Isolating the Effects of Mg²⁺, Mn²⁺ and Sr²⁺ Ions on Osteoblast Behavior from those Caused by Hydroxyapatite Transformation

Abstract

Ionic trace elements such as Mg²⁺, Mn²⁺ and Sr²⁺ are very difficult to stabilize in the hydroxyapatite lattice, and they can induce phase transformations when subjected to thermal treatments. The effects of the ions themselves are often confused with the effects of the newly formed phases. Therefore, the objective of this work was to evaluate the effects of the ions isolated from the effects caused by the inherent phase transformations observed in these systems. WDXRF, XRD, FTIR and biological assays using mouse preosteoblastic cells (MC3T3-E1) showed the Mg²⁺ ions can stimulate cell differentiation even when segregated from the HA structure in the form of MgO. However, the presence of MgO considerably retards cell proliferation. At the same time, Mn²⁺ ions are able to increase cell proliferation and induce the production of high levels of ALP, regardless of whether they are inserted into the HA structure or segregated in the form of Mn₃O₄. Finally, the presence of Sr²⁺ in the HA lattice does not appear to directly affect cell behavior, since both the proliferation and production of ALP are comparable to those observed in the nondoped HA sample.

Keywords:
hydroxyapatite; trace element; osteoblast behavior; MC3T3-E1; phase transformation

1. Introduction

The insertion of trace elements into the hydroxyapatite structure [Ca₁₀(PO₄)₆(OH)₂] is widely proposed as an important way to improve the biocompatibility of bone grafts and implants used in the treatment of bone diseases^1-3. Its structure can accommodate doping ions of different sizes and charges, either individually or simultaneously^4-6. The family of apatite minerals have the general formula M₁₀(XO₄)₆(Y)₂, where M refers to mono-, di- or trivalent ions, such as Ag⁺, Na⁺, Ca²⁺, Sr²⁺, Mg²⁺, Mn²⁺ and Zn²⁺; XO refers to PO₄^3-, CO₃^2-, HPO₄^2- or SO₄^2-; and Y refers to OH^-, CO₃^2-, Cl^- or F^-6-8. Apatite minerals can be found in nature from metamorphic transformations in the Earth's crust^9-11 or from biomineralized deposits in living organisms^12-14. The biomineralized hydroxyapatite found in bone tissue is also highly substituted with trace elements, making it a large reservoir of ionic nutrients necessary for life^5,12.

Being multisubstituted, biomineralized hydroxyapatite is very different from typical hydroxyapatites synthesized in the laboratories, and it is easier to evaluate the effect of one element on the cells if this element is the only dopant in the structure. For this reason, the isolated effects of trace elements on cells, specifically cells in bone tissue, have been continuously studied. Mg²⁺ influences bone metabolism, stimulating osteoblast proliferation, especially at the beginning of osteogenesis^2,15,16. Mn²⁺ is involved in the regulation of bone remodeling and activates integrins, improving cellular adhesion^5,17,18. Sr²⁺ exhibits beneficial effects on bone formation and resorption processes through osteoblast stimulation and inhibition of osteoclast activity^2,19-21. However, these effects are often attributed to one specific element without accounting for the contributions of the phase transformations that occur in the studied system. The presence of trace elements in hydroxyapatites can produce small phase transformations during thermal treatments, which are required due to the difficulty of introducing these elements into the apatitic lattice^15,22-27. Therefore, in many cases, the effects attributed to one element in the doped hydroxyapatites are mixed with those caused by the presence of phases that are unaccounted for. For instance, Webler et al.²⁸ observed that the level of Mg²⁺ and the concentration of phases in biphasic ceramics (β-TCP/hydroxyapatite) had no effect on macrophage viability after 24 h of culture. In this case, two factors were simultaneously influencing this system: Mg²⁺ and the β-TCP/hydroxyapatite concentration. Bhattacharjee et al.²⁹ attributed the differences observed in the in vivo behavior of hydroxyapatite implants to the presence of Zn²⁺ but did not consider the presence of the β-TCP phase generated when these implants were calcined at 800 and 1150°C. Likewise, Ullah et al.³⁰ studied the role of Fe/Sr-doped dielectric hydroxyapatites on the adhesion and proliferation of human mesenchymal stem cells (hMSCs). The singular Fe-substituted group showed better cell proliferation than the singular Sr-substituted group and the pristine group. This difference was attributed to the biological role of the Fe and Sr ions released from the ceramic over time. However, the insertion of Fe and/or Sr into hydroxyapatite induced a phase transformation at 1100°C, yielding a multiphasic ceramic with at least 52% β-TCP and different amounts of Fe₂O₃. Again, the effect of the dopant itself was overlaid with the effect of the presence of different phases in the sample. Therefore, many of the effects attributed to one specific element in the doped hydroxyapatites on cellular behavior or in vivo performance are not adequately explained.

