Effects of SiO₂, SrO, MgO and ZnO dopants in TCP on osteoblastic Runx2 expression

Abstract

Calcium phosphate materials share a compositional similarity to natural bone which makes them excellent for use in orthopedic applications. Although these materials are osteoconductive, they lack strong osteoinductive capabilities and recent research has focused on the addition of biologics and pharmacologics with varying successes. In this study, trace elements that have been proven to play important roles in bone health and bone formation were incorporated into β-tricalcium phosphate compacts in their oxide forms (SiO₂, ZnO, SrO, and MgO). Cell material interactions were characterized using human fetal preosteoblastic cells. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was used to evaluate cellular proliferation. Cellular differentiation was evaluated using an enzymatic colorimetric alkaline phosphatase assay as well as immunohistochemistry for Runt-related transcription factor 2 (Runx2) expression. Results prove ZnO and MgO to be effective mitogenic factors and SiO₂, ZnO and SrO to be capable of inducing rapid cellular differentiation. MgO was found to have little effect on the modulation of osteoblastic differentiation, likely due to more aggressive inherent cellular regulation of Mg²⁺. In addition to the results from the study, a signaling mechanism is proposed as to the action of the dopants for further consideration.

1. Introduction

Calcium phosphate (CaP) ceramics are amongst the most widely studied materials for use in orthopedic implant applications due to their excellent biocompatibility, tailorable bioresorbability and compositional similarity to natural bone [1–3]. Current clinical uses include, but are not limited to, coatings on metallic implants, grafting materials and bone cement applications. [4]. The physical properties of CaPs are largely dependent on composition and phases present in the material. Of particular interest are the CaPs with a calcium-to-phosphorus ratio of 1.5:1, known as tricalcium phosphates (TCP). TCP has a solubility product (K_sp = 2.83 × 10⁻³⁰) that allows it to degrade over the course of months under physiological conditions, allowing it to provide mechanical support while the natural healing process can take place [1,5]. Our group and others have shown that these degradation kinetics as well as the overall material strength can be further tailored by the addition of dopants or sintering additives [6–8]. Additionally, by choosing dopants that can play important biological roles in the bone healing process, such as strontium, magnesium, silicon and zinc, the biological interaction between the material and natural bone can be enhanced [9–11].

Strontium is a non-essential element that has the ability to replace calcium in natural bone tissue, so it is said to have bone seeking behavior[12]. It is thought to displace Ca²⁺ ions in osteoblastic mediated processes. Researchers have identified that strontium likely stimulates bone formation by activating the calcium sensing receptor (CaSR) [13,14] and simultaneously increasing osteoprotegerin (OPG) production and inhibiting receptor activator of nuclear factor kappa B ligand (RANKL) expression in osteoblasts [15]. OPG is a protein that inhibits RANKL induced osteoclastogenesis by operating as a decoy receptor for RANKL [16]. The OPG/RANKL ratio, then, can be a powerful regulator of bone resorption and osteoclastogenesis. Phase III clinical trials that began in 2000 investigated the efficacy of strontium ranelate in reducing vertebral fractures and peripheral fractures, including hip fractures. After 3 years, patients treated with strontium ranelate showed significant reduction in vertebral fractures (41%) and hip fractures (36%) compared with patients treated with placebo [17].

Magnesium is one of the most abundant cations in the human body, with about 65% of it being contained in bone and teeth [18]. It has the ability to influence intracellular Ca²⁺ homeostasis via the parathyroid hormone pathway [19]. Furthermore, studies have shown that magnesium deficiency can result in reduced bone growth, bone formation and mineralization [20,21].

Silicon has been proven to be an important trace element in bone and connective tissue formation and can stimulate biological activity by increasing the solubility of the material, generating a more electronegative surface and creating a finer microstructure resulting in transformation of the material surface to a biologically equivalent apatite [22]. Recent studies have also demonstrated that silicate containing bioceramics have the added benefit of inducing angiogenesis, which is a crucial aspect of bone defect healing [23,24].

Zinc, also an important trace element in bone formation, has been shown to have stimulatory effects on bone formation when added to CaP [25]. Zinc is released during skeletal breakdown and has shown to inhibit osteoclastic bone resorption [26]. It does this by inhibiting osteoclast-like cell formation from bone marrow cells and inducing apoptosis of mature osteoclast [27]. Osteoporotic patients have also been shown to have lower levels of skeletal zinc than that of control groups [28]. Zinc has also been shown to induce osteoblastogenesis, osteoblastic differentiation and mineralization [29,30].

