Influence of monocalcium phosphate on the properties of bioactive magnesium phosphate bone cement


 Background: The bone defects caused by different reasons led to deformity and dysfunction of human body. Considering the need for clinical application, it was essential for bone regeneration to exploit a scaffold with bioactive bone cement. In this paper, we fabricated bioactive magnesium phosphate bone cement (BMPC) at room temperature, then it was set at 37℃ and 100% humidity for 2h. Methods: The process was as follows. MgO was formed by calcining Mg2(OH)2CO3, MgO, KH2PO4 and carboxymethyl chitosan were mixed to form magnesium phosphate bone cement(MPC), then Ca(H2PO4)2 was added to neutralize alkaline product after MPC hydration to fabricate bioactive magnesium phosphate bone cement (BMPC). The influence of doped content of Ca(H2PO4)2 on the properties of bone cement was discussed. Results: The results showed that Ca(H2PO4)2 and carboxymethyl chitosan can adjust the setting time of bone cement within the scope of 8 minute and 25 minute. The compressive strength increased first and then decreased. After 48h without additional pressure, the compressive strength reached the maximum value, so it was about of 38.6 MPa. Ca(H2PO4)2 and carboxymethyl chitosan can play a synergistic role in regulating the BMPC properties. BMPC was degradable in the simulated body fluid (SBF). The results of cytotoxicity experiment and laser confocal microscopy experiment indicated that BMPC fabricated at room temperature had better biocompatibility and degradability, which was more consistent with clinical operation requirement.Conclusions: BMPC can be used as a promising orthopedic material, and it can meet with the needs for repairing bone defects.


Introduction
The treatment of bone tissue defect was a common problem in clinics. At present, the traditional autologous bone transplantation was still the "golden standard" for the clinical treatment of bone defects. However, autologous bone transplantation could increase patients' additional trauma. It was lack of bone source and was limited by donors. Besides, it could also increase the number of operations and prolong the healing period. Its future development prospect was not ideal [1,2] . Compared with traditional bone transplantation, degradable bioactive bone cement was prepared to repair bone defects had the advantages of minor damage by bone tissue engineering technology. It was necessary for proper repair of bone morphology in the defect area and no obvious antigenicity [3,4] . Monocalcium phosphate (Ca(H 2 PO 4 ) 2 ), also known as acidic calcium phosphate, was a colorless, granular, or crystalline powder. It existed in the form of Ca (H 2 PO 4 ) 2 •H 2 O at room temperature. The aqueous solution was acidic and lost crystalline water after heating. It was widely used as buffer and food additive [5,6] . Chitosan was the product of deacetylation of chitin. It was the most important derivative of chitin. The surface of chitosan was hydrophilic. As an essential water-soluble chitosan derivative, carboxymethyl chitosan had good biocompatibility and biodegradability. It was widely used in hydrogels, wound healing biomaterials, and tissue engineering scaffold materials [7] . Previous studies showed that carboxymethyl chitosan had no antigenicity in animals and was used as an additive for bone defect repair materials and as a fabrication of bone cement [8,9] . Magnesium phosphate cement (MPC) was an inorganic non-metallic bioceramic material generated by slightly soluble salinization reaction. It was usually used in the industrial eld.
Based on the characteristics of high early strength and rapid curing, it had good biocompatibility and biodegradability. In the recent years, it had attracted the attention of bioactive bone scaffold researchers [10,11] . MPC was fabricated by mixing magnesium oxide (MgO) and ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ) as solid components. The main reaction product was magnesium ammonium phosphate (MgNH 4 PO 4 ), commonly known as guano stone, a natural crystal [12,13] . Accordingly, this process led to the release of NH 3 during the degradation of bone cement, which was easy to contaminate environment and was toxic to the implanted tissue. [14] Potassium dihydrogen phosphate (KH 2 PO 4 ) was applied to replace NH 4 H 2 PO 4 in the fabrication of industrial cement [15,16] . Compared with NH 4 H 2 PO 4 , KH 2 PO 4 had a smaller dissociation constant and lower solubility, which was easier to control the reaction rate. Also, it did not produce NH 3 when reacting with water. The nal product was magnesium potassium phosphate (MgKPO 4 ), which was isomorphic with guanite [17,18] . However, in the research eld of bioactive bone cement, there were few reports on the determination of pH of MgKPO 4 in the aqueous phase and the effect of Ca (H 2 PO 4 ) 2 on the properties of MPC bone cement.
As for the temperature conditions for the fabrication of bone cement, there were several reports on the fabrication of bone cement below 0℃ [19,20] . It was reported that the mechanical properties of bone cement fabricated at low temperature in industry were not optimistic [21] . In clinical practice, the temperature in the operating room was generally controlled at 20~26℃ [22,23] . It was necessary that solid and liquid phases of bone cement was mixed and stirred in proportion at room temperature and implanted into the bone defect quickly. Therefore, it was signi cative to carry out more researches on the fabrication of bone cement at room temperature.
In this essay, bioactive magnesium phosphate bone cement was investigated. Firstly, MgO, KH 2 PO 4 and carboxymethyl chitosan were mixed at 25℃ to fabricate bioactive magnesium phosphate bone cement (BMPC), and pH of MPC hydration product MgKPO 4 was measured. Then Ca(H 2 PO 4 ) 2 was added to MPC. The acidity produced by the degradation of Ca(H 2 PO 4 ) 2 was utilized to neutralize the alkalinity of MgKPO 4 , the main hydration product of MPC. Finally, the effects of Ca(H 2 PO 4 ) 2 and carboxymethyl chitosan on pH, preserving time, compressive strength, degradability, cell morphology, and biocompatibility of bone cement were discussed.

