Physicochemical properties and in vitro activity of SrHPO4 modified magnesium oxychloride bone cement

In response to the shortcomings of magnesium oxychloride cement (MOC), which has excellent with outstanding mechanical properties and favorable bioactivity but poor water resistance, strontium hydrogen phosphate (SrHPO4) was used as a water resistance modifier for MOC in the paper, and the effects of SrHPO4 on the strength, water resistance, in vitro degradation and bioactivity of MOC were investigated; the results showed that SrHPO4 could enhance the water resistance of MOC, in which the initial strength of MOC containing 4.0 wt% SrHPO4 was 92.3 ± 2.5 MPa, and the strength was still 8.2 ± 0.9 MPa after 84 d of immersion in SBF solution; the degradation experiments of the samples in SBF solution showed that the degradation of SrHPO4-MOC was controlled, and the low alkaline environment created by the degradation promoted the deposition of hydroxyapatite on the cement surface, it indicated that SrHPO4-MOC material had good degradation properties and bioactivity; cell experiments showed that compared with MOC, SrHPO4-MOC was noncytotoxic and could promote cell proliferation, which was expected to be a new material for bone repair.


Introduction
Traffic accidents, trauma, infections, and aging may cause bone defects.Although bones have a certain ability to heal themselves, bone grafting is required for bone defects that exceed a critical size [1][2][3][4].Autologous bone is the best bone repair material, but due to its limited source, it does not meet the growing demand well.However, the use of allogeneic bone can't completely avoid immune rejection, risk of disease transmission, etc.So there is an urgent need for artificial bone repair materials with outstanding mechanical properties, and favorable bioactivity, which are also harmless to the human body to fill the gap [5,6].Bone cement is a class of artificial bone repair materials that can be shaped arbitrarily, and is widely used in clinical practice.At present, the main ones used are calcium phosphate bone cement, polymethylmethacrylate bone cement, calcium sulfate bone cement and magnesium phosphate bone cement [7][8][9][10].
Polymethyl methacrylate (PMMA) has been widely used in orthopedic and trauma surgery with its good mechanical properties and plasticity, but in the process of clinical use, PMMA has also revealed some problems, such as poor bioactivity and biocompatibility, easily burns surrounding tissue as to it's a large amount of heat dissipated by the curing process [11][12][13].Calcium phosphate bone cement has good bioactivity and biocompatibility because it can form hydroxyapatite (HA) after solidification and osseointegration with bone tissue, but it also has disadvantages such as poor adhesion and low mechanical strength [14][15][16].Magnesium phosphate bone cement has good mechanical properties, degradability and bioactivity.However, its short setting time and rapid exotherm limit its clinical use [17][18][19].Calcium sulfate bone cement has good biocompatibility, osteoconductivity, and bone healing promotion, but its application is limited due to its poor mechanical properties and excessive degradation rate [20][21][22].
Magnesium oxychloride cement (MOC) is a kind of MgO-MgCl 2 -H 2 O ternary composite system mixed with active MgO, MgCl 2 and H 2 O [23][24][25].Compared to the bone cement typically utilized in clinical settings, MOC bone cement has excellent mechanical properties and is biodegradable, and the Mg 2+ produced during degradation can promote the absorption of Ca 2+ in the body, which is beneficial to bone repair [26][27][28].Therefore, researchers modified MOC to explore its application in bone repair; for example, Wen et al [28] found that the implantation of MOC into animal bodies was able to induce the formation of new bone; Lowther et al [29] compounded silver nitrate with MOC and found that MOC possessed good antibacterial properties and was non-cytotoxic.These findings indicate that MOC has potential as a new biodegradable bone repair material.Compared with the significant advantages, MOC also has a disadvantage that can't be ignored-its poor water resistance.After the erosion of water, MOC will be rapid hydrolysis, the mechanical properties of a sharp decline.To address this shortcoming, it is mainly through the addition of admixtures to improve its water resistance, commonly used admixtures are phosphoric acid and phosphate, citric acid and citrate, etc [30][31][32].
Strontium (Sr), an essential trace element for the human body, belongs to the same group as calcium in the periodic table of elements, which has similar elemental properties to calcium.Besides, its physiological functions are also closely related to bone formation , and have the functions of promoting the proliferation of proosteoblasts, differentiation of bone marrow mesenchymal stem cells to osteoblasts, mineralization of bone matrix, and inhibition of osteoclast differentiation [33,34].In addition, Liu et al showed that the synergistic effect of Sr 2+ and Mg 2+ release favors cell proliferation and new osteogenesis [35].
In this study, strontium hydrogen phosphate (SrHPO 4 ) was used as a modifier to prepare SrHPO 4 -modified MOC (SrHPO 4 -MOC).On the one hand, phosphate was used to enhance the water resistance of MOC, while Sr was introduced into the MOC system to enhance its biological activity and promoted osteoblast proliferation.The paper investigated the strength, mass loss and microstructural changes of SrHPO 4 on MOC before and after soaking in simulated body fluid (SBF), the changes of Ca 2+ and pH of SBF solution, and the cell viability of SrHPO 4 -MOC.= SrHPO 4 was added in the amounts of 0.0 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%,4.5 wt% and 5.0 wt% of the total weight of MgO and MgCl 2 .The detailed mixture proportions of SrHPO 4 -MOC are shown in table 1 The preparation process of MOC: Weigh each raw material in proportion, added MgCl 2 and SrHPO 4 to H 2 O in turn to get mixed suspension A. Then, added MgO powder to suspension A and stirred uniformly for 5 min, poured the homogeneous slurry mixture into stainless steel cubic molds (10 mm × 10 mm × 10 mm) and The SrHPO 4 -MOC specimens (10 mm × 10 mm × 10 mm, each group was assigned six specimens.)were tested for compressive strength R 0 using a universal material testing machine (CMT-20, Jinan Lian Gong Test Technology Co., Ltd, China), with the loading speed set to 0.5 mm min −1 .Another part of the specimens (six specimens per group) was placed in centrifuge tubes and filled with SBF solution (formulated according to a previous study [36]), the volume ratio of specimens to SBF solution was 1:10.Then the centrifuge tube was placed in a water bath thermostatic shaker (37 °C, 60 rpm) and changed SBF solution every three days.The specimens were taken out after 7 d, 14 d, 28 d, 56 d and 84 d.The compressive strength R n of the specimens after soaking was tested using a universal material testing machine (n was the soaking time).The softening factor I f of the specimens was then calculated by equation (1) to assess their water resistance.