Recently, we studied mechanisms by which it is possible to stabilize several cations in a multisubstituted hydroxyapatite³¹. We verified that some ions that are more difficult to insert into the hydroxyapatite lattice can be more easily incorporated if they are in the presence of other cations or counterions such as carbonate^31,32. Using the Rietveld refinement on the X-ray diffraction data, the insertion of the ions into the hydroxyapatite phase or their segregation in other phases during calcination could be confirmed. Therefore, our objective in this work was to start from similar systems comprising Mg²⁺-, Mn²⁺- and Sr²⁺-doped hydroxyapatite to isolate the effects on osteoblast behavior caused by the doping element itself from those caused by the presence of other phases formed during the heat treatment.

2. Materials and Methods

2.1. HA synthesis

2.1.1. Pure HA

The pure HA powder was obtained by a aqueous precipitation method through a reaction between phosphoric acid (0.220 mol L^-1) and calcium hydroxide (0.334 mol L^-1). The reaction was conducted at 60°C under stirring, and the pH was maintained at 10 with the addition of sodium hydroxide. After 24 h of aging, the precipitate was filtered, washed with distilled water, dried at 120°C for 24 h, and sieved (106 μm).

2.1.2. Doped HA

The same procedure described above was employed to synthesize the doped HA samples. Sr²⁺, Mg²⁺ and Mn²⁺ ions were added to the reaction medium as follows: magnesium and strontium chloride were dissolved in the calcium hydroxide suspension, while manganese chloride was dissolved in the phosphoric acid solution to avoid Mn²⁺ oxidation. The relative ratio between the doping ions and Ca²⁺, i.e., M²⁺/(Ca²⁺ + M²⁺), was kept at 0.04 (4 mol %). The reagents were purchased from Sigma-Aldrich and had a purity above 99%.

2.2. Powder processing

The thermal stability of the samples was studied after calcining the obtained powders for 2 h at 1000°C under a heating rate of 2.8°C/min. For the cell culture assays, pellets 10 mm in diameter and 1.0 mm in height were made by uniaxial pressing of the prepared HA powders under a load of 237 MPa. After pressing, the obtained tablets were calcined under the conditions described above for the powders.

2.3. Physicochemical characterization

2.3.1. Elemental composition

The elements in the samples were identified and quantified by wavelength-dispersive X-ray fluorescence spectroscopy (WDXRF) using a Bruker S8-Tiger 4 kW spectrometer equipped with LiF 200, PET, XS-5 and XS-C crystals. The calcined powders were pressed into pellets and directly analyzed. A calibration curve was established using a standard hydroxyapatite (Ca/P = 1.67) powder mixed in 6 different proportions with standard strontium, manganese and magnesium nitrate powders. The mixtures were homogenously ground in a ball mill apparatus for 30 min at 350 rpm, and a curve was constructed by analyzing each mixture in tablet form. All reagents were of high purity (≥ 99.5%) and were purchased from Sigma-Aldrich.

2.3.2. Structural changes

The structural changes were studied by X-ray diffraction (XRD) using a Shimadzu XRD 6000 diffractometer (CuKα λ = 1.5405 Å; 60 kV, 55 mA, Ni filter, scintillation detector). The samples were scanned from 10º to 60º with a step size of 0.02º and an acquisition time of 2 s per point. The diffraction patterns were refined using the Rietveld method (HighSchore Plus), which allowed us to follow the phase transformations and lattice parameter changes in the hydroxyapatite phase.

