The stability mechanisms of an injectable calcium phosphate ceramic suspension

Calcium phosphate ceramics are widely used as bone substitutes in dentistry and orthopedic applications. For minimally invasive surgery an injectable calcium phosphate ceramic suspension (ICPCS) was developed. It consists in a biopolymer (hydroxypropylmethylcellulose: HPMC) as matrix and bioactive calcium phosphate ceramics (biphasic calcium phosphate: BCP) as fillers. The stability of the suspension is essential to this generation of “ready to use” injectable biomaterial. But, during storage, the particles settle down. The engineering sciences have long been interested in models describing the settling (or sedimentation) of particles in viscous fluids. Our work is dedicated to the comprehension of the effect of the formulation on the stability of calcium phosphate suspension before and after steam sterilization. The rheological characterization revealed the macromolecular behavior of the suspending medium. The investigations of settling kinetics showed the influence of the BCP particle size and the HPMC concentration on the settling velocity and sediment compactness before and after sterilization. To decrease the sedimentation process, the granule size has to be smaller and the polymer concentration has to increase. A much lower sedimentation velocity, as compared to Stokes law, is observed and interpreted in terms of interactions between the polymer network in solution and the particles. This experimentation highlights the granules spacer property of hydrophilic macromolecules that is a key issue for interconnection control, one of the better ways to improve osteoconduction and bioactivity.     

Premixed macroporous calcium phosphate cement scaffold

Calcium phosphate cement (CPC) sets in situ to form resorbable hydroxyapatite and is promising for orthopaedic applications. However, it requires on-site powder-liquid mixing during surgery, which prolongs surgical time and raises concerns of inhomogeneous mixing. The objective of this study was to develop a premixed CPC scaffold with macropores suitable for tissue ingrowth. To avoid the on-site powder-liquid mixing, the CPC paste was mixed in advance and did not set in storage; it set only after placement in a physiological solution. Using 30% and 40% mass fractions of mannitol porogen, the premixed CPC scaffold with fibers had flexural strength (mean ± sd; n = 5) of (3.9 ± 1.4) MPa and (1.8 ± 0.8) MPa, respectively. The scaffold porosity reached (68.6 ± 0.7)% and (74.7 ± 1.2)%, respectively. Osteoblast cells colonized in the surface macropores of the scaffold and attached to the hydroxyapatite crystals. Cell viability values for the premixed CPC scaffold was not significantly different from that of a conventional non-premixed CPC known to be biocompatible (P > 0.1). In conclusion, using fast-dissolving porogen and slow-dissolving fibers, a premixed macroporous CPC scaffold was developed with strength approaching the reported strengths of sintered porous hydroxyapatite implants and cancellous bone, and non-cytotoxicity similar to a biocompatible non-premixed CPC. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States.     

可注射磷酸钙骨水泥及其应用

可注射磷酸钙骨水泥作为一种新型人工骨替代材料,以其良好的生物相容性和骨传导性被广泛应用于临床骨缺损和牙缺损的修复.本文介绍了可注射磷酸钙骨水泥的种类和特性,指出了存在的问题和应用前景.     

一种可注射可降解磷酸钙骨水泥的结构与性能

通过采用部分结晶磷酸钙和磷酸氢钙制备了新型可注射可降解磷酸钙骨水泥.研究表明:该材料具备优良的可注射性能,并通过添加变性淀粉,显著改善了材料的抗溃散性能,骨水泥的水化产物是直径和长度在分别约为100和1000nm左右的棒状类骨羟基磷灰石.所研制的骨水泥在体温(37℃)条件下凝结较快,而在室温(25℃)和冷藏温度(5℃)可在较长时间保持不固化,这就为骨水泥的临床应用提供了很有利的条件.体外溶血试验、体外细胞毒试验、热原性试验、小鼠的急性毒性试验、微核试验、豚鼠的致敏性试验、小鼠的肌内埋植试验及兔的骨内埋植试验等一系列毒性及生物相容性试验表明该材料无毒副作用,具有良好的生物相容性.复合rhBMP-2的可注射磷酸钙骨水泥植入猕猴椎体后的近远期影像学和组织学观察表明,骨水泥可降解且降解和新骨长入基本同步.     