Materials and methods
( )

Degradation assay in vitro
The SrHPO 4 -MOC cylindrical (Ф10 mm × 2 mm) specimens were rinsed using anhydrous ethanol, the specimens were then dried in an electric blast dryer for three hours at a temperature setting of 60 °C.The dried specimens were weighed and the weight was recorded as M 0 .The specimens were then placed in centrifuge tubes containing SBF solution in a 1:10 volume ratio of specimens to SBF solution.The SBF solution was refreshed every 3 d.The specimens were immersed for 7 d, 14 d, 28 d, 42 d, 56 d, 70 d and 84 d, removed, rinsed with anhydrous ethanol.The specimens were then dried in an electric blast dryer for three hours at a temperature setting of 60 °C.The dried specimens were weighed and the weight was recorded as M t .The weight loss rate (M L ) of the samples was calculated according to equation (2).
pH value The 10 mm × 10 mm × 10 mm SrHPO 4 -MOC specimens were soaked in SBF with a volume ratio of 1:10 between specimens and SBF solution.pH values were tested daily using a pH meter (pH meter type PHS-3E, Shanghai Raycom, China) and the SBF solution was refreshed.

The concentration change of Ca 2+ and Mg 2+
The 10 mm × 10 mm × 10 mm SrHPO 4 -MOC specimens were immersed in SBF with specimens to SBF solution volume ratio of 1:20, and the concentrations of Ca 2+ and Mg 2+ in the SBF solution were tested daily using a fully automatic titrator (T960 Automatic Titrator, Shandong Haineng Instruments, China) without refreshing the SBF solution during the soaking period.