2.3.3. Molecular groups in the HA phase

The changes in the functional groups in the HA phase, such as PO₄^3- and OH^-, were observed by Fourier transform infrared (FTIR) spectroscopy on a Perkin-Elmer 1720X apparatus. The insertion of HPO₄^2-, CO₃^2- and other species was investigated. The spectra were acquired in the range of 4000 to 400 cm^-1 with a resolution of 1 cm^-1. The samples were prepared by mixing with anhydrous KBr and pressing into pellets.

2.4. Biological assays

2.4.1. Osteoblast culture

Mouse preosteoblastic cells (MC3T3-E1) were cultured in 75-cm² flasks containing alpha-MEM culture medium (Sigma) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) in an incubator at 37ºC under a humidified atmosphere of 95% air and 5% CO₂. After reaching confluence, the cells were trypsinized, resuspended in complete medium and counted in a Neubauer chamber. The ceramic samples were sterilized by heating in air at 180ºC for 2 h. Sterilization by autoclaving was avoided because of the possible induction of surface phase transformations and dissolution. The samples were placed into a culture plate and immersed for 1 h in 1000 μL of alpha-MEM culture medium (Sigma-Aldrich) supplemented with 10% FBS and antibiotics. Then, the medium was discarded, and 15 μL of cell suspension containing 1.0×10⁴ cells was seeded on each sample, and the samples were left for 1 h in an incubator to allow cell adhesion onto the sample instead of the bottom of the well. The wells were then filled with 1000 μL of the culture medium supplemented with antibiotics. The cells were monitored after 7, 14 and 21 days of culture.

2.4.2. Cellular growth: MTT assay

At each time point, the samples were gently rinsed with PBS solution to remove nonadherent cells. Five hundred microliters of 5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] solution (Sigma-Aldrich) was added to the remaining cells, and they were incubated for 3 h at 37°C under a humidified atmosphere of 95% air and 5% CO₂. Viable cells convert the yellow tetrazolium salt (MTT) into blue formazan crystals. Afterwards, the MTT was removed, and the cells were immersed in 500 μL of acidic isopropanol to dissolve the intracellular formazan crystals produced by live cells. The absorbance was determined at 570 nm using an ELISA reader. The cell number was obtained based on the linear correlation between the absorbance and the MC3T3-E1 cell concentration (from 1×10⁴ to 5×10⁴ cells/mL). The cell number was adjusted to the surface area of the sample and is expressed in cells/mm². The experiment was conducted in triplicate.

2.4.3. Cellular differentiation: ALP activity

Alkaline phosphatase (ALP) activity was assessed by examining the hydrolysis of p-nitrophenyl phosphate (Sigma-Aldrich) in an alkaline buffer solution (substrate). At each time point, the cells plated on the surfaces were permeabilized in 0.5% Triton X-100 (octylphenol ethoxylate) (Sigma-Aldrich) in water and incubated for 30 min with the substrate. The reaction was ended by the addition of 0.1 mol L^-1 EDTA in a 1 mol L^-1 NaOH solution. Colorimetric determination of the product (p-nitrophenol) was carried out at 405 nm (ELISA reader). ALP activity was calculated from a standard curve (from 50 up to 3200 nmol p-nitrophenyl phosphate/L), and the results were expressed in nanomols (nmol) of p-nitrophenol produced per 10⁴ cells.

2.5. Statistical analyses

All values are expressed as the mean ± standard deviation. The statistical significance of the obtained data was assessed using one-way ANOVA followed by Tukey’s test. Differences at p ≥ 0.05 were considered not statistically significant.