Study on injectable and degradable cement of calcium sulphate and calcium phosphate for bone repair

Injectable calcium sulphate/phosphate cement (CSPC) with degradable characteristic was developed by introduction of calcium sulphate (CS) into calcium phosphate cement (CPC). The setting time, compressive strength, composition, degradation, cells and tissue responses to the CSPC were investigated. The results show that the injectable CSPC with optimum L/P ratio exhibited good injectability, and had suitable setting time and mechanical properties. Furthermore, the CSPC had good degradability and its degradation significantly faster than that of CPC in Tris–HCl solution. Cell culture results indicate that CSPC was biocompatible and could support MG63 cell attachment and proliferation. To investigate the in vivo biocompatibility and osteogenesis, the CSPC were implanted in the bone defects of rabbits. Histological evaluation shows that the introduction of CS into CPC enhanced the efficiency of new bone formation, and CSPC exhibited good biocompatibility, degradability and osteoconductivity with host bone in vivo. It can be concluded that the injectable CSPC had a significant clinical advantage over CPC, and might have potential to be applied in orthopedic, reconstructive and maxillofacial surgery, especially for minimally invasive techniques.     

Development of a fully injectable calcium phosphate cement for orthopedic and dental applications

A study on the development of a fully injectable calcium phosphate cement for orthopedic and dental applications is presented. The paper describes its characteristic properties including results of biocompatibility studies. A conventional two-component calcium phosphate cement formulation (having a powder part containing dry mixture of acidic and basic calcium phosphate particles and a liquid part containing phosphate solution) is modified with a biocompatible gelling agent, to induce flow properties and cohesion. The quantity of the gelling agent is optimized to get a viscous paste, which is smoothly injectable through an 18-gauge needle, with clinically relevant setting parameters. The new formulation has a setting time of 20 min and a compressive strength of 11 MPa. The X-ray diffraction, Fourier transform infrared spectrometry, and energy dispersive electron microprobe analyses showed the phase to be hydroxyapatite, the basic bone mineral. Scanning electron microscopy revealed a porous structure with particle sizes of a few micrometers. The cement did not show any appreciable dimensional or thermal change during setting. The injectability is estimated by extruding through needle and the cohesive property is assessed by water contact method. The cement passed the in vitro biocompatibility screening (cytotoxicity and haemolysis) tests.     

Optimisation of the mechanical and handling properties of an injectable calcium phosphate cement

Calcium phosphate cements have the potential to be successful in minimally invasive surgical techniques, like that of vertebroplasty, due to their ability to be injected into a specific bone cavity. These bone cements set to produce a material similar to that of the natural mineral component in bone. Due to the ceramic nature of these materials they are highly brittle and it has been found that they are difficult to inject. This study was carried out to determine the factors that have the greatest effect on the mechanical and handling properties of an apatitic calcium phosphate cement with the use of a Design of Experiments (DoE) approach. The properties of the cement were predominantly influenced by the liquid:powder ratio and weight percent of di-sodium hydrogen phosphate within the liquid phase. An optimum cement composition was hypothesised and tested. The mechanical properties of the optimised cement were within the clinical range for vertebroplasty, however, the handling properties still require improvement.     

Structural stability of calcium phosphate cement during aging in water

A calcium phosphate cement (CPC) has been prepared by mixing dicalcium phosphate anhydrate (DCPA, CaHPO₄) and calcium hydroxide (Ca(OH)₂) with a sodium phosphate (Na₂HPO₄) solution. After setting and hardening, the cement is aged in water. High resolution structural and microstructure analyses are carried out to evaluate the stability of the CPC in water over a period of 150 days. The lattice parameters of the apatite crystal remain the same throughout the aging process. The size of apatite crystallites is not changed either; nevertheless, the shape of the particles changes from equiaxed to rod-like.     

