The effect of autoclaving on the physical and biological properties of dicalcium phosphate dihydrate bioceramics: Brushite vs. monetite
Introduction
Calcium phosphate biomaterials are of special interest in bone regeneration due to their similar composition to bone. Dicalcium phosphate dihydrate, mineral name brushite, is a calcium phosphate biomaterial that can be prepared in the form of hydraulic cements with a wide range of applications [1]. Besides their ability to regenerate bone, brushite biomaterials can resorb in vivo faster than most calcium phosphates, enabling the replacement of the bioceramic by newly regenerated tissues. However, in vivo studies have shown that even though brushite is initially resorbable after implantation, the bioceramic tends to react with the surrounding medium, forming insoluble hydroxyapatite [1], [2]. This reaction results in a severe reduction in the resorption rate of the biomaterial, limiting its clinical applications.
Monetite is the anhydrous form of brushite, and it is also a useful biomaterial for bone regeneration. Monetite bioceramics can be prepared by modifying the precipitation conditions of brushite cements [2]. For instance, setting brushite cements in excessively low pH conditions, in water-deficient environments, or in the presence of metallic ions would disrupt brushite crystals favouring monetite formation [2], [3], [4], [5]. Another method of preparing monetite bioceramics is by thermal dehydration of already set brushite cements [6]:
Thermal dehydration of brushite bioceramics can cause shrinkage of the material and damage its mechanical properties. However, by maintaining high pressure and humidity during the dehydration process, overall shrinkage can be prevented [7]. Sterilizing pre-set brushite cements by autoclaving provides the adequate temperature, pressure and humidity conditions that result in their dehydration into monetite without altering the overall macroscopic geometry of the material. Monetite bioceramics prepared by this method have been shown to stimulate vertical bone augmentation, and regeneration of bone defects in animals as well as in human patients [6], [8], [9], and can achieve higher volumes of bone regeneration than hydroxyapatite-based biomaterials [6], [10].
Monetite bioceramics prepared by autoclaving of brushite preset cements have inferior mechanical strength to that of their brushite precursor, and similar levels of cytotoxicity [11]. However, monetite bioceramics prepared by this method release ions at a slower rate than their brushite precursors, do not form insoluble hydroxyapatite in vivo [6], [10], and upon subcutaneous implantation they resorb much faster than brushite bioceramics [12].
These crucial differences between the two biomaterials could result in variations in their behaviour as bone regeneration biomaterials. However, within the limit of our knowledge, there is no direct comparison in the literature between these two materials regarding their capacity to regenerate bone. This information will not only help identify which of this materials is more suitable for bone regeneration procedures, but it will also help us understand better what are the most relevant physical properties needed for bone regeneration with calcium phosphate biomaterials.
Accordingly, this study was designed to compare the physical–chemical properties, and the in vitro and in vivo behaviour of monetite and brushite bioceramics regarding their ability to regenerate bone. This included accurate characterization of the bioceramics, osteogenic gene expression assays for bone marrow cells cultured on the bioceramics, and in vivo bone implantation and assessment of both biomaterials.
Section snippets
Synthesis
Monocalcium phosphate monohydrate (MCPM), calcium carbonate (CC) and dicalcium phosphate dihydrate (DCPD) were purchased from Sigma–Aldrich (St Louis, MO). β-TCP was prepared by heating an stoichiometric homogeneous mixture of DCPD and CC (Sigma–Aldrich) at 800 °C for 14 h [13].
Brushite cements were prepared as previously reported [6]. Briefly, 1.2 g of β-TCP was mixed with 1.0 g of MCPM before adding water in a powder-to-liquid ratio of 2.0. The cement paste was left to set in Teflon moulds with
Physical and chemical analyses
X-ray diffraction confirmed that the two groups of prepared bioceramics were brushite and monetite respectively (data not shown). Elemental analysis of the bioceramics revealed that both the brushite and monetite samples had a similar calcium-to-phosphate ratio, slightly higher than 1.0 (Table 1). This confirms that their composition was mainly of dicalcium phosphate, although the small amount of excess calcium seems to indicate that there are traces of unreacted β-TCP in the final composition
Discussion
Dehydration of brushite bioceramics by autoclaving results in a material with the same calcium phosphate content but with very different physical properties; these translated into important differences in the biological behaviour that are discussed below.
Conclusion
Autoclaving brushite bioceramics results in their transformation into a monetite-based biomaterial with higher density, specific surface area, and porosity, and lower compressive strength than their brushite precursors. Our experiments indicated that the differences in the physical and chemical characteristics of brushite and monetite bioceramics had a profound effect on their in vitro and in vivo behaviour as biomaterial for bone regeneration. Monetite bioceramics were able stimulate bone
Acknowledgements
The authors acknowledge financial support from a “COBO” MDEIE (PSR-SIIRI) grant, The Faculty of Dentistry of McGill University, RSBO-FODQ program, and the “Harry Rosen” salary award (F.T., the provision of a Canadian Research Chair (Jake Barralet)).
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