Fabrication of BMPC samples
BMPC was composed of solid powder and liquid phase (deionized water). The solid powder was made from MgO, KH 2 PO 4 , carboxymethyl chitosan and Ca (H 2 PO 4 ) 2 . Among them, MgO was prepared by heating and decomposition of Mg 2 (OH) 2 CO 3 . All powder materials were obtained from Sinopharm Chemical Reagent Co., China. Mg 2 (OH) 2 CO 3 was calcined in mu e furnace to 1500 ℃, the heating rate was 10 ℃/min, kept warm for 2h, cooled with the furnace, wet ball milled with alcohol for 2 h, and sieved through 300 mesh nylon sieve to prepare MgO. KH 2 PO 4 and Ca (H 2 PO 4 ) 2 were respectively screened through a 300 mesh nylon screen after ball milling. Then carboxymethyl chitosan powder was added by 1.5% of mass fraction [24] . At 25℃, deionized water was added at a solid-liquid ratio of 1.6g/ml, after being thoroughly mixed, made into bioactive cement paste, and placed in a 3D printing polyethylene mold (size 10×10×5 mm) without additional pressure. The BMPC sample was collected after restoring at 37℃ and 100% relative humidity for 48h. The composition of BMPC samples was analyzed by X-ray crystal diffraction (XRD, D8-Advance, Germany). The surface morphology and microstructure of BMPC were observed and studied by scanning electron microscope (SEM, JSM-7800, Japan).

pH determination of MPC hydration product
Four different mass ratios of MgO and KH 2 PO 4 , i.e., 1:2, 1:3, 1:4 and 1:5, were selected. carboxymethyl chitosan was added by a 1.5% mass fraction and synthesized MPC with the participation of deionized water. The main hydration product MgKPO 4 was analyzed by XRD. The MPC sample was immersed in normal saline with a solid-liquid ratio of 0.2g/ml and placed in a constant temperature oscillator for 24h. The supernatant was taken, and the pH was measured. Ca(H 2 PO 4 ) 2 was introduced at the mass ratio of 1:2 (MgO and KH 2 PO 4 ) in order to prepare different mass fractions of Ca(H 2 PO 4 ) 2 bone cement samples (0wt.%, 20wt.%, 40wt.%, 60wt.%), which were recorded as BMPC0, BMPC20, BMPC40 and BMPC60 respectively. The effects of different content of Ca(H 2 PO 4 ) 2 on the properties of BMPC were examined.

Characterization of BMPC samples
In order to determine the pH of BMPC soaking solution with different content of Ca(H 2 PO 4 ) 2 , BMPC samples were immersed in normal saline at a solid-liquid ratio of 0.2g/mL and stored in a constant temperature oscillator for 24 h. The supernatant was taken and pH was determined by a pH meter. The setting time of BMPC was measured using a Vicat meter. The Vicat meter which was used, had a sliding metal round rod with a weight of 300g and a test needle of 1mm diameter and 50 mm length at the lower end of the rod. The setting time was the time required from mixing the solid and liquid phases of the composite bone cement to the time when the test needle failed to penetrate more than 1 mm into the specimen. The test was repeated three times, and the average value was calculated. After 48h setting of the bone cement, the compressive strength was measured with a loading rate of 2mm/min using the MTS810 universal mechanical testing machine, and ve samples were taken at each group. The degradation of different BMPC in SBF was determined by the degradation rate at different time points. BMPC samples (10mm×10mm×5mm) were dried for 2h as initial weight (W 0 ). Then, BMPC samples were immersed in SBF at 37℃ in a thermostatic shaker with a solid-to-liquid mass ratio of 1:20 g/ml. The solution was renewed every two days. The weights of BMPC were determined at days 3, 5, 7, 14, 21 and 28, respectively. The operation was performed by removing the specimen from the liquid after immersion, rinsing it with deionized water, drying it for 2 h, and recording the new weight of all specimen. All values were the average of three tests. The degradation rate was calculated by the following formula (1):