3. Results and Discussion

In a previous work³¹, we demonstrated that the amounts of Sr²⁺, Mg²⁺ and Mn²⁺ ions present in the precipitated hydroxyapatite phase were not similar. Since their concentrations in the reaction medium were not equal, it was not possible to precisely evaluate the competition among the elements for sites in the hydroxyapatite lattice or their isolated effects on the physicochemical and biological properties. In addition, two different counterions were used during the synthesis (chloride and nitrate), making the obtained results more complex, as discussed in another work³². Therefore, in the present work, we made significant changes to the methodology to specifically evaluate the isolated effects of each element on the physicochemical and biological properties, as follows: a) the relative ratio between the doping ions and Ca²⁺, i.e., M²⁺/(Ca²⁺+M²⁺) was kept at 0.04 (4 mol %) for all conditions and b) only chloride was used as a counterion. After making these changes, we considerably reduced the number of variables, making it possible to isolate the effects of each element on the hydroxyapatite properties.

3.1. Effect of the doping elements on the hydroxyapatite structure

The results obtained by WDXRF demonstrated that the presence of one element did not affect the capture of others during the precipitation process. The amount of Mg present in the samples was not significantly influenced by the presence of Sr or Mn (Figure 1a). Similarly, the amount of Mn was not influenced by the presence of Mg or Sr (Figure 1b), and the amount of Sr was not influenced by the presence of Mg or Mn (Figure 1c). However, the amount of Sr was always lower than the measured amounts of Mg and Mn. This means that, in competitive terms, the capture of Sr²⁺ during the precipitation process was less favorable than the capture of Mg²⁺ and Mn²⁺ when they were present at the same concentration in the reaction medium.

Figure 1
Amount of each dopant in the samples as measured by WDXRF. The effect of Mn²⁺ and Sr²⁺ on the amount of Mg²⁺ (a), the effect of Mg²⁺ and Sr²⁺ on the amount of Mn²⁺ (b), and the effect of Mg²⁺ and Mn²⁺ on the amount of Sr²⁺ (c). *Significantly different from the amount of the element in the initial mixture: Mg²⁺ (a), Mn²⁺ (b) and Sr²⁺ (c).

The Gibbs free energies of hydration (Δ_hydG°/kJ mol^-1) for Sr²⁺, Ca²⁺, Mn²⁺ and Mg²⁺ ions are 1380, 1505, 1760 and 1830, respectively³³. In general, the higher the energy of hydration is, the stronger the interaction between the ion and water molecules in the solvation shell³⁴. This strong interaction hinders further interactions between the ion and interfaces, for instance, in an adsorption process. Thus, it was expected that the capture of Mn²⁺ and Mg²⁺ by the hydroxyapatite clusters during nucleation and growth would be less pronounced than that observed for Sr²⁺ ions. In this case, the energy of hydration should not be the most important factor influencing the accommodation of these ions in the HA structure. In fact, the HA structure presents two Ca²⁺ sites (Ca[I] and Ca[II]) with different chemical environments that accommodate ions in different ways. Therefore, the affinity of these ions for each site must be the dominant factor controlling the amount of ions incorporated into the structure.

Precipitation occurred without atmospheric control, and under such conditions, CO₂ can be dissolved in the reaction medium, resulting in CO₃^2- ions³⁵. This means that, in addition to the doping elements (Mg²⁺, Sr²⁺ and Mn²⁺), CO₃^2- ions were also available to be captured during the precipitation process. The presence of CO₃^2- in the obtained sample was monitored by FTIR spectroscopy. The relative intensity changes in the ν₂ CO₃^2- and ν₄ PO₄^3- absorption bands made it possible to estimate the relative levels of CO₃^2- captured by the precipitates. The results demonstrated that the presence of the doping elements affected the capture of CO₃^2-. In general, the presence of Mg²⁺, Sr²⁺ and Mn²⁺ inhibited the capture of CO₃^2- ions (Figure 2), which was verified by the remarkable decreases in the ν₂ CO₃^2-/ν₄ PO₄^3- intensity ratios observed in the spectra of the as-synthesized samples. The calcination process decreases these intensity ratios even more, meaning that CO₃^2- groups were released during the thermal treatment in the form of CO₂^31,32,36. This release was more pronounced for the Mn²⁺-containing samples and less pronounced for the pure and Sr²⁺-containing samples. The inherent inclusion of CO₃^2- into the apatite lattices during precipitation is very well known and has also been observed in biomineralization processes^35,37. In several cases, the simultaneous inclusion of this ion along with other doping elements into an apatite lattice can thermally stabilize the structure^31,32,38,39.