While many benefits have been attributed to these particular elements in healthy bone biology, the mechanisms of action to which they may influence bone and the healing process is still largely a mystery. The aim of this study is to identify potential signaling pathways by which strontium, silicon, magnesium and zinc may play a role in osteoblastic differentiation. Runt related transcription factor 2 (Runx2) is essential for osteoblastic differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression [31]. It is vital for the maturation of osteoblasts and both intramembranous and endochondral ossification [32]. Alkaline phosphatase (ALP) has long been identified as an osteoblast differentiation marker, signaling the maturation of the extracellular matrix (ECM) produced by osteoblasts; the final step before terminal differentiation or apoptosis for osteoblast cells. By studying these two targets, which are seemingly unrelated in the differentiation process, insight may be gained as to which signaling pathways should be studied further.

2. Methods

2.1 Sample preparation

β-tricalcium phosphate powder (β-TCP) was obtained from Berkeley Advanced Biomaterials (Berkeley, CA) with an average particle size of 550 nm. High purity strontium oxide (SrO) (99.9% purity) was purchased from Sigma Aldrich ( St. Louis, MO) and magnesium oxide (MgO, 99.998%) was procured from Alfa Aesar, MA, USA. Silicon dioxide (SiO₂) (99%+ purity) and zinc oxide (ZnO) (99.9%+ purity) were purchased from Fisher Scientific (Fair Lawn, NJ). Samples were prepared by mixing 50 g of β-TCP powder and appropriate amounts of dopants (1 wt% SrO, 1 wt% MgO, 0.5 wt% SiO₂ and 0.25 wt% ZnO) in 250 mL polypropylene Nalgene bottles containing 75 mL of anhydrous ethanol and 100 g zirconia milling media with 5mm diameter. Dopant concentrations were chosen based on previous optimization research [6,7,9,10] The mixtures were then milled for 6 h at 70 rpm to minimize the formation of agglomerates and increase homogeneity. After milling, powder was dried in an oven at 60 °C for 72 h and pressed to discs (12 mm diameter and 2.5 mm thickness) using a uniaxial press at 145 MPa. Green compacts were then cold isostatically pressed at 414 MPa for 5 min and sintered at 1250 °C for 2 h in a muffle furnace.

2.2 Sample Density

Density was measured using Archimedes method. Samples were weighed initially dry and then submerged in boiling water for 3 minutes to remove any excess air that may be trapped in the porous structure. The samples were then transferred from the boiling water to room temperature water, where the weight was recorded again (n=3).

2.2 Cell culture

All samples were sterilized by autoclaving at 121 °C for 20 min. In this study, established human preosteoblast cell line hFOB 1.19 (ATCC, Manassas, VA) were used. Cells were seeded onto the samples in 24-well plates at a density of 10⁵ cells/sample. The base medium for this cell line was a 1:1 mixture of Ham's F12 Medium and Dulbecco's Modified Eagle's Medium (DMEM/F12, Sigma, St. Louis, MO), with 2.5 mM L-glutamine (without phenol red). The medium was supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 0.3 mg/ml G418 (Sigma, St. Louis, MO). Cultures were maintained at 34 °C under an atmosphere of 5% CO₂ as recommended by ATCC for this particular cell line. Medium was changed every 2 days for the duration of the experiment.

2.4 Cellular proliferation

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was used to evaluate cell proliferation. The MTT (Sigma, St. Louis, MO) solution of 5 mg/ml was prepared by dissolving MTT in sterile filtered PBS. 10% MTT solution was then added to each sample in 24-well plates. After 2 h of incubation, 1 ml of solubilization solution made up of 10% Triton X-100, 0.1N HCl and isopropanol was added to dissolve the formazan crystals. 100 μl of the resulting supernatant was transferred into a 96-well plate, and read by a plate reader at 570 nm. Triplicate samples were used in MTT assay experiments to insure reproducibility. Data was normalized to the absorbance values for the pure TCP control.

2.4 Cellular morphology

Samples for testing were removed from culture after 3 days of incubation. All samples for SEM observation were fixed with 2% paraformaldehyde/2% glutaraldehyde in 0.1M cacodylate buffer overnight at 4 °C. Post-fixation was performed with 2% osmium tetroxide (OsO₄) for 2 h at room temperature. The fixed samples were then dehydrated in an ethanol series (30%, 50%, 70%, 95% and 100% three times), followed by a hexamethyldisilane (HMDS) drying procedure. After gold coating, the samples were observed under field emission scanning electron microscope (FESEM) (FEI 200F, FEI Inc., OR, USA) for cell morphologies.

