Brushite (dicalcium phosphate dihydrate) and monetite (dicalcium phosphate anhydrous) are of considerable interest in bone augmentation owing to their metastable nature in physiological fluids. The anhydrous form of brushite, namely monetite, has a finer microstructure with higher surface area, strength and bioresorbability, which does not transform to the poorly resorbable hydroxyapatite, thus making it a viable alternative for use as a scaffold for engineering of bone tissue. We recently reported the formation of monetite cements by a simple processing route without the need of hydrothermal treatment by using a high concentration of sodium chloride in the reaction mix of β-tricalcium phosphate and monocalcium phosphate monohydrate. In this paper, we report the biological responsiveness of monetite formed by this method. The in vitro behaviour of monetite after interaction and ageing both in an acellular and cellular environment showed that the crystalline phase of monetite was retained over three weeks as evidenced from X-ray diffraction measurements. The crystal size and morphology also remained unaltered after ageing in different media. Human osteoblast cells seeded on monetite showed the ability of the cells to proliferate and express genes associated with osteoblast maturation and mineralization. Furthermore, the results showed that monetite could stimulate osteoblasts to undergo osteogenesis and accelerate osteoblast maturation earlier than cells cultured on hydroxyapatite scaffolds of similar porosity. Osteoblasts cultured on monetite cement also showed higher expression of osteocalcin, which is an indicator of the maturation stages of osteoblastogenesis and is associated with matrix mineralization and bone forming activity of osteoblasts. Thus, this new method of fabricating porous monetite can be safely used for generating three-dimensional bone graft constructs.
The treatment of ‘critical’ bone defects (i.e. bone deficiency that precludes the spontaneous healing of the natural tissue leading to severe functional impairments) represents a challenge for orthopaedic and oral-maxillofacial surgeons owing to lack of therapeutic approaches that are able to fully restore the lost bone . Moreover, the poor functionality (i.e. quality) of the damaged tissue surrounding the defect because of trauma, tumour or infection increases the complexity of the bone restoration procedures. In this scenario of clinical need, research on new materials for bone repair is growing at a rapid rate and today, calcium orthophosphates are extensively investigated on account of their compositional similarity to that of the bone mineral phase, biocompatibility and osteconductive properties [2–5]. Among the different calcium orthophosphates, brushite (dicalcium phosphate dihydrate, DCPD: CaHPO4 · 2H2O) and monetite (dicalcium phosphate anhydrous, DCPA: CaHPO4) are of special interest as they can partake in enhancing bone formation owing to their thermodynamic metastability under physiological conditions . However, brushite once in contact with biological fluids tends to dissolve and re-precipitate into a less soluble calcium phosphate (CaP) which limits its clinical application . By contrast, DCPA bioceramics implanted in vivo have been found to conserve their chemical composition and degradability, allowing replacement by the newly formed bone tissue [8,9]. However, it is imperative to understand the in vitro behaviour of potential scaffolds to effectively be used as scaffolds for engineering of bone tissue. Thus far, there is limited in vitro data on the ability of monetite to maintain its chemical composition in vitro and interaction with bone forming cells. This information is extremely important when monetite is used as a ‘scaffold’ for tissue regeneration, namely when bone cells obtained from the patient are seeded on the material to form bone matrix in vitro before in vivo implantation. Moreover, the cellular change required to promote bone formation in vitro, mainly depends on the overall material properties (i.e. chemistry, surface area, charge, roughness, porosity and mechanical properties) and is significantly influenced by the preparation method [9,10]. In the fabrication of monetite bioceramics, a multi-step process is generally performed where a DCPD cement is dehydrated by thermal treatment [9,11] or an α-tricalcium phosphate (α-TCP: (Ca3(PO4)2 matrix) is chemically transformed by ageing in acid solution . Nevertheless, the use of brushite as a precursor has been reported to have detrimental effects on the mechanical properties of the monetite formed while, the method of ageing in acid solution requires at least 3 days of soaking in acid to achieve a full chemical conversion into monetite. Recently, we reported the direct formation of micro and macroporous monetite cements  using sodium chloride to control the supersaturation of the components in the setting of the CaP cement. The formation of a pure monetite phase occurred within 24 h from mixing of the reagents that resulted in maximum compressive strengths higher than that reported in literature for monetite bioceramics and comparable with cancellous bone (2–12 MPa) . Futhermore, the direct formation of monetite as a cement, opens up the possibility to inject it into three-dimensional custom-made moulds to produce patient-tailored bone grafts with improved mechanical properties. In this work, we report a direct comparison between the new monetite cement and a traditional brushite cement, comparing their ability to support human osteoblast cells (HOBs) proliferation and differentiation. In addition, ageing of monetite was performed to study the fate of the chemical phase and evaluate if the monetite cement is able to retain its chemical phase in vitro as already reported in vivo. With this study, a first evaluation of the potential use of monetite made by our innovative fabrication method in the field of bone tissue engineering is presented and discussed.