Figure 2
FTIR spectra and intensity ratios for the ν₂ CO₃^2- and ν₄ PO₄^3- absorption bands before and after calcination at 1000°C. The effect of Mn²⁺ and Sr²⁺ on Mg²⁺ (a), the effect of Mg²⁺ and Sr²⁺ on Mn²⁺ (b), and the effect of Mg²⁺ and Mn²⁺ on Sr²⁺ (c).

The (Ca+M)/P ratios measured for all samples were always above 1.67 (Table 1), indicating the formation of nonstoichiometric hydroxyapatites. (Ca+M)/P ratios above 1.67 could mean either an excess of cations or insufficient phosphate groups^13,40. As observed by FTIR spectroscopy (Figure 2), the sample obtained in the absence of a dopant (HA) exhibited the highest ν₂ CO₃^2-/ν₄ PO₄^3- ratio, with an evident B-type substitution (replacement of the PO₄^3- groups with CO₃^2-)⁴¹. Therefore, the high (Ca+M)/P ratio measured for the HA sample was likely caused by the insertion of CO₃^2- into the PO₄^3- sites. As mentioned before, the ν₂ CO₃^2-/ν₄ PO₄^3- ratio was significantly lower for the samples prepared in the presence of the dopants. In these cases, the high values observed for the (Ca+M)/P ratios could mean the effective insertion of cations into the hydroxyapatite structure, especially if one considers that no phases other than hydroxyapatite were present in the as-synthesized samples, as demonstrated by the XRD analyses (Figure 3a). The presence of the doping elements did not induce the precipitation of phases other than hydroxyapatite. However, a high (Ca+M)/P ratio for the hydroxyapatite structure means that the excess cations are likely segregated in the form of oxides during the thermal treatments. Indeed, after calcination at 1000°C, the hydroxyapatite phase became more crystalline (Figure 3b), and small phase transformations were observed, which were appropriately quantified by Rietveld refinement.

Table 1
Ca/P and (Ca+M)/P ratios obtained from WDXRF. M = Mg, Sr and/or Mn.

Samples	Ca/P	(Ca+M)/P
			HA	1.74	1.74
HA-Mg	1.68	1.75
HA-Sr	1.66	1.71
HA-Mn	1.65	1.72
HA-MgSr	1.63	1.74
HA-MgMn	1.65	1.78
HA-SrMn	1.63	1.74
HA-MgSrMn	1.60	1.77

Figure 3
XRD patterns of the a) as-synthesized samples and b) samples after calcination at 1000°C.

The HA sample was altered by thermal treatment, producing 5.3 wt. % calcium oxide (CaO) (Figure 4). The insertion of Mg²⁺ reduced the CaO formation to 0.3 wt. % but induced the appearance of 1.8 wt. % magnesium oxide (MgO). This indicates the segregation of approximately 2.7 mol % of Mg²⁺ from the as-synthesized hydroxyapatite phase. Considering the standard deviation observed in the WDXRF results (Figure 1), one can conclude that all the Mg²⁺ ions were segregated from the apatite phase, having been transformed into MgO after calcination.

Figure 4
Phase composition of each sample after calcination at 1000°C as a function of the doping elements. The effect of Mn²⁺ and Sr²⁺ on Mg²⁺ (a), the effect of Mg²⁺ and Sr²⁺ on Mn²⁺ (b), and the effect of Mg²⁺ and Mn²⁺ on Sr²⁺ (c).

The effect of the simultaneous insertion of Mg²⁺ and Sr²⁺ did not appear to be different from the effect of Mg²⁺ alone (Figure 4a). However, the simultaneous insertion of Mg²⁺ and Mn²⁺ inhibited the formation of CaO and MgO and resulted in the appearance of 3.6 wt. % Mn₃O₄. If we take into account the 2.3 mol % Mn²⁺ measured in the HA-MgMn sample and the error associated with the WDXRF analysis, it is possible to say that all Mn²⁺ ions were segregated from the apatite phase after calcination. The same behavior was observed for the simultaneous insertion of the three elements (Figure 4a), indicating that Sr²⁺ had a negligible effect on this phase transformation. On the other hand, the insertion of Mn²⁺ alone (Figure 4b) completely inhibited the formation of CaO relative to that seen in pure HA, and Mn₃O₄ was also not formed.