2.5 Alkaline phosphatase activity

The alkaline phosphatase (ALP) activity of the cells was determined by a spectrophotometric endpoint assay that determined the conversion of colorless p-nitrophenyl phosphate to colored p-nitrophenol. Cell media was aspirated from each sample at 7 and 11 days and samples were subsequently transferred to new 24 well plates. 350 μl 2-amino-2-methyl-propanol buffer and 500 μl p-nitrophenyl phosphate (4 mg/ml) were added. Samples were incubated for 15 min at 37° C and stopped by the addition of 500 μl of 2M NaOH to each well. The solution was then transferred to a 96 well plate in triplicate and absorbance was read at 405 nm. Triplicates of each sample were examined to ensure reproducibility. Data was normalized to MTT data to account differences in cellular proliferation/expression.

2.6 Runx2 activity

Samples were fixed in 3.7% paraformaldehyde/ phosphate buffered solution with a pH of 7.4 at room temperature for 10 min. Samples were then washed in PBS 3 times (5 min each) and cells were permeabilized with 0.1% Triton X-100 (in PBS) for 4 min at room temperature. Next, samples were rinsed in TBST 3 times (5 minutes each) and incubated in TBST-BSA (Tris-buffered saline with 1% bovine serum albumin, 250 mM NaCl, pH 8.3) blocking solution for 1h at room temperature. Primary antibody against Runx2 (Abcam, Cambridge, MA) was added at a 1:100 dilution and incubated at room temperature for 2 h and kept at 4 °C overnight. Samples were then washed with TBST 3 times (10 min each). The secondary antibody, goat anti-mouse (GAM) Oregon green 488 (Molecular Probes, Eugene, OR), was diluted 1:100 in TBST and was used to incubate the cells for 1h. After rinsing three times for 10 minutes each with TBST the samples placed in 24 well plates with Vectashield mounting medium (Vector Labs, Burlingame, CA) with propidium iodide (PI). Samples were immediately analyzed by use of fluorescent area scans using a 5×5 grid pattern on a Synergy Hybrid multi-mode microplate reader (Biotek, Winooski, VT) with excitation wavelength of 488 nm and emission wavelength of 540 nm. Data was normalized to account for differences in cellular proliferation/expression. Afterwards, samples were placed on glass coverslips and confocal micrographs were taken using a Zeiss 510 laser scanning microscope (LSM 510 META, Carl Zeiss MicroImaging, Inc., NY, USA).

2.7 Statistical analysis

Statistical analysis was performed using a one way ANOVA and p<0.05 was considered statistically significant.

3. Results

3.1 Density

All samples exhibited very high density, >95%, as seen in Table 1. None of the samples demonstrated a significant difference in density in the group.

Table 1.

Relative densities of sintered samples

Sample	Relative Density
Pure TCP	96.17 ± 0.89
Si – TCP	97.02 ± 1.47
Zn – TCP	98.07 ± 0.52
Sr - TCP	95.02 ± 1.39
Mg - TCP	98.61 ± 0.97

3.1 Cellular proliferation

At day 3 (Figure 4), all samples showed decreased cellular proliferation when compared to the pure TCP control with ZnO-TCP samples having significantly lower living cells. At day 7 (Figure 1), both ZnO-TCP and MgO-TCP samples had significantly greater amount of live cells when compared to the pure TCP controls, while the SiO₂-TCP and SrO-TCP samples where comparable in live cell density to the pure TCP controls. By day 11 (Figure 2/5) SiO₂-TCP and SrO-TCP had lower live cell density, ZnO-TCP had comparable cell density and MgO-TCP showed increased cellular density when compared to pure TCP controls.

Figure 4.

MTT measurements for cellular proliferation and Runx2 fluorescent measurements for cellular differentiation at day 3 for SiO₂, ZnO, SrO, MgO doped and pure TCP samples. Data is normalized to pure TCP control. Runx2 data is normalized to MTT measurements. *P < 0.05 (n=3)

Figure 1.