2. Material and methods
2.1. Sample preparation and compressive testing
Macroporous cements were prepared with a solid phase consisting of equimolar quantities of β-tricalcium phosphate (β-TCP, assay greater than 96%; FLUKA, Germany) and monocalcium phosphate monohydrate (MCPM, assay min. 98%; Sharlau, Spain) wherein sucrose (Sigma-Aldrich; greater than or equal to 99.5%) or sodium chloride crystals (NaCl, Merk; greater than or equal to 99.5%) previously sieved to keep the diameter in the range of 150–600 µm were dispersed at 60% by weight with respect to the CaP powders to function as the porogen. The liquid phase consisted of a sodium citrate (C6H7O7Na) solution of molar concentration C equal to 0.5 M saturated with sucrose or salt in order to avoid an anticipated dissolution of the porogen. The ratio of the CaP powders and the liquid phase (R = P/L) was kept fixed at a value of 3 g · ml−1. The cement mix was stirred manually for approximately 2 min under ambient conditions (20–23°C and 50–60% humidity) and then poured into cylindrical (d = 6 mm, h = 12 mm) Teflon moulds. The casts were allowed to mature for 24 h, cut in order to obtained disc-shaped samples (d = 6 mm, h = 4.2 mm) or left uncut (for mechanical evaluation) and subsequently stored in water for 5 days to completely remove the porogen. After this time, the still wet samples (n = 12) were subsequently tested in uniaxial compression using an electromechanical testing machine Instron 5569A, with a load cell of 500 N and a cross-head rate of 1 mm min−1.
2.2. X-ray diffraction and ATR/FTIR analysis
X-ray diffraction (XRD) on the powder samples were carried out on a Bruker D8 Advance diffractometer equipped with a Lynx Eye detector, in flat plate geometry using Ni-filtered Cu kα radiation. Data were collected in the range 2θ = 10–100° with a step size of 0.02° and a count time of 12 s. ATR/FTIR analysis was performed at room temperature (20–23°C) on dried samples by using a Perkin Elmer Spectrum One FTIR spectrometer with a resolution of 4 cm−1.
2.3. Micro-CT and scanning electron microscopy analysis
The porous cement blocks were scanned using micro-CT (Locus SP, micro-CT scanner, GE Healthcare, USA) set at a resolution of 7 µm. The microstructure of the materials was observed using scanning electron microscopy (SEM, a Hitachi S-3500).
2.4. Interaction of the macroporous cements with phosphate buffer saline and tissue culture medium
The macroporous cements (d = 6 mm, h = 4.2 mm, n = 4) were immersed in 3 ml (for each sample) of phosphate-buffered saline (PBS) or cell culture media (Dulbecco's modified Eagle's media: DMEM D6046, Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS), 1% non-essential amino acids, l-ascorbic acid (0.150 g l−1), 1% of 200 mM l-glutamine, 2% of 1 M HEPES, penicillin (100 U ml−1) and streptomycin (0.1 mg ml−1) (all from Sigma, UK) up to 21 days at 37 ± 1°C. The media were refreshed after 24 h from the first immersion and every 3 days subsequently. At a fixed time point (1, 7, 14 and 21 days), the pH (pH electrode Fisherbrand FB68797) and free calcium ion concentration (calcium ion selective electrode Sartorius PY-107) in the immersion media and the wet mass (Mettler Toledo AT250 readability 0.1 mg) of the samples were measured. Cement water content was determined by dehydration at room temperature until a stable weight was measured.