The small influence of Sr²⁺ on these phase transformations was confirmed by the fact that the insertion of this ion alone reduced the formation of CaO from 5.3 wt. % (HA sample) to 2.0 wt. % but the transformations previously attributed to the Mg²⁺ and Mn²⁺ ions were unchanged (Figure 4c). In fact, the formation of strontium oxide (SrO) was not observed, suggesting that the Sr²⁺ ions are adequately stabilized in the apatite structure, regardless of the insertion of the other ions.

To confirm whether the doping elements were inserted into the apatite lattice or were completely segregated, as suggested in the previous discussion, we analyzed the changes in the hydroxyapatite unit cell volume before and after calcination for all conditions (Figure 5). Because it is smaller than Ca²⁺ (1.00 Å)^13,33, Mg²⁺ (0.72 Å) should induce a decrease in the unit cell volume. However, no significant changes were observed for the HA-Mg sample compared to the HA sample (Figure 5a). This behavior can be evaluated by comparing the samples before and after calcination. Before calcination, one can suppose that the unit cell volume measured for the HA-Mg sample already included the volume contraction caused by the insertion of the Mg²⁺ ions into the hydroxyapatite lattice. Similarly, the insertion of CO₃^2- ions into the apatite lattice is also known to decrease the unit cell volume^42,43. If one considers that the amount of CO₃^2- inserted in the HA sample was considerably higher than that inserted in the HA-Mg sample, one can conclude that the volume contraction induced by the Mg²⁺ insertion in the HA-Mg sample was comparable to the contraction induced by the CO₃^2- insertion in the HA sample. This seems to be confirmed because after calcination, the volume remained the same for both the HA and HA-Mg samples. That is, after the release of the Mg²⁺ and CO₃^2- ions, the hydroxyapatite phase had a composition closer to its standard stoichiometry in both the HA and HA-Mg samples.

Figure 5
Unit cell volume calculated from the HA phase before and after calcination at 1000°C as a function of the doping elements. The effect of Mn²⁺ and Sr²⁺ on Mg²⁺ (a), the effect of Mg²⁺ and Sr²⁺ on Mn²⁺ (b), and the effect of Mg²⁺ and Mn²⁺ on Sr²⁺ (c). *Significantly different from the HA samples. **Significant difference between the as-synthesized and calcined samples in the same group.

The incorporation of Mn²⁺ ions tended to decrease the unit cell volume (Figure 5b). As discussed before, the insertion of Mn²⁺ alone or with Sr²⁺ ions was favorable when no phase transformation was observed. However, with associated with Mg²⁺, the Mn²⁺ ions were completely segregated as Mn₃O₄ after calcination. For this reason, the volume contraction caused by the insertion of Mn²⁺ ions into the HA lattice was clearly observed.

In contrast to Mg²⁺, Sr²⁺ ions significantly increased the unit cell volume of the hydroxyapatite phase (Figure 5c). Because it is larger than Ca²⁺, Sr²⁺ (1.28 Å) tends to increase the unit cell volume⁴⁴. When Sr²⁺ is no longer segregated during the calcination process in the form of SrO or other Sr-rich phases (Figure 4), its presence in the lattice did not cause any volume increase even after calcination. Therefore, it is possible to say that all the Sr²⁺ ions inserted into the samples are included in the hydroxyapatite phase.

3.2. Biological behavior

To isolate the effect of each element on cellular behavior, only the samples containing Mg²⁺, Mn²⁺ or Sr²⁺ ions alone and the samples containing all three ions were compared with the nondoped HA sample.