Cell culture and cytotoxicity experiment
Mouse osteoblasts 3T3E1 were selected and cultured in complete Roswell Park Memorial Institute 1640 (RPMI) containing 10% foetal bovine serum (FBS), 1% antibiotics (penicillin, streptomycin) and at 37℃ in a humidi ed incubator with 5% CO 2 . Cells were harvested after the con uence with 0.25% trypsin and inoculated individually on culture dishes at an initial density of 2000 cells per well, placed in 96 well culture dishes, and incubated at 37℃/CO 2 . The culture medium was changed every 3 days. BMPC extracts with different mass fractions of Ca(H 2 PO 4 ) 2 were used as the experimental group, and a normal cell culture medium was used as the control group. The BMPC extracts were prepared according to the method in the literature [25] . First, the bone cement raw material was added to serum-free RPMI at a solidliquid ratio of 0.2 g/ml under aseptic conditions to obtain the solution, which was incubated at 37℃ for 24h and then centrifuged, and the supernatant was collected, refrigerated at 4℃ and stored for further use. The cells were cultured for 1 day, 3 days and 5 days, respectively. The cytotoxicity of BMPC was estimated by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. To each well, 20µl of MTT solution was added, and incubation was continued at 37 ℃ and 5% CO 2 for 4 h. The supernatant in the wells was then discarded, and 200µL of dimethyl sulfoxide (DMSO) was added to fully dissolve the purple crystals. On days 1, 3, and 5, respectively, the absorbance value of each well at 490 nm was measured by an enzyme maker OD. The relative growth rate (RGR) was calculated according to the measured OD value by the following formula (2), and the test results were evaluated according to the cytotoxicity grade standard (Table 1) [26] .

Laser confocal microscopy experiment
Mouse osteoblasts 3T3E1 in logarithmic growth stage were taken at 37 ℃, 5% CO 2 in a constant temperature oven, and treated with 4×10 5 /ml in four groups BMPC extracts with different levels of Ca(H 2 PO 4 ) 2 , respectively. After 5 days of culture, the cells were xed with 0.2 ml of 4% paraformaldehyde, and the wells were placed in the thermostat for 10 min. The xative solution was pipetted out, rinsed with PBS, and 0.5% Triton x-1000 5 ml was added, and the membrane was permeabilized in the thermostat for 20 min, after which the permeabilized solution was pipetted out, rinsed with PBS, and 0.5 ml of staining solution was added to re-stain the plates for 5min, and the re-staining solution was pipetted out and rinsed with PBS. 200μL of rhodamine ghostpen ring peptide was added and treated in a dark room at room temperature for 40 min and then sealed with uorescence quencher after PBS rinsing. After material treatment, cell morphology and growth were observed using laser confocal microscopy. setting for 48 hours. It was found that BMPC0 contained prismatic crystals. It was speculated that these crystals were KMgPO 4 as well as clay materials with dense morphology and structure, as shown in Figure   2(a, b). With the increase of Ca(H 2 PO 4 ) 2 content, prismatic crystals disappeared and clay materials increased, which was consistent with the XRD results. KMgPO 4 disappeared with the increase of Ca(H 2 PO 4 ) 2 . It could be seen that clay materials accumulated together, resulting in high strength of the specimens, as shown in Figure 2(c,d) to Figure 2(e, f). Moreover, there were many pores in the clay materials, which were consistent with the experimental results observed by Wang s, et al(2019) [27] . The existence of these pores was not only conducive to the degradation of bone cement and phosphate deposition, but also conducive to the adhesion and proliferation of osteoblasts, so it induced the growth of new bone. Therefore, it could result in the degradation of BMPC specimens (Figure 2 (g, h)).