2.5. In vitro cell culture
A primary HOBs cell model was used for the in vitro tests. Briefly, cells were isolated from the femoral heads of patients undergoing surgery for total joint replacement, as previously described by Di Silvio & Gurav . HOB cells were cultured in DMEM, supplemented with 10% FCS, 1% non-essential amino acids, l-ascorbic acid (0.150 g l−1), 1% of 200 mM l-glutamine, 2% of 1 M HEPES, penicillin (100 U ml−1) and streptomycin (0.1 mg ml−1) (all from Sigma, UK) at 37°C in a controlled humidified atmosphere with 5% CO2.
2.6. Cell proliferation: alamar Blue assay
The macroporous cement (d = 6 mm, h = 4.2 mm, n = 4) was sterilized by gamma irradiation at a dose of 31.8 kGy. Cell proliferation was determined using the alamar Blue assay (Life Technologies) which is a redox indicator that measures proliferation quantitatively. Cells were micro-seeded at a total density of 1 × 105 cell per scaffold placed in a 24-well plate. The time periods studied were 1, 3, 7, 14 and 21 days. The cell culture media was changed after 24 h and every 3 days subsequently to minimize cell disturbance. Thermanox discs (Nalge NUNC International, Rochester, USA) and commercial sintered macroporous hydroxyapatite ((HA 0621), a gift from DyTech, Sheffield, UK) (d = 6 mm, h = 4.2 mm) were used as standard cell culture and material controls.
2.7. Quantitative RT-PCR analyses
Total RNA was extracted using TRI reagent (Ambion, Warrington, UK) and Phase Lock Gel Heavy tubes (5 prime, VWR, Leicestershire, UK) according to the manufacturer's instructions. RNA purity and quantity was assessed by nanodrop (Fisher Scientific; A260/A280 1.8–2 was considered suitable for further analysis), possible contaminating DNA was removed and cDNA prepared from 1 µg RNA using QuantiTect Reverse Transcription Kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. qRT-PCR was performed on a rotor gene 6000 thermal cycler (Qiagen) using Brilliant III Ultra-Fast SYBR Green qPCR Master mix (Stratagene, Agilent Technologies, Cheshire, UK) and the following primer pairs: (5′ to 3′) RPL13a (GGATGGTGGTTCCTGCTG and TGGTACTTCCAGCCAACCTC); Runx-2 (AATGGTTAATCTCCGCAGGTC and TTCAGATAGAACTTGTACCCTCTGTT); ALP (AACACCACCCAGGGGAAC and TGGCATGGTTCACTCTCGT) and osteocalcin (CATGAGAGCCCTCACACTCC and ACCTTTGCTGGACTCTGCAC). PCR conditions consisted of one cycle of 95°C for 3 min and 40 cycles of 95°C for 10 s and 60°C for 10 s followed by melting analysis of one cycle with gradual increase from 65°C to 95°C. RPL13a was used as an invariant housekeeping gene.
2.8. Statistical analysis
Statistical comparisons between means were made by Student's t-test (SPSS 16, SPSS). A p-value of less than 0.05 was considered statistically significant.