After 7 days of culture, all the samples had essentially the same number of cells, except for the HA-Mg sample, which had a lower number of cells (Figure 6). This indicates that up to 7 days, the HA-Mg sample did not favor proliferation. However, at 14 days of culture, the number of cells significantly increased for all the samples. The proliferation profiles showed that the cells grew almost linearly over time. Thus, the cell growth rate for each sample could be calculated by deriving the number of cells as a function of time. The results showed that the highest proliferation rate occurred on the Mn²⁺-containing samples (HA-Mn and HA-MgMnSr). The HA-Mn sample had the highest proliferation rate among all the samples (91 ± 26 cells mm^-1 day^-1), followed by the HA-MgMnSr sample (76 ± 2 cells mm^-1 day^-1). The lowest rate was observed on the HA sample (10 ± 2 cells mm^-1 day^-1). Although the HA-Mg sample had the lowest number of cells after 7 days of culture, the proliferation rate was even higher than that of the cells on HA until the end of 21 days (36 ± 2 cells mm^-1 day^-1). According to the phase composition data (Figure 4), all the Mn²⁺ ions in the HA-Mn sample were inserted into the apatite, but in the HA-MgMnSr sample, they were segregated as Mn₃O₄; nevertheless, the insertion of Sr²⁺ and Mg²⁺ was confirmed. One can assume that manganese plays a more significant role in cell proliferation, probably due to its vital role in the binding of integrins to ligands, which is the same process by which cells bind to substrates⁴⁵, stimulating osteoblast activity⁴⁶. Huang et al.⁴⁷ cultivated MC3T3-E1 cells onto titanium discs coated with Mn²⁺-doped hydroxyapatite and demonstrated a higher proliferation rate compared to cells on a pure hydroxyapatite coating after 7 days, corroborating our findings.

Figure 6
Typical time profile for MC3T3-E1 growth on the original and doped samples. *Significantly different from the HA sample at the same time point. **Significant difference between similar samples relative to the first time point (7 days).

The beneficial effects of individual dopants in the apatite structure seem different when this element is associated with other elements. Inhibitory and stimulatory effects on cellular activity can be produced from this combination of factors^48,49. These changes in activity are caused by physicochemical changes in the calcium phosphate samples, such as phase transformation and changes in crystallinity and solubility, which are induced by the presence of the ions⁴⁸. Therefore, the effect of a dopant competes with other factors rather than being the only factor influencing cellular behavior. For this reason, our results need to be interpreted as a function of the phase composition observed for each sample after calcination. According to the results presented in Figure 4, several samples showed a small amount of oxide in their composition. Unlike Mn₃O₄, CaO and MgO are highly reactive with water, generating Ca(OH)₂ and Mg(OH)₂ as products, respectively. The solubility of the products, Ca(OH)₂ (pK_sp = 5.18) and Mg(OH)₂ (pK_sp = 11.14), at 25°C is much lower than that of Mn₃O₄ (pK_sp = 54.15), indicating that Ca(OH)₂ and Mg(OH)₂ can be readily dissolved in aqueous environments after formation.

It is possible to observe a close correlation between the cell proliferation rate and the presence of CaO and MgO in the samples. The samples that did not present these oxides showed higher proliferation rates (Figure 6). Therefore, one can assume that the oxide reaction would cause a local pH increase, significantly diminishing cellular proliferation. However, it is important to note that all the samples were immersed in supplemented medium for 1 h prior to seeding the cells. This medium was replaced with new medium before seeding the cells. Therefore, the majority of the reactive oxides were no longer on the surface of the samples when the cells attached. In addition, CaO may not have a damaging effect because it was most abundant on the HA sample, and after 7 days of culture, this sample had the highest number of cells among all the studied samples. Therefore, it does not seem reasonable to attribute the decrease in the proliferation rate to the presence of these oxides. Most likely, the dopant played an important role in proliferation. For example, if one excludes the possible negative effect attributed to CaO, the increase in the rate of cell proliferation observed for the Mn²⁺-containing samples can be easily correlated with the presence of the Mn²⁺ ions in the samples.