MTT measurements for cellular proliferation and ALP measurements for cellular differentiation at day 7 for SiO₂, ZnO, SrO, MgO doped and pure TCP samples. Data is normalized to pure TCP control. ALP data is normalized to MTT measurements. *P < 0.05 (n=3)

Figure 2.

MTT measurements for cellular proliferation and ALP measurements for cellular differentiation at day 11 for SiO₂, ZnO, SrO, MgO doped and pure TCP samples. Data is normalized to pure TCP control. ALP data is normalized to MTT measurements. *P < 0.05 (n=3)

Figure 5.

MTT measurements for cellular proliferation and Runx2 fluorescent measurements for cellular differentiation at day 11 for SiO₂, ZnO, SrO, MgO doped and pure TCP samples. Data is normalized to pure TCP control. Runx2 data is normalized to MTT measurements. *P < 0.05 (n=3)

3.2 Cellular morphology

Micrographs depicting cellular morphology are given in Figure 3. At day 3 all samples demonstrated a barely visible confluent monolayer of cells. At day 7, a second layer of cells was reaching confluency on all samples. By day 11, all samples showed multiple layers of cells with evidence of early stages of mineralization.

Figure 3.

FESEM micrographs depicting hFOB cell morphology after 3, 7 and 11 days in culture

3.3 Alkaline phosphatase activity

Expression of ALP was evaluated for hFOB cells cultured on SiO₂-TCP, ZnO-TCP, SrO-TCP, MgO-TCP and pure TCP samples. Results after 7 and 11 days of culture are shown in Figure 1 and Figure 2, respectively. At day 7, ALP activity was significantly lower in all doped samples when compared to the pure TCP samples. At day 11, ZnO-TCP and SrO-TCP samples demonstrated higher ALP activity, while SiO₂-TCP samples had decreased activity and MgOTCP samples had comparable activity to the pure TCP control samples.

3.4 Runx2 activity

Expression of Runx2 in osteoblast cells cultured on doped and pure TCP samples were evaluated at days 3 and 11. Results for fluorescent area scans for day 3 are given in Figure 4 and results for day 11 are given in Figure 5. Confocal micrographs for each day are shown in Figure 6, where cellular nuclei fluoresce in red and green indicates Runx2 expression. At day 3, SiO₂-TCP, ZnO-TCP and SrO TCP all showed increased Runx2 expression when compared to pure TCP samples, with SiO₂-TCP and ZnO-TCP samples being significantly greater. MgO doped samples showed less Runx2 activity, but not at a significant level. At day 11, samples containing SiO₂ and ZnO had comparable Runx2 expression to pure TCP control samples, while both SrOTCP and MgO-TCP samples had decreased Runx2 expression. The MgO-TCP samples demonstrated significantly lower Runx2 expression than the pure TCP samples. In general, when compared with day 3 data, Runx2 expression decreased for all samples with respect to pure TCP. Confocal micrographs confirm the results of the quantitative fluorescent area scans, showing similar patterns of Runx2 expression.

Figure 6.

Confocal micrographs showing Runx2 expression in hFOB cells at 3 and 11 days. Green fluorescence indicates active Runx2; red fluorescence indicates propidium iodide bound to the nuclei of cells.

4. Discussion

Previous studies have well characterized the inherent material properties associated with these dopants [6,7,9,10]. The use of isostatic pressing, eliminates most of the inherent density differences associated with dopant addition, resulting in a very high average density ranging from 95% - 98% (Table 1) with no statistically significant differences detected. Studies have also shown that the addition of any single dopant used in this study has the ability to reduce α phase formation associated with a high sintering temperature when compared to the pure TCP samples [6,7,9,10]. These two properties are extremely important when thinking about potential differences in dissolution between sample types. The pure control composition has higher alpha phase so its dissolution rate will be higher than its doped experimental samples if the densities are similar, however, dissolution rate will be minimal considering the time frame of the study as seen in previous studies [33,34]. It is well documented that increased Ca²⁺ and P⁵⁺ can positively affect cellular differentiation and proliferation [35–37], but the results observed in this study demonstrated that samples containing dopants had significant positive effects when compared to the pure composition. It is then very likely, then, that the cells are responding to the dopants rather than Ca²⁺ or P⁵⁺ due to differences in dissolution.