Microscopic morphology and physical composition
A portion of Ф10 mm × 2 mm SrHPO 4 -MOC specimens was immersed in SBF solution for different cycles, removed, and the surface was rinsed with anhydrous ethanol and dried in an electric blast dryer for 3 h at a temperature setting of 60 °C.Determination of the phase composition of samples using a rotary target x-ray diffractometer (Smrtlab, 9KW, RIKEN, Japan), and the surface micromorphology of the specimens before and after soaking was observed by a scanning electron microscope (Merlin Compact, Carl Zeiss NTS GmbH, Germany).

Cell viability experiments 2.4.1. The preparation of modified MOC extracts
The Ф10 mm × 2 mm SrHPO 4 -MOC specimens were sterilized by UV irradiation for 24 h.The cell culture medium was added at a surface area to volume ratio of 1.25 cm 2 ml −1 , and then extracted at 37 °C for 24 h.The extracts were filtered with a 0.22 μm needle filter and set aside.To simulate in vivo conditions more accurately, the extracts were diluted twice for cellular experiments [37][38][39].

MTT method to test cell viability
The Complete medium (containing 10% FBS, 1% PS) was set up and labeled as control.The extract (containing 10% FBS, 1% PS) was added to the 96-well plate after inoculation of cells with complete medium, and 100 μL was added to each well.Viability tests were performed after 1, 3 and 7 days of incubation, and the extract and complete medium were changed every two days during the incubation period, and five rewells were set in each group.For the assay, 10 μL of MTT assay solution was added to each well and incubated for 4 h under light-proof conditions.100 μL of DMSO solution was added after aspirating and discarding the medium, and the 96-well plates were placed on a shaker and shaken at 80 rpm for 10 min.The 96-well plates were then placed in an ELISA and their absorbance (Optical density, OD) values at 492 nm were measured, and the data was processed.

Cell activity was determined by live/dead assay
Calcein-AM, PI and assay buffer were mixed well according to the volume ratio of 1:1:1000 to obtain Calcein-AM/PI assay buffer.The diluted extract was added with complete medium to the 96-well plate after inoculation of cells, and 100 μL was added to each well.After incubation in a CO 2 incubator at 37 °C for 24 h, the culture medium was aspirated and discarded, and the cells were washed once with PBS; 100 μL of the prepared assay buffer was added to each well, and the cells were incubated in a CO 2 incubator for 30 min protected from light and then staining was observed by fluorescence microscopy.The whole process was operated under light avoidance.

Results
3.1.Mechanical strength change, mass loss, pH value and ion concentration of samples Figure 1 showed the changes in compressive strength, mass loss, pH value, and ion concentration of SrHPO 4 -MOC before and after immersion in SBF solution.From the compressive strength and softening coefficient of SrHPO 4 -MOC at different ages of immersion in SBF solution (figure 1(a)), it could be seen that the initial compressive strength of MOC was 112.5±3.9MPa.The addition of SrHPO 4 decreased the initial compressive strength of MOC, and the compressive strength gradually decreased with the increase of incorporation.MOC (without SrHPO 4 ) showed a large number of cracks on the surface and collapsed after soaking in SBF solution for 12 h (Macroscopic images of samples after immersion in SBF solution for 0 and 12 h and corresponding microscopic images in the supplementary data Files figures S2, S3), and its strength rapidly decreased to 0 MPa, while the compressive strength of SrHPO 4 -MOC gradually decreased after soaking in SBF solution, but its softening coefficient was still greater than 0.3 after 28 d.The compressive strength of 4.0 wt% and 4.5 wt% SrHPO 4 before soaking was 92.3 ± 2.6 MPa and 90.9 ± 4.5 MPa, after soaking for 28 days, the compressive strength was 48.9 ± 2.2 MPa and 50.1 ± 1.9 MPa, and the softening coefficient was 0.53 and 0.55.The softening coefficients of the 4.0 wt%, 4.5 wt%, and 5.0 wt% SrHPO 4 groups were 0.18, 0.24, and 0.4 after 56 d of soaking, 0.09, 0.07, and 0.13 after 84 d.This indicated that the addition of SrHPO 4 could significantly enhance the water resistance of MOC, and the shape structure remained intact during the immersion in SBF solution, although the compressive strength gradually decreased.
(Figure 1(b)) showed the mass loss of SrHPO 4 -MOC after immersion in SBF solution.In (figure 1(b)), all groups of samples lost mass during soaking, but the addition of SrHPO 4 was able to slow down the rate of mass loss of MOC, and the slowing down effect was better as the amount of incorporation increased; However, when the incorporation amount reached 5.0 wt%, it did not further reduce the mass loss of MOC, but accelerated the mass loss compared to 4.5 wt%.
From figure 1(c) pH values of SrHPO 4 -MOC in SBF solution after immersion, it could be seen that SrHPO 4 addition caused a rapid increase in pH value of SBF solution during the first 12 h of soaking and gradually decreased after reaching a high value at 24 h; pH value dropped to a low value at 48 h and then gradually increased; after 192 h, pH values of all groups fluctuated between 8.7 and 9.1.
Figure 1(d) showed the changes in Ca 2+ and Mg 2+ concentrations of SBF solution after immersion in SBF solution; we could observe that Ca 2+ concentration gradually decreased with the extension of immersion time, and the decrease increased with the increase of SrHPO 4 doping; while the Mg 2+ concentration increased with the increase of immersion time.