The results of characterization of BMPC samples
The pH of BMPC extracts were shown in Fig. 3 which was close to the pH value of simulated body uid (SBF) used in general cell experiments. This condition of pH environment promoted cell growth and proliferation.
The effect of different amounts of Ca(H 2 PO 4 ) 2 on the setting time was shown in Fig. 4. With increasing Ca(H 2 PO 4 ) 2 content, the setting time of BMPC was increased from 8 min to 25 min, a time range that was consistent with the operating time needed for general clinical bone defect repair. When the ratio of MgO to KH 2 PO 4 was 1:2, the content of MgO was relatively overreacted (Table. 2). In contrast, as the Ca(H 2 PO 4 ) 2 content increased, the acidity of the reaction system increased, more crystalline products were generated, and the main product KMgPO 4 disappeared, prolonging the time for the reaction to reach equilibrium and resulting in a longer setting time of bone cement, which facilitated implantation of bone cement. than that of magnesium ammonium phosphate bone cement prepared at low temperature [20] . The increase of compressive strength was due to the decrease of pH in the reaction system, then it resulted in more hydration products similar to clay particles (Fig. 2). These granular materials were closely staggered and stacked together to form high compressive strength. Therefore, the specimens not only performed satisfactory mechanical strength, but also met the needs of on-site fabrication of bone cement at room temperature. Compared with BMPC40, the compressive strength of BMPC60 decreased. It was the reason that crystal structure became irregular as the crystallinity of hydration products decreased (Fig. 2), which cannot form a high compressive strength and resulted in a decrease of compressive strength with BMPC60. It should be pointed out that BMPC lled with Ca(H 2 PO 4 ) 2 produced MgHPO 4 after setting for 48h. After degradation for 28 days, MgHPO 4 still existed, while other products were transformed into magnesium phosphate, pyrophosphate and HAP (Table 2). Thus, MgHPO 4 could be regarded as buffering agent in the reaction system, and the regulation of pH with BMPC depended on the existence of MgHPO 4 . Figure 6 showed the degradation rate of BMPC samples soaked in simulated body uid (SBF) at different time. Obviously, BMPC degraded in SBF as time went on and the degradation rate was related to the amount of products (Table 2). It can be found that after degrading for 28 days, the product amount of BMPC40 samples had the most signi cant reduction and the highest degradation rate. The results showed that the more products disappeared, the faster the degradation was.

The results of cytotoxicity with BMPC by MTT assay
MTT assay was widely applied to detect the bioactivity or cytotoxicity of biomaterials. MTT assay was selected to detect the extracts of BMPC samples to determine the cytotoxicity to mouse osteoblasts 3T3E1. According to RGR and toxicity grade standard (Table 3), when Ca(H 2 PO 4 ) 2 content was less than 40%, the toxicity grade of BMPC samples was grade 1. When Ca(H 2 PO 4 ) 2 content reached 40% or more, the toxicity grade of BMPC samples was grade 0 (Fig. 7, Table 3). It indicated that BMPC40 and BMPC60 samples had good biocompatibility, which were consistent with the pH results of BMPC extracts (Fig. 3). It was the reason that addition of Ca(H 2 PO 4 ) 2 reduced the pH of BMPC extracts, which was close to the pH of simulated body uid, thereby it was bene t to improve the biocompatibility of bone cement. It also proved that the alkaline environment of KMgPO 4 was not suitable for the growth, adhesion and proliferation of mouse osteoblasts 3T3E1.

The results of laser confocal microscopy experiment
The results of laser confocal microscopy experiment were shown in Fig. 8. The morphology of mouse osteoblasts 3T3E1 in bone cement extracts was irregular, mostly triangular and polygonal, with many protrusions, mononuclear and oval nucleus. The cell matrix was wrapped around the nucleus, and the pseudopodia between cells fused with each other. It indicated that the cells grew on the matrix of all bone cement samples. In terms of different Ca(H 2 PO 4 ) 2 contents, compared with BMPC0 ( Fig. 8(a)) and BMPC20 ( Fig. 8(b)), the number of osteoblasts with BMPC40 and BMPC60 increased signi cantly (Fig.  8(c,d)). It suggested that with the increase of Ca(H 2 PO 4 ) 2 content, the changes of bone cement degradation products and alkaline environment promoted cell proliferation and differentiation. This was consistent with the cytotoxicity results of BMPC by MTT assay. Compared with the other groups, the largest number of cells was detected in BMPC60 extracts (Fig. 8 (c, d)), which could be interpreted for two reasons. On the one hand, the pH of BMPC40 and BMPC60 were close to the pH of simulated body uid (Fig. 3), which was suitable for cell growth and proliferation. On the other hand, the contents of calcium ions, magnesium ions and phosphate in the solution were high, which provided a suitable environment for the growth of osteoblasts.