Preparation and characterization of injectable calcium phosphate cement paste modified by polyethylene glycol-6000

An ICPC with high structure recoverability and paste stability was successfully developed directly incorporating PEG-6000 into the liquid phase of CPC. The rheological behavior of ICPC was investigated with rheometric scientific ARES902-30004 controlled strain rheometer. Novel approaches of flow rate, shear thinning index (SI), shear stress slowdown (Δτ) and thixotropy loop area have been applied to assess the injectability and structure recoverability of the ICPC paste. The addition of PEG-6000 to ICPC resulted in a thixotrophic structure with shortened setting time, slightly increased viscosity, larger thixotropic hysteresis loop area and lower Δτ, with the improvement largely dependent on the PEG-6000 content. With acceptable injectability and shortened setting time, ICPC (1%) showed the lowest Δτ and the highest SI, endowing the paste good structure recoverability and paste stability. The ICPC (1%) was bioactive and facilitated cell attachment and proliferation. The optimized ICPC (1%) paste with a relatively good structure stability and paste stability may serve as a good candidate for tooth root-canal fillings and percutaneous vertebroplasty in microinvasive surgery.     

Addition of sodium hyaluronate and the effect on performance of the injectable calcium phosphate cement

An injectable calcium phosphate cement (CPC) with porous structure and excellent anti-washout ability was developed in the study. Citric acid and sodium bicarbonate were added into the CPC powder consisting of tetracalcium phosphate (TTCP) and dicalcium phosphate dihydrate (DCPD) to form macro-pores, then different concentrations of sodium hyaluronate (NaHA) solution, as liquid phase, was added into the cement to investigate its effect on CPC’s performance. The prepared CPCs were tested on workability (injectable time and setting time), mechanical strength, as well as anti-washout ability. The experimental results showed that addition of NaHA not only enhanced the anti-washout ability of the CPC dramatically but also improve its other properties. When NaHA concentration was 0.6 wt%, the injectable time elongated to 15.7 ± 0.6 min, the initial and final setting times were respectively shorten to 18.3 ± 1.2 and 58.7 ± 2.1 min, and the compressive strength were increased to 18.78 ± 1.83 MPa. On the other hand, Addition of NaHA showed little effect on porous structure of the CPC and enhanced its bioactivity obviously, which was confirmed by the apatite formation on its surface after immersion in simulated body fluid (SBF). In conclusion, as an in situ shaped injectable biomaterials, the CPC with appropriate addition of NaHA would notably improve its performance and might be used in minimal invasive surgery for bone repair or reconstruction.     

Production of in-situ macropores in an injectable calcium phosphate cement by introduction of cetyltrimethyl ammonium bromide

In the present study, cetyltrimethyl ammonium bromide (CTAB) was introduced to an injectable calcium phosphate cement (CPC) to produce macropores during the setting process to accelerate the absorbing ability in vivo. The effects of CTAB on the rheological properties, injectability, setting time, compressive strength, phase evolution, microstructure and degradation rate of CPC were studied. The results showed that the addition of CTAB increased the viscosity and yield stress, and decreased the injectability of the cement pastes. The macroporosity and total porosity increased and the compressive strength of the cement obviously decreased with the increase of CTAB. The macroporosity of the CPC prepared at 5 mM CTAB solution reached 44.2 +/- 2.5% and the mass loss of the cement increased almost 50% as compared with the cement without CTAB. Considering the injectability, compressive strength and degradation rate of CPC, the preferred CTAB concentration was 5 mM. The injectable CPC with macropores is promising to be used in minimally invasive approach.     