The osteoblastic cell cycle is a process comprised of three distinct stages (Figure 7): proliferation, maturation and termination. During the proliferation stage and part of the maturation stage, Runx2 plays a stimulatory role in differentiation of mesenchymal stem cells (MSCs), osteochondrogenic precursors and preosteoblasts. During the latter portion of the maturation stage and the terminal differentiation stage, Runx2 plays an inhibitory role in osteoblastic differentiation [38]. It is, therefore, necessary for the upregulation of Runx2 initially and the downregulation of Runx2 during the latter part of the cell cycle to obtain mature bone growth. Results from the MTT assay at day 3 (Figure 4) suggest an expeditive transition from the proliferation stage of the osteoblast life cycle to the maturation stage in samples containing SiO₂, ZnO and MgO as evidenced by the decrease in proliferation rates. Samples doped with SrO are comparable to that of the pure TCP samples. Research has shown that Sr is able to augment proliferation of osteoblast cells while maintaining increased differentiation rates [39], so it is possible that cells seeded on the SrO doped samples are also in the maturation phase. SEM micrographs (Figure 3) of cells at day 3 show characteristics of mature osteoblasts in all samples manifested by the broadening and flattening of cells as well as the extension of cellular processes and cellular aggregation. At day 7 (Figure 1), MTT results show increased proliferation in samples containing ZnO and MgO compared to pure TCP. The other samples had similar values to pure TCP, indicating that cells on the pure TCP samples had reached advanced maturity and the resultant slowing of proliferation. The increased level of viable cells in the MgO and ZnO doped samples can be attributed to the fact that Zn²⁺ and Mg²⁺ are both mitogenic factors and may prevent terminal apoptosis of osteoblast cells [40,41]. SEM micrographs show the formation of multiple layers of mature osteoblast cells on all samples. Day 11 MTT results (Figure 2/5) indicate an affinity for active terminal differentiation in samples containing SiO₂ and SrO demonstrated by decreased cellular viability (increased terminal apoptosis). Samples containing MgO had increased proliferation rates, while ZnO doped samples were comparable to pure TCP indicating a lasting effect of Mg²⁺ as a mitogenic factor. SEM images (Figure 3) for day 11 show all doped samples demonstrate advanced characteristics of cellular differentiation when compared to pure TCP samples, embodied by the sheet-like formation of bone lining cells, where single cells are often difficult to distinguish, as well as apoptosis and mineralization.

Figure 7.

Diagram depicting the osteoblast cell lifecycle and influence of Runx2 [52,53].

Results from the ALP assay reveal that at day 7 (Figure 1), all samples show significantly lower activity when compared to pure TCP. At day 11 (Figure 2), ALP activity in SiO₂ doped samples was less than pure TCP, MgO doped samples comparable and ZnO and SrO doped samples significantly greater. Alkaline phosphatases are glycosyl-phosphatidylinositol anchored, Zn²⁺ metallated glycoproteins that are released during the beginning of the termination phase of the osteoblastic cell life cycle and help to catalyze the hydrolysis of phosphomonoesters into inorganic phosphates [42]. They create an alkaline environment that favors the mineralization of these inorganic phosphates. These results suggest a prolonged maturation phase in the ZnO and MgO doped samples through day 7, likely caused by increased Runx2 activity in the samples through this time period. Researchers have also shown that the Zn²⁺ binding sites in ALP may be easily substituted by other cations, where differing cationic radii and charge can act as destabilizing forces in the enzyme causing decreased functionality [43]. This phenomenon becomes apparent when considering day 11 results. ZnO and SrO have a large increase in functional enzymatic ALP activity while SiO₂ is continuing to show destabilization when compared to the pure TCP samples. Zn²⁺, being the native cation in ALP, is expected to show increased activity. While the ionic radii of Sr²⁺ is dissimilar to Zn²⁺ (132pm and 88 pm, respectively), Sr²⁺ seems to act as an agonist for ALP activity. Si⁴⁺, likely due to vast differences in ionic radii (54 pm) and charge, may cause destabilization of the ALP enzyme. Mg²⁺ is very similar in size (86 pm) and charge to Zn²⁺ and has been demonstrated to have very little effect on the stability of ALP [43], so it was expected that results would be similar to samples doped with ZnO. This was not the observation, however. It is apparent that MgO doped samples are only producing as much ALP as the pure samples at day 11where we expected to see an increase in activity. Two different mechanisms may be responsible for these findings. The first, we still see significant increase in proliferation rates with samples doped with MgO, so it may be that samples remain in the early maturation stage. Runx2 findings, however, do not correlate well with this. They should remain at high levels during states where proliferation is prevailing. The second mechanism would suggest that Mg²⁺ is well regulated along the differentiation pathways (as we still see increases in cellular proliferation). This seems to be the more likely scenario as Mg²⁺ is one of the most abundantly available cations in the human body and is expected to be more highly regulated.