3.1. X-ray diffraction and ATR/FTIR spectroscopy
The XRD pattern of monetite and brushite cements formed in the study exhibited a 95% or higher degree of conversion as shown in figure 1a(A) and b(A), respectively. Considering that both composition as well as pH of the immersion media may affect transformation of the chemical phase of the macroporous brushite and monetite cements over time, it was decided to study the effect of ageing both in DMEM and PBS solutions. DMEM, is a medium that is used in in vitro cell-culture tests, which contains inorganic salts, amino acids, vitamins and glucose, while PBS is frequently used to investigate the ageing of CaP materials [15–18]. The osmolarity and ion concentration of PBS solutions match those of the human body, which is a water-based salt solution mainly containing sodium phosphate and sodium chloride without calcium salts present in the grade used in this study. The XRD were determined for both cements in DMEM with and without cell seeding after three weeks, which showed that monetite retained its chemical phase as shown in figure 1a(B) and (C). By contrast, brushite exhibited the formation of octacalcium phosphate (OCP, confirmed by the doublet at 31.6°) and hydroxyapatite (figures 1b(B) and (C) and 2a) in DMEM both in presence and absence of the cells. However, brushite cements that were immersed in PBS solution did not exhibit transformation in their chemical phase (figure 2b). A further analysis of the chemical composition of the cements was performed and the results are reported in figures 3 and 4. The FTIR spectrum of brushite in drastically changed after immersion cell culture medium with the appearance of new peaks at 1018, 914 and 872 cm−1 which could be ascribed to the stretching mode of the PO and P–OH group of the OCP phase (ν PΟ: 1023, ν P–OH: 914 and 874 cm−1) . However, the peak at 1018 cm−1 may also be assigned to non-stoichiometric HA (ν PO: 1020 cm−1) . The spectrum of monetite powder instead did not change except for the presence of a small distinct peak at 1026 cm−1 ascribed to an OCP or HA phase.
3.2. Chemical evaluation of the immersion media
The DMEM immersion medium in which the brushite cements were stored exhibited a decrease in the pH values and the concentration of the calcium ions with respect to the control medium (figures 5 and 6, respectively) owing to the acidic dissolution products releasing HPO42− and H+ ions and the formation of OCP (Ca8(HPO4)2(PO4)4 · 5H2O) which requires the consumption of calcium ions from the ageing medium. These observations are in agreement with the results reported by Mandel & Tas , where the precipitation of OCP from brushite powders (equation (3.1)) immersed in DMEM were attributed to a decrease of the pH from 7.4 to approximately 6.8. 3.1 The pH values of the immersion media in which monetite cements were placed did not change significantly with respect to the control (figures 5 and 7) although an increase of the calcium ions in the PBS solution was observed (figure 8) and a decrease of the latter in DMEM (figure 6) significant at 7 and 21 days, suggesting that dissolution of the material occurred locally together with a re-precipitation process in the cell culture medium as clearly shown by the appearance of a new distinct peak in the FITR spectrum of monetite sample aged in DMEM medium (figure 4).
3.3. Micro-CT, gravimetric changes of the cements aged in liquid media and compression test
The micro-CT analyses showed an average porosity of 40 and 45% for brushite and monetite, respectively, with pore diameters between 200 and 650 µm. The pores were distributed throughout the cement while the structure maintained sound continuity along its multiple iso-surfaces in all directions (X, Y, Z) (figure 9). The maximum compressive strength (σc) and Young's modulus (E) measured for monetite and brushite were σc = 1.5 ± 0.1 MPa, E = 191 ± 34.3 MPa and σc = 0.9 ± 0.2 MPa, E = 287 ± 72.6 MPa, respectively. The gravimetric changes of the cements recorded showed a significant increase in weight by approximately 40% (tables 1–2), which may be attributed to presence of pores that increase the net surface area (first data recorded after 24 h of immersion). The weight changes recorded over 21 days showed that the brushite cements lost 15 and 19% of their initial dry weight in PBS and DMEM solution, respectively. By contrast, monetite samples appeared to be physically stable without any significant decrease in their initial dry weight after 21 days of immersion. These results support earlier findings related to the development of a significant degradation/dissolution process of the brushite cements only .
3.4. Scanning electron microscopy analysis
The microstructure of monetite and brushite cements are shown in figure 10a and b, respectively. The morphology of the brushite grains was characterized by the typical thick platelet shapes that were relatively non-homogeneous in size. On the contrary, monetite showed a different microstructure consisting of thinner platelets that were homogeneous in size. After 21 days in culture the microstructure of the brushite samples changed with formation of crystals having almost the same size and morphology (figure 11c–f). In several cases, the growth of needle-like crystals from the walls to the centre of the macropores (figure 11f) was also detected. The macrostructure of monetite cements did not alter in the cell culture medium and appeared similar to those recorded before immersion with no observable changes in the microstructure post-ageing in DMEM medium (figure 12). The micrographs of brushite, however, clearly show evidence of a transformation in the microstructure (figure 11a–f), which is supported by the XRD data of the aged brushite cements exhibiting the formation of a more stable CaP phase.
