustained delivery of an angiogenic factor by the substitute itself. Recently, we reported on functionalization of Biocement D (BioD) and several modifications of this calcium phosphate bone cement with vascular endothelial growth factor (VEGF). Maintenance of biological activity of VEGF after release from the cement was improved by modification of BioD with mineralized collagen type I (BioD/coll). However, BioD/coll composites showed a higher initial burst of VEGF release than do the unmodified BioD. In the present study, VEGF release from BioD/coll composites modified with different amounts of heparin was investigated. We found a distinct reduction of the initial burst of release by adding heparin in a concentration-dependent manner. Moreover, the heparin modification had a positive impact on the biological activity of released VEGF. An advancement of biological properties of BioD/coll by addition of heparin was further shown by improved adhesion of endothelial cells on the cement surface. Characterization of material properties of the heparin-modified BioD/coll composites revealed a finer microstructure with smaller HA-particles and a higher specific surface area than heparin-free BioD/coll. However, higher amounts of heparin resulted in a reduced compressive strength. The rheological properties of these cement pastes have been found to be favorable for good handling particularly with regard to their clinical application.     

Evaluation of calcium phosphates and experimental calcium phosphate bone cements using osteogenic cultures

In this study, rat bone marrow cells (RBM) were used to evaluate two biodegradable calcium phosphate bone cements and bioactive calcium phosphate ceramics. The substances investigated were: two novel calcium phosphate cements, Biocement F and Biocement H, tricalcium phosphate (TCP), surface-modified alpha-tricalcium phosphate [TCP (s)] and a rapid resorbable calcium phosphate ceramic consisting of CaKPO(4) (sample code R5). RBM cells were cultured on disc-shaped test substrates for 14 days. The culture medium was changed daily and also examined for calcium, phosphate, and potassium concentrations. Specimens were evaluated using light microscopy, and morphometry of the cell-covered substrate surface, scanning electron microscopy, and energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. Areas of mineralization were identified by tetracyline labeling. Except for R 5, rat bone-marrow cells attached and grew on all substrate surfaces. Of the different calcium phosphate materials tested, TCP and TCP (s) facilitated osteoblast growth and extracellular matrix elaboration to the highest degree, followed by Biocements H and F. The inhibition of cell growth encountered with R 5 seems to be related to its high phosphate and potassium ion release.     

Effect of added gelatin on the properties of calcium phosphate cement

This study investigates the effect of gelatin on the setting time, compressive strength, phase evolution and microstructure of calcium phosphate cement. The composite cement powder (about 18 wt% gelatin, and 82 wt% alpha-tricalcium phosphate) was prepared from the solid compound obtained by casting a gelatin aqueous solution containing alpha-tricalcium phosphate. 5 wt% of CaHPO(4) x 2H(2)O were added to the powder before mixing with the liquid phase. Two cement formulations were prepared using two different liquid/powder ratios, and their properties compared with those of control samples, prepared without gelatin. The final setting time increases from 10 min to more than 45 min when the L/P ratio increases from 0.3 to 0.4 ml/g. The presence of gelatin accelerates the setting reaction, and improves the mechanical properties of the cements. The compressive strength increases with the setting reaction up to 10.7-14.0 MPa for the gelatin cements, whereas the control samples exhibit much lower values. The improved mechanical properties of the composite cements with respect to the controls can be related to their reduced total porosity and more compact microstructure.     

Effect of substrate temperature on mechanical properties of calcium phosphate coatings

The effect of substrate temperature and processing parameters on mechanical properties of nanoscale calcium phosphate coatings are being studied in order to refine the processing technique for Functionally Graded Hydroxyapatite (FGHA) coatings. Coatings were deposited on titanium substrates with a set substrate temperature of 450, 550, 650, or 750 degrees C in an Ion Beam Assisted Deposition (IBAD) system using a sintered hydroxyapatite (HA) target. Mechanical properties of the coatings deposited with a set substrate temperature such as, bonding/adhesion strength to the substrate, nanohardness, and Young's Modulus as well as coating thickness were evaluated and compared with commercial plasma spray HA coatings. It is concluded that depositing FGHA coatings would better be started at 550-650 degrees C to maintain superior properties of the film at the interface. It can also be concluded that the residual stresses caused by different Coefficient of Thermal Expansions (CTEs) between the substrate and coatings are not the only factor controlling the bonding strength and mechanical properties of these samples. Other parameters such as the nature of the interface layers and their bonding to each other as well as the density and grain structure of the coatings must be taken into consideration for an appropriate evaluation of mechanical properties of calcium phosphate coatings deposited on heated substrate.