Discussion
Here we developed novel bioactive magnesium phosphate bone cement (BMPC) with improved physicochemical properties by incorporating different ratios of Ca(H 2 PO 4 ) 2 into magnesium phosphate bone cement (MPC). It was revealed by X-ray diffraction (XRD) that the mass ratio of magnesium oxide (MgO) to potassium dihydrogen phosphate (KH 2 PO 4 ) was 1:2. The major hydration reaction of MPC made from MgO and KH 2 PO 4 was: K + +Mg 2+ +PO 4 3− =MgKPO 4 Besides the MgKPO 4 and unreacted MgO, XRD analysis did not reveal any other hydration products. It indicated that carboxymethyl chitosan mainly performed as micro-ller in the MPC reaction system, therefore, it does not involve in the formation process of hydration products. After adding different concentrations of Ca(H 2 PO 4 ) 2 , the typical peaks of MgO and KMgPO 4 gradually disappeared in XRD analysis, indicating that all samples were transformed into other hydration products.
The setting time was one of the vital properties which could re ect the polymerization time for repairing bone defects [28] . The setting time of MPC was greatly affected by the conditions of powder size, surface area, MgO content, powder-to-liquid ratio, etc. [29] . In the preparation process of inorganic salt bone cement, the acid-base reaction rate was fast and di cult to control, so that the setting time was very short [30] . Several former studies had revealed that a setting time of 8 to 20 minutes was suitable for implanting bone cement in surgery [30] . This study showed that the setting time could be prolonged from 8 min to 25min by adjusting the content of Ca(H 2 PO 4 ) 2 in MPC, with the carboxymethyl chitosan at a xed ratio. Therefore, it was su cient long for repairing the bone defects by injecting and shaping the cement. The setting time prolonged because Ca(H 2 PO 4 ) 2 as an acid salt, it played a certain buffering role in the reaction system, which can decelerate the rate of hydration reaction. Moreover, the carboxymethyl chitosan can forms coatings to cover the surface of MgO and KH 2 PO 4 to reduce its hydration, slowing down the hydration reaction. We speculate that Ca(H 2 PO 4 ) 2 can coordinated with the carboxymethyl chitosan in this process. Without a buffer, the hydration reaction can take a violent exothermic effect [31] . The prolonged setting time indicated the composite cement had a moderate hydration reaction, generating less heat during the setting process. It can be helpful to avoid tissue damage and apoptosis [32] .
Bone cement needs to achieve a certain mechanical strength in clinical applications, at least to meet the compressive strength of the cancellous bone [33] . We found that the compressive strength rst increased and later decreased with the Ca(H 2 PO 4 ) 2 concentration increasing. The maximum value of compressive strength was 38.6 MPa, with BMPC40. The increase of compressive strength was due to the decrease of pH value in the reaction system, resulting in more hydration products similar to clay particles. These granular materials were closely staggered and stacked together to form high compressive strength. Therefore, the specimen not only had good mechanical strength, but also met the needs of on-site preparation of bone cement at room temperature. Compared with BMPC40, the compressive strength of BMPC60 decreased. This was because the crystallinity of the hydration product of BMPC60 reduced, the crystal structure became irregular, and the high compressive strength cannot be formed, resulting in the decrease of compressive strength. In addition, carboxymethyl chitosan, as a hydrophilic polymer, it may adsorb deionized water in the liquid phase and forms a high viscosity coating on the cement surface.
This can be observed from SEM images. MPC showed many brittle crystal cracks, while carboxymethyl chitosan lled these cracks, forming a dense microstructure, which had a certain degree of fracture resistance.
pH should also be considered because it can signi cantly affect osteogenesis of bone cement [34] .
Generally, surplus MgO in MPC composites can lead to a large amount of OH − . In order to decrease the alkalinity of hydration products, a relatively low Mg/P ratio of 1:2 was adopted. We also utilized the The degradation rate or biodegradability was another important property of bone cement. There was evidence that lower degradation rate can be caused by lower porosity [35] . We found that the degradation ; Ca 10 (PO 4 ) 6 (OH) 2 Table 3 The relative growth rate(RGB) and toxicity grade(TG) of BMPC samples.    The setting time of BMPC specimens with Ca(H 2 PO 4 ) 2 .

Figure 6
The degradation rate of BMPC samples with Ca(H 2 PO 4 ) 2 .

Figure 7
The cytotoxicity of BMPC samples.