Controllable release of salmon-calcitonin in injectable calcium phosphate cement modified by chitosan oligosaccharide and collagen polypeptide

The aim of this research is to study the effect of the controlled releasing character of the salmon calcitonin (S-CT) loaded injectable calcium phosphate cement (CPC) modified by adding organic phase, chitosan oligosaccharide (CO) and collagen polypeptide (CP). The uniform design was used to determine the basic formulation with suitable injectable time for clinical application, and then the changes of the physical characters, the controlled releasing character of the modified CPC along with the ratio of the organic phase were also evaluated in vitro. The surface morphous of the modified CPC been implanted in the abdominal cavity or soaked into the serum of rat was also observed by scanning electron microscope (SEM). The result shows that a suitable formulation of modified CPC could be got, and the injectable time is 12 min, the compressive strength is 12 MPa, and the final setting time is 40 min. Comparing with the CPC without organic phase, the releasing rate of S-CT would increase along with the increase of the organic phase after 7th day. Therefore, a novel S-CT loaded bioactive injectable CPC for treating osteoporosis induced bone defect was obtained, and the release of the containing S-CT was controlled easily through adjusting the ratio of CO and CP.     

Development of a novel calcium phosphate cement composed mainly of calcium sodium phosphate with high osteoconductivity

Two novel calcium phosphate cements (CPC) have been developed using calcium sodium phosphate (CSP) as the main ingredient. The first of these cements, labeled CAC, contained CSP, α-tricalcium phosphate (TCP), and anhydrous citric acid, whereas the second, labeled CABC, contained CSP, α-TCP, β-TCP, and anhydrous citric acid. Biopex^®-R (PENTAX, Tokyo, Japan), which is a commercially available CPC (Com-CPC), and OSferion^® (Olympus Terumo Biomaterials Corp., Tokyo, Japan), which is a commercially available porous β-TCP, were used as reference controls for analysis. In vitro analysis showed that CABC set in 5.7 ± 0.3 min at 22 °C and had a compressive strength of 86.0 ± 9.7 MPa after 5 days. Furthermore, this material had a compressive strength of 26.7 ± 3.7 MPa after 2 h in physiologic saline. CAC showed a statistically significantly lower compressive strength in the presence of physiologic saline and statistically significantly longer setting times than those of CABC. CABC and CAC exhibited apatite-forming abilities in simulated body fluid that were faster than that of Com-CPC. Samples of the materials were implanted into the femoral condyles of rabbits for in vivo analysis, and subsequent histological examinations revealed that CABC exhibited superior osteoconductivity and equivalent bioresorbability compared with Com-CPC, as well as superior osteoconductivity and bioresorbability compared with CAC. CABC could therefore be used as an alternative bone substitute material.     

Air-foamed calcium aluminate phosphate cement for geothermal wells

Air-foamed low-density calcium aluminate phosphate (CaP) cement slurry was prepared by mixing it with chemical foaming reagent at room temperature without any pressure, followed by autoclaving at 200 °C. When the porosity, compressive strength, and water permeability of the autoclaved CaP foam cement made from a 1.22 g/cc slurry density was compared with those of N₂ gas-foamed Class G cement made from a slurry of similar density under high pressure and hydrothermal temperature at 288 °C, the CaP cement revealed some advanced properties, such as a higher compressive strength and lower porosity. These advanced properties were due to the hybrid formation of three crystalline hydrothermal reaction products; hydroxyapatite, boehmite, and hydrogarnet phases. However, one shortcoming was an increase in water permeability because of the formation of an undesirable continuous porous structure caused by coalesced air bubble cells, suggesting that an appropriate lesser amount of foaming reagent be used to create a conformation in which fine discrete air-bubble cells are uniformly dispersed throughout the slurry. For non-foamed cement, three major factors contributed to protecting carbon steel against corrosion: (1) good adherence to steel, reflecting a high extent of coverage by the cement layer over the steel’s surfaces; (2) retardation of cathodic corrosion reactions; and, (3) minimum conductivity of corrosive ionic electrolytes. However, adding an excessive amount of foaming reagent did not offer as effective corrosion protection as that of non-foamed cement.     