The production of ALP by the cells reached its highest value after 7 days of culture for all samples (Figure 7). The behavior was the inverse of that observed for proliferation, which is in agreement with the literature. Preosteoblasts, such as MC3T3-E1 cells, differentiate into osteoblasts in a three-step process. The early stage (the proliferation phase) is characterized by substantial DNA synthesis and cell division, resulting in an increase in the cell number. The second stage (matrix maturation stage) is characterized by intense ALP expression, an early marker of osteoblastic differentiation. During the last phase (matrix mineralization), the cells demonstrate both ALP activity and mineralized nodules^50-53. The ALP protein is secreted from the cells after synthesis, and its expression signals the phase at which differentiation is thought to be irreversible in most systems studied⁵⁴.

Figure 7
Cell differentiation of MC3T3-E1 on original and doped samples. *Significantly different from the HA sample at the same time point. **Significant difference between similar samples relative to the last time point (21 days).

Herein, the presence of mature osteoblasts after the proliferation phase is indicated by the production of ALP. The exact mechanism by which this enzyme functions is not completely elucidated, but it seems to both increase the concentration of inorganic phosphates, a mineralization agent, and reduce the amount of extracellular pyrophosphates, an inhibitor of mineral formation⁵⁵. The HA-Mg sample presented the highest level of ALP while exhibiting one of the lowest proliferation rates. Therefore, one can assume that Mg²⁺ stimulated differentiation faster than the other doping ions. In a previous report, Xue et al.⁵⁶ demonstrated that Mg²⁺-doped calcium phosphate favors osteoblastic cellular attachment, proliferation, and ALP production. At the same time, the HA-Mn sample presented the highest proliferation rate and had a lower level of ALP relative to the Mg²⁺-doped HA. One can conclude that Mn²⁺-doped HA increases osteoblast differentiation more than nondoped HA but less than Mg²⁺-doped HA. This indicates that although the Mn²⁺ ions accelerated cell growth, they also improved ALP production (osteoblast differentiation) compared to nondoped HA.

It is also important to note that the increase in cell proliferation promoted by the Mn²⁺ ions in HA-Mn was maintained in the multisubstituted HA sample (HA-MgMnSr). Most likely, the presence of Mn₃O₄ in both samples resulted in these similar effects. However, the differentiation increase induced by the presence of MgO in the HA-Mg sample was remarkably weaker in the HA-MgMnSr sample. Note that in this last sample, all the Mg²⁺ ions are preferentially included in the HA lattice and are not in the form of MgO. This indicates that the effect of Mg²⁺ on differentiation is quite dependent on the phase in which the Mg²⁺ ions are present, most likely because of the differences in solubility and, therefore, the release of the ions into the culture medium.

Considering both proliferation and differentiation assays, all the doped HA samples presented satisfactory results compared to the nondoped HA samples. The Mn²⁺ ions stand out since their incorporation into the apatite was more efficient and led to an outstanding proliferation rate at 21 days and an early differentiation peak at 7 days, indicating that mature osteoblasts were proliferating. Nevertheless, Mg²⁺ ions, which cause the opposite cell behavior, deserve special attention. However, it is clear that the Mg²⁺ ions were not inserted into the apatite lattice.

4. Conclusions

Mg²⁺ ions can stimulate cell differentiation, especially when segregated from the apatite structure in the form of MgO. However, the presence of MgO appears to considerably retard cell proliferation. At the same time, Mn²⁺ ions can increase cell proliferation and induce the production of high levels of ALP, regardless of whether the Mn²⁺ ions are inserted into the apatite structure or segregated in the form of Mn₃O₄. Finally, the presence of Sr²⁺ in the apatite lattice does not appear to directly affect cell behavior, since both proliferation and the production of ALP are comparable to those observed for the nondoped HA sample.

5. Acknowledgments

The authors acknowledge financial support from the Brazilian research agencies CNPq, FAPITEC/SE and CAPES in the form of the grant PROMOB CAPES/FAPITEC-SE (Nº 88881.157913/2017-01).

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Publication Dates

• Publication in this collection
  29 May 2020
• Date of issue
  2020

History

• Received
  27 Feb 2020
• Reviewed
  25 Mar 2020
• Accepted
  07 Apr 2020