3.5. Biological tests: alamar Blue and gene expression
The alamar Blue data showed a steady significant increase in cell number from days 1 to 21 for Thermanox (a tissue culture plastic), hydroxyapatite and monetite except for the brushite cements. The brushite cement showed a significant decrease in cell number within 7 days indicating that dissolution products may have affected cell viability (figure 13). The SEM micrographs post-cell seeding at 3 days showed the ability of the cells to attach and spread onto the different materials (figure 14a–d) with confluency over the entire surfaces at day 21, which was distinctly different from the cell attachment on the brushite cements. After 21 days, the cells were seen to cover the external surface of the monetite and HA materials which was not observed with the brushite samples (figure 15a–c).
Furthermore, to determine whether monetite and the other materials studied (HA and brushite) had osteogenic potential the gene expression associated with the process of osteogenesis was assessed and compared to cells cultured on Thermanox. HOB cells seeded on monetite cement showed significantly higher expression of Runx-2 (1.4-fold) and osteocalcin (sevenfold) mRNA after 10 days of culture when compared to cells seeded on Thermanox or HA (figure 16). Higher expression of osteocalcin (twofold) was also observed after 20 days of culture (figure 17). ALP levels remained unchanged in monetite cement compared with Thermanox, however, both were higher than HA at 10 days (figure 16) of differentiation. Runx-2 and ALP expression were lower when compared with Thermanox at the late time point (figure 17, 20 days).
In the last decade, many studies have focused on comparing the chemical, physical and biological properties of monetite with those of its precursor brushite in order to identify which of these materials is more suitable for bone regeneration [7,9,21]. Monetite and brushite are acidic CaP phases characterized by the same calcium to phosphorus ratio (Ca/P = 1) and a chemical formula (monetite: CaHPO4 and brushite: CaHPO4 · 2H2O) that only differs in the water of crystallization of the brushite lattice. However, despite their chemical similarity, the in vivo degradation rate has been reported to be different [7,9]. Monetite bioceramics, once implanted in the body, degrade over time encouraging new bone formation, whereas brushite tends to dissolve and re-precipitate into a less soluble CaP phase, which limits its replacement by new bone tissue . In our study, monetite bioceramics were fabricated using the typical reactants for formulation of brushite cements (equation (4.1)) with the addition of NaCl crystals (equation (4.2)) [10,22] that led to a method of forming monetite without the use of heat and harsh acidic environment.
The absence of any chemical transformation of brushite and monetite in PBS suggests that both are stable, however, with the transformation of brushite in cell culture medium under similar conditions is evidence of the participation of the free calcium ions arising from the cell culture medium to form a supersaturated solution leading to the formation of a mixture of OCP and HA. The total concentration of calcium ions when brushite and monetite are at equilibrium in aqueous solution at neutral pH are known to be 0.90 and 1.7 mM, respectively . If it is assumed that dissolution of the CaP phases occurs if the solubility product of the material is higher than the corresponding ion concentration (approx. 1.4 mM for the DMEM as shown in figure 6) in the surrounding medium, this process should be favoured only for monetite.
However, the dissolution of brushite may still occur, even if the ionic concentration is higher than the solubility product if the pH value of the media is low enough, so that the solubility of DCPD at that pH value is larger than the calcium and also phosphate concentration in the solution . Moreover, to meet this condition, (for calcium ions) the pH value should be below 5.9 . In the light of what has been reported by Bohner et al. , we hypothesize that the transformation of brushite to OCP occurred due to the formation of an acidic environment determined from the measurable lowering of the pH of the DMEM medium. In vivo studies have shown the ability of monetite to be resorbed allowing its replacement by newly regenerated tissue; thus the interest in monetite as a bone repair material has risen considerably in the last 5 years. However, lack of information regarding the in vitro ageing of monetite makes its use uncertain in a bone tissue engineering approach where the biomaterial should support and enhance the regeneration of bone tissue in the body. This latter consideration is motivated by the fact that, metastable CaP phases as brushite and monetite dissolve once immersed in liquid media, releasing calcium and phosphate ions that can inhibit or enhance positive cellular responses depending on their concentration in the surrounding environment [24,25]. Moreover, the acidic dissolution may have adverse effects on the biological activity of the cells both in vitro and in vivo . As a consequence, the evaluation of the ageing properties of metastable bioceramics appears to be mandatory, in particular, when the material is intended to be used as cell-seeded scaffold in vitro where the ability of the environment of adapting itself to external changes is limited.