Non-decay type fast-setting calcium phosphate cement using chitosan

A non-decay type fast-setting calcium phosphate cement (nd-FSCPC) has been described, which did not decay but set within approximately 5–6 min even when the paste was immersed in serum immediately after mixing, and which forms hydroxyapatite as its end product. nd-FSCPC was produced by adding sodium alginate to the liquid phase of the base cement FSCPC. Sodium alginate forms a water-insoluble gel, and reduces the process of fluid penetration into the paste which is the cause of decay. The aim of this investigation was to confirm the mechanism of the non-decaying behaviour of nd-FSCPC proposed in a previous paper, using another chemical with properties similar to those of sodium alginate. Also, it was intended to further improve both the mechanical properties and tissue response of nd-FSCPC. Chitosan, which also forms a water-insoluble gel in the presence of calcium ions and has been reported to have pharmacologically beneficial effects on osteoconductivity, was added to the liquid phase of the base cement FSCPC. The cement thus prepared showed behaviour similar to that of nd-FSCPC using sodium alginate. The cement paste did not decay but set within approximately 5–6 min even when immersed in serum immediately after mixing. DTS value of the set mass was approximately 3–4 MPa, slightly lower than that of nd-FSCPC using sodium alginate, and no inhibitory effect was observed for the transformation of cement component to apatite within the range used in this investigation (up to 1.5%). Therefore, it was concluded that the mechanism of non-decaying behaviour was, at least in part, reduction of fluid penetration into the cement paste. nd-FSCPC using chitosan showed slightly poorer mechanical properties than that using sodium alginate. However, pharmacological effects such as osteoconductivity could be expected in nd-FSCPC using chitosan. Thus, this cement may be useful as a more sophisticated bioactive cement than nd-FSCPC using sodium alginate.     

Formation of macropores in calcium phosphate cement implants

A calcium phosphate cement (CPC) was shown to harden at ambient temperatures and form hydroxyapatite as the only end-product. Animal study results showed that CPC resorbed slowly and was replaced by new bone. For some clinical applications, it would be desirable to have macropores built into the CPC implant to obtain a more rapid resorption and concomitant osseointegration of the implant. The present study investigated the feasibility of a new method for producing macropores in CPC. Sucrose granules, NaHCO₃, and Na₂HPO₄ were sieved to obtain particle sizes in the range of 125 m to 250 m. The following mixtures of CPC powder (an equimolar mixture of tetracalcium phosphate, Ca₄(PO_{4 2}O, and dicalcium phosphate anhydrous, CaHPO₄) and one of the above additive granules were prepared: control–no additive; mixture A–0.25 mass fraction of sucrose; mixture B–0.25 mass fraction of NaHCO₃; mixture C–0.25 mass fraction of Na₂HPO₄, and mixture D–0.33 mass fraction of Na₂HPO₄. Cement samples were prepared by mixing 0.3 g of the above mixtures with 0.075 ml of the cement liquid (1 mol/l Na₂HPO₄). After hardening, the specimens were placed in water for 20 h at about 60 °C to completely dissolve the additive crystals. Well-formed macropores in the shapes of the entrapped crystals were observed by scanning electron microscope (SEM). The macroporosities (mean±standard deviation; n = 6) expressed as volume fraction in % were 0, 18.9 ± 1.7, 26.9 ± 1.6, 38.3 ± 4.4 and 50.3 ± 2.7 for the control, A, B, C and D, respectively. The diametral tensile strengths (mean±standard deviation; n = 3) expressed in MPa were 10.1 ± 0.7, 3.7 ± 0.3, 2.4 ± 0.2, 1.5 ± 0.5 and 0.4 ± 0.1, respectively, for the five groups. The results showed that macropores can readily be formed in CPC implants with the use of water-soluble crystals. The mechanical strength of CPC decreased with increasing macroporosity. © 2001 Kluwer Academic Publishers     