Physical phase composition and micromorphology
Figure 2 showed the XRD patterns of SrHPO 4 -MOC at 1 d of air conditioning and at different cycles of immersion in SBF solution.Figure 2(a) showed that the hydration products of MOC were mainly phase 5 and a few of Mg(OH) 2 after 1 d of air conditioning, while the phase composition of SrHPO 4 -MOC was mainly the hydration product phase 5 and unreacted MgO. Figure 2(b) showed that the phase composition of SrHPO 4 -MOC was phase 5 and MgO after 7 d of soaking in SBF solution, where 3.0 wt% and 3.5 wt% SrHPO 4 -MOC groups with weak MgO peaks, indicating a high degree of reaction of MgO with water and poor water resistance; figure 2(c) showed that after 14 d of soaking in SBF solution, the peaks of MgO in SrHPO 4 -MOC all decreased and weak HA peaks appeared; by 28 d (figure 2(d)), the MgO peaks of all samples were no longer obvious, the 5-phase peaks weakened, and the Mg(OH) 2 peaks were significantly enhanced, among which only Mg(OH) 2 peaks appeared in the 3.0 wt% group of samples, and 5-phase is not obvious; indicating that with the prolongation of soaking time, MgO reacted almost completely with water to form Mg(OH) 2 , while part of 5-phase was also decomposed into Mg(OH) 2 , which caused the weakening of the peak, and the appearance of HA indicated that SrHPO 4 -MOC had good bioactivity SrHPO 4 doping of 4.0 wt%, 4.5 wt% and 5.0 wt% groups after 56 d, 84 d of immersion (figures 2(e), (f)) showed that the main crystalline phase was still phase 5 and Mg(OH) 2 , while the HA content increased.
Figure 3 shows the SEM images of SrHPO 4 -MOC after curing in air for 1 day and after immersion in SBF for different periods.After 1 d of air conditioning, a large number of intertwined needle-like 5-phase crystals appeared on the surface of both MOC and SrHPO 4 -MOC, but compared with MOC (figure S1 ), the needle-like crystals on the surface of SrHPO 4 -MOC (figure 3 a 1 , b 1 , c 1 , d 1 , e 1 ) were more densely intertwined, and with the increase of SrHPO 4 , these needle-like crystals intertwined on the surface of SrHPO 4 -MOC formed a dense platelike structure and made the SrHPO 4 -MOC surface denser.
After immersion in SBF for 7 d, the surface of SrHPO 4 -MOC was still densely interwoven with needle-like 5-phase crystals, but some tiny spherical crystals could be observed deposited on the sample surface, and EDS showed that the spherical material contained Ca and P elements, and it was speculated that these spherical crystals might be HA formed by the deposition of Ca 2+ and PO 4 3-plasma from SBF solution onto the surface of SrHPO 4 -MOC in combination with XRD patterns.After 14 days of immersion in SBF solution, the amount of HA deposited on the surface of SrHPO 4 -MOC increased, but the surface of the specimens with less SrHPO 4 incorporation (figure 3 a 3 , b 3 ) showed pores due to severe water erosion, and the denseness decreased.Cracks and voids appeared on the surface of both groups after 28 d of immersion (figure 3 a 4 , b 4 ), while the surface of the three groups with more SrHPO 4 doping was a denser structure (figure 3 c 4 , d 4 , e 4 ).
SrHPO 4 doping 4.0 wt%, 4.5 wt% and 5.0 wt% groups showed a large accumulation of spherical material and pores on the surface at 56 d of soaking SEM images (figure 3 c 5 , d 5 , e 5 ).After 84 d of soaking, denser structures were again observed on the surfaces of the two groups (figure 3 c 6 , d 6 ), except for the 5.0 wt%.This phenomenon indicated that SrHPO 4 -MOC might be degraded layer by layer, firstly the surface in contact with SBF solution was degraded but the internal structure was still dense at this time, with the erosion of water, the surface dense structure gradually degraded and became loose and porous, exposing the internal dense structure; when the surface of SrHPO 4 -MOC degraded, the internal dense structure would act as the 'surface' again, thus preventing the erosion of water.