The monetite cements immersed in media were found to be chemically stable and showed little tendency to undergo dissolution. Thus on the basis of these results, the degradation process most likely occurs exclusively by the cell activity, which is in agreement with the observations reported by Grobard et al.  where, monetite scaffolds were found not to degrade when immersed in culture media but, once cultured with osteoclastic cells (which provide a local acid environment) the solubility of the material was found to increase. However, in contrast to monetite, brushite cements were observed to undergo a degradation process that may have occurred due to their higher solubility , also responsible for the higher amount of free calcium ions measured in the PBS ageing solution. The decrease in the metabolic activity of the cells cultured on brushite may be attributed to an acidic environment. A study by Kaysinger et al.  on the effect of extracellular pH on the activity of cultured human osteoblasts showed that cells in HEPES-buffered media at initial pH adjusted from 7.0 to 7.8 exhibited an increase in cell proliferation with increasing pH as well as a decrease in the biosynthetic and mitogenic activity of the cells with decreasing extracellular pH. Interestingly, our biological results are in contrast to those reported by Klammert et al.  where the authors studied the cytocompatibility of brushite matrix and monetite scaffolds. The latter were produced by autoclaving the brushite matrix. The biological evaluation was carried out over 12 days and the brushite cements were shown to be suitably biocompatibile as cell-culture scaffolds for hard tissue regeneration. The reason why our results are not in agreement with those reported by Klammert et al.  may reside in the presence of brushite macropores which generate a structure with higher surface area and consequently with a better fluid exchange within the scaffold and an higher acid dissolution capacity for equal amounts of liquid medium. The reduction in Runx-2 expression could be explained by time-dependent expression of osteogenic phenotypic genes, with loss of early markers and up-regulation of late markers at the more mature stage of matrix mineralization. Gene expression analysis on cells seeded on brushite cement was unsuccessful due to reduced cell number as shown by alamar Blue data. This study on monetite bioceramics showed the supportive nature of the material for bone formation and osteogenic activities. Literature data on gene expression of osteoblast cultured on monetite surfaces suggested that the pattern of osteogenic gene expression in bone marrow cells is similar to that observed on HA. Although some association may be drawn by comparing separate studies, the data need to be confirmed within a single study. Here we reveal by direct comparison that monetite made by our method  could stimulate osteoblasts to undergo osteogenesis and accelerate osteoblast maturation earlier than cells cultured on HA scaffolds (porosity of approx. 70% with a diameter of pores ranging between 250 and 500 µm) or Themanox discs. Osteoblasts cultured on monetite cement also showed higher expression of osteocalcin, which is an indicator of the maturation stages of osteoblastogenesis and associated with matrix mineralization and bone forming activities of osteoblasts. In addition, our recent study  suggested the possibility of using monetite cements in a bioreactor for induction of MSC proliferation and differentiation. Taken together, the data indicate a strong potential for the use of monetite cement in tissue engineering approaches for bone regeneration.
The new method adopted to produce monetite bioceramics from a chemical formulation typical of hydraulic cements generates three-dimensional porous structures able to support the proliferation and differentiation of human osteoblast cells. Although monetite is metastable under physiological condition, the chemistry of the material did not change in contrast to brushite where the transformation into a more chemically stable CaP phase negatively affected cell viability. The fabrication method presented in this work may be considered as an alternative simple technique to produce monetite-based potential candidate materials for bone tissue engineering applications.
This work was supported by research funding from the Wellcome/EPSRC Centre of Excellence in Medical Engineering. The authors would also like to thank the Guy's and St Thomas Charity.
- Received July 4, 2014.
- Accepted September 18, 2014.
- © 2014 The Author(s) Published by the Royal Society. All rights reserved.