In vitro aging of a calcium phosphate cement

Cement samples made of -tricalcium phoshate (-TCP), phosphoric acid (PA) and water mixtures were incubated in several aqueous solutions to determine their stability over time. The effects of the cement composition and the incubating temperature were investigated in more detail. The cement samples contained mostly dicalcium phosphate dihydrate (DCPD) and remnants of -TCP crystals. Depending on the initial cement composition, a certain amount of dicalcium phosphate (DCP) crystals were formed. The larger the initial PA concentration, the larger the DCP amount. After setting, the cement composition was stable for at least 16 days up to 60 °C. Above that temperature, the DCPD crystals decomposed into DCP crystals. The latter reaction provoked a decrease of the pH of the incubation solution, phenomenon expected for a cement sample containing an excess of PA. As the cement samples contained an excess of -TCP, it was postulated that -TCP crystals became so covered by DCP or DCPD crystals during setting that the setting reaction was stopped prematurely. The latter phenomenon gave a good explanation for the low pH values measured in the incubation solutions. ©©2000 Kluwer Academic Publishers     

Fabrication of calcium phosphate–calcium sulfate injectable bone substitute using hydroxy-propyl-methyl-cellulose and citric acid

In this study, an injectable bone substitute (IBS) consisting of citric acid, chitosan, and hydroxyl propyl methyl cellulose (HPMC) as the liquid phase and tetra calcium phosphate (TTCP), dicalcium phosphate dihydrate (DCPD) and calcium sulfate dehydrate (CSD, CaSO₄·2H₂O) powders as the solid phase, were fabricated. Two groups were classified based on the percent of citric acid in the liquid phase (20, 40 wt%). In each groups, the HPMC percentage was 0, 2, and 4 wt%. An increase in compressive strength due to changes in morphology was confirmed by scanning electron microscopy images. A good conversion rate of HAp at 20% citric acid was observed in the XRD profiles. In addition, HPMC was not obviously affected by apatite formation. However, both HPMC and citric acid increased the compressive strength of IBS. The maximum compressive strength for IBS was with 40% citric acid and 4% HPMC after 14 days of incubation in 100% humidity at 37°C.     

Reinforcing of a calcium phosphate cement with hydroxyapatite crystals of various morphologies

A series of biocomposite materials was successfully prepared by reinforcing advanced calcium phosphate cement with hydroxyapatite fibrous and elongated plate-like particles. Powder X-ray diffraction showed that ball-milled biocomposite precursors (dicalcium and tetracalcium phosphates) entirely transform to a single phase hydroxyapatite end product within 7 h at 37 °C. Electron microscopy showed that the resultant biocomposites are constituted of nanoscaled cement particles intimately associated with the reinforcement crystals. The influence of shape, size, and concentration of the hydroxyapatite filler on the compression strength of reinforced cements is discussed. The best compression strength of 37 ± 3 MPa (enhancement of ～50% compared to pure cement) was achieved using submicrometer-sized hydroxyapatite crystals with complementary shapes. Nanoindentation revealed that averaged elastic modulus and hardness values of the cements are consistent with those reported for trabecular and cortical human bones, indicating a good match of the micromechanical properties for their potential use for bone repair. The stiffness of the biocomposites was confirmed to gradate-compliant cement matrix, cement-filler interface, and stiff filler-as a result of the structuring at the nanometer-micrometer level. This architecture is critical in conditioning the final mechanical properties of the functional composite biomaterial. In vitro cell culture experiments showed that the developed biomaterial system is noncytotoxic.     

Crystallization mechanisms of calcium phosphate cement for biological uses

Self-setting calcium phosphate cement for dental or surgical applications can be prepared by the addition of a liquid to a mixture of acidic and basic calcium phosphate. After hardening, the final compound becomes hydroxyapatite. Using an orthogonal central composite plan, the main factors which control the setting and the final hardness of the cement were defined and models are proposed. The mechanisms of crystallization, the role of free and linked water, and the nature of the final and intermediate compounds are described.This paper was accepted for publication after the 1995 Conference of the European Society of Biomaterials, Oporto, Portugal, 10–13 September.