Cell toxicity test
Figure 4 showed the staining results of Calcein/PI after co-culture of MOC extract with BMSC cells.Since 3.0 and 3.5 wt% SrHPO 4 -MOC had faster strength loss and poor water resistance in SBF solution, we chose 4.0, 4.5, and 5.0 wt% SrHPO 4 -MOC, which had better compressive strength and water resistance, for the cell experiments.It could be seen from the figure that the number of live cells in the MOC group was less than that in the SrHPO 4 -MOC groups, and the number of dead cells was more than that in the SrHPO 4 -MOC group.The MTT assay results of BMSC and SrHPO 4 -MOC extracts cultured in vitro for different times were shown in figure 5. From the figure, we could observe that relative cellular activities of the MOC group cultured in the extracts for 1 d and 7 d were lower than 75%, while the relative cellular activities of the SrHPO 4 -MOC groups were higher than 75% and even higher than the control group, this drew the same conclusion the Calcein/PI staining results, suggesting that SrHPO 4 addition contributed to increased cell activity.

Discussion
MOC is usually used in the construction material industry because of its good mechanical properties and flameretardant performance, but its poor water resistance restricts its large-scale application [40][41][42].However, on the other hand, MOC is an easy reaction with water which can be used as a biodegradable bone repair material, and by improving the water resistance of MOC and controlling its leaching rate, it can be used as a bone repair material that can control the rate of degradation.
The compressive strength of artificial bone repair materials is a key factor in whether they can be used in humans [43].The compressive strength of human cancellous bone and cortical bone is about 2-45 MPa and 90-230 MPa, and the compressive strength of medical inorganic bone cement used in clinical practice is generally between the two.[28,[43][44][45].The initial compressive strength of MOC was 112.5 ± 3.9 MPa, but it collapsed after only 12 h of soaking in SBF; while SrHPO 4 -MOC had a gradual decrease in compressive strength after soaking in SBF, the structure was intact and the strength still reached 48.9 ± 2.2 MPa after 28 d (containing 4.0 wt% SrHPO 4 ), and the compressive strength of the specimens was still 8.2 ± 0.9 MPa after 84 d of immersion.It meets the strength requirement of bone cement for clinical use.The loss of MOC strength is mainly due to its 5-phase hydrolysis and hydration of MgO after erosion by water in SBF, with the following reaction equation [46]:

+ 
When MOC is soaked in aqueous solutions, on the one hand, the residual MgO and phase 5 will continuously hydrolyze to produce Mg(OH) 2 , resulting in a gradual decrease in the content of phase 5. Since phase 5 is the phase that contributes the most to the strength enhancement of MOC [47], a decrease in phase 5 causes a subsequent decrease in the strength of MOC; while residual MgO is the skeleton phase of MOC, the reaction of MgO will destroy the structure of MOC, which will also reduce its compressive strength.On the other hand, an increase in the amount of Mg(OH) 2 produced by the reaction causes an increase in OH -and a gradual increase in pH value (figure 1(c)).The XRD results (figure 2) also showed that the intensity of the characteristic peaks of phase 5 gradually decreased and the characteristic peaks of Mg(OH) 2 appeared and continuously increased with the increase of immersion time.And the improvement of water resistance of MOC by SrHPO 4 may be due to the dense surface of SrHPO 4 -MOC.The dense structure reduces the channels for water to enter the MOC and slows down water erosion, which can reduce the 5-phase hydrolysis and hydration of MgO.
Biodegradability is another requirement for bone cement as this can lead to faster in vivo resorption and cellular infiltration [48].However, biodegradable bone repair materials should have a suitable degradation rate, on the one hand, they should not degrade too fast, and new bone will not be generated yet if they degrade too fast so that there will be a hole structure in the defect and lower strength, which is not conducive to supporting human activities [49]; On the other hand, if the degradation rate is too slow, the material will not fully integrate with the surrounding tissue and may pose a risk associated with chronic foreign bodies [50].Clinically, bone grafting for bone defects usually takes 4 to 6 months to recover [51].After SrHPO 4 -MOC was immersed in SBF for 12 w (figures 1(a), (b)), the mass loss rate of SrHPO 4 -MOC was 46%-50% and retained some strength, so it could continue to provide support strength in the late stage of bone healing.
A successful bone repair material also needs to have good bioactivity.In vitro, bioactivity can be evaluated by immersing the biomaterial in SBF and evaluating its osseointegration ability by the ability to form a bone-like HA layer on the surface [52,53].As seen in figure 1   Figure 2 showed that spherical substances containing Ca, P, O and Sr elements were generated on the surface of each group of SrHPO 4 -MOC samples after immersion in SBF solution, both the combination of XRD patterns (figures 2 (c), (d)) and the gradual decrease of Ca 2+ concentration (figure 1 (d)) showed that the spherical substances were Sr-doped HA.SEM images showed that the deposition of HA was observed at different cycles of immersion, which indicated that new HA was continuously generated with the layer-by-layer degradation of MOC, meaning that SrHPO 4 -MOC has good bioactivity.
The cellular activity and cell proliferation of the materials could be examined by fluorescence staining and the MTT method.As seen in figures 4 and 5, the addition of SrHPO 4 could improve the cell survival of MOC, reduce cytotoxicity and promote cell proliferation, which may be due to the release of Sr 2+ ions from SrHPO 4 -MOC during the leaching process to promote cell proliferation [33,56].

Conclusions
In this paper, SrHPO 4 was used to modify MOC, and the following conclusions were obtained by studying the mechanical strength change, degradation performance, and cellular activity of MOC in different cycles of immersion in SBF.
(1) After SrHPO 4 is added to MOC, its mechanical properties are decreased, but water resistance is improved, in which the strength of 4.0 wt%-SrHPO 4 -MOC before immersion is 92.3 MPa, and after 84 d of immersion, the strength is 8.2 MPa, which can satisfy the use of bone cement.
(2) The microscopic morphology of MOC was changed by the addition of SrHPO 4 , but not its physical phase composition.The improvement of water resistance was mainly because the surface structure became denser, preventing moisture erosion of MOC.
(3) Spherical HA was formed on the surface of SrHPO 4 -MOC after immersion in SBF solution; cellular experiments showed that the relative cellular activities of SrHPO 4 -MOC were all above 75%, which was harmless to the cells.
In a word, SrHPO 4 -MOC has a suitable degradation rate and biological activity and is expected to be a new biodegradable bone repair material.

Figure 1 .
Figure 1.Compressive strength, mass loss, pH value and ion concentration changes of SrHPO 4 -MOC before and after immersion in SBF solution (a) Compressive strength and softening coefficient; (b) Mass loss; (c) pH value; (d) Concentration changes of Ca 2+ and Mg 2+ .

Figure 2 .
Figure 2. XRD patterns of the SrHPO 4 -MOC after curing in air for 1 day and immersion in SBF for different periods.

Figure 3 .
Figure 3. SEM images of SrHPO 4 -MOC after curing in air for 1 day and immersion in SBF for different periods.
(c), the pH value of the SBF solution in which SrHPO 4 -MOC specimens were soaked fluctuated between 8 and 9.1, indicating that the degradation of MOC can create an alkaline environment for the surrounding body fluid, which is conducive to the formation of HA [54, 55].

Figure 4 .
Figure 4. Calcein/PI staining results of MOC and modified MOC extracts co-cultured with m-BMSCs for 1 day.

Figure 5 .
Figure 5. Relative cell activity of SrHPO 4 -MOC extracts co-cultured with m-BMSCs at different time.