Results from this study indicate that at day 3 (Figure 4), Runx2 expression is significantly upregulated in samples containing SiO₂ and ZnO. SrO doped samples also showed upregulation of Runx2, but not quite at a significant level. At day 11 (Figure 5), samples containing SiO₂ and ZnO show Runx2 expression to be about the same as pure TCP. Samples containing MgO and SrO show downregulation of Runx2, with MgO doped samples giving significant results. Confocal microscopy (Figure 6) confirms these findings. Overall results signify that samples containing SiO_2, ZnO and SrO can appropriately modulate Runx2 activity leading to accelerated differentiation of osteoblast cells when compared to pure TCP samples. A proposed signaling mechanism for the effect of dopants on Runx2 expression is offered in Figure 8. One of the major regulators of osteoblastic cellular differentiation, and in particular Runx2, is the canonical Wnt signaling pathway[32,44]. The non-canonical Wnt5 calcium-dependent signaling pathway may also play an important role in the osteoblastic differentiation process and has been shown to cross paths with other Wnt isoform signaling pathways [45]. Several studies have presented that dopants, such as Sr²⁺, can interact with the calcium sensing receptor (CaSR) in osteoblastic cells to moderate cellular processes [13,46]. In the presence of high extracellular Ca²⁺, Wnt5 is activated through the CaSR and binds to the Frizzled G protein-coupled receptor and starts the intracellular Ca²⁺ signaling cascade [47]. There are extensive reviews available concerning this cascade [44,48]. Briefly, once Wnt is bound, the cytosolic G protein breaks off into its α and β/γ subunits. The β/γ then activates membrane bound phospholipase C (PLC) which, in turn, hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then diffuses through the cytosol and activates calcium channels located in the cellular membrane and endoplasmic reticulum causing an influx of intracellular calcium. Studies have shown that calcium channels can be non-selective to certain ions, including Sr²⁺, which may allow entrance of the dopants into the cell[49]. Other studies also suggest that the endoplasmic reticulum may also allow for the storage of cations such as Mg²⁺, Sr²⁺ and Zn²⁺ [50–52]. The influx of Ca²⁺ into the cell leads to the activation of calcineurin (CaN) and Ca²⁺-calmodulin-dependent protein kinase II (CamKII), while DAG activates protein kinase C (PKC). PKC activates the translocation of cyclic adenosine monophosphate (cAMP) response element-binding (CREB) and nuclear factor kappa B (NFκB) from the cytoplasm to the nucleus. CamKII also plays a role in the translocation of NFκB. CaN is responsible for the permutation of Nuclear factor of activated T-cells (NFAT) into the nucleus. CREB, NFκB and NFAT have all been shown to play important roles in the regulation of Runx2 [53–55]. The ability of cations to act as a competitive agonist with Ca²⁺ for the activation of CaN or CamKII has been established with strontium. Fromique et al. demonstrated that strontium ranelate induced NFATc1 nuclear translocation and was completely abrogated with CaN inhibitors[56]. It is likely that other dopants may interact in a similar manner, although confirmation studies are still required to confirm this.

Figure 8.

Activation of the Wnt/Fz ligand-receptor leads to the production of the second messengers IP3 and DAG from membrane-bound PIP2 via the action of membrane-bound enzyme PLC. IP3 causes release of Ca² from the ER and extracellular Ca²⁺ influx through transmembrane Ca²⁺ channels; CaN and CamKII are activated which in turn activate NFAT and NFkB. DAG is also activated by increased inctracellular Ca²⁺, which activates PKC. PKC activates NFkB and CREB. NFAT, NFkB, and CREB translocate to the nucleus and transcribe downstream regulatory genes such as Runx2.

A recapitulating theme throughout this study is the apparent inactivity of Mg²⁺ in the osteoblastic cellular differentiation process as evidenced by results from Runx2 and ALP analysis. In the human body, Mg²⁺ is the second most abundant cation and is known to be a cofactor in over 300 enzymatic reactions ranging from energy metabolism to nucleic acid synthesis [57]. Because it is so abundant, it is also highly regulated and can even act as a Ca²⁺ ion channel blocker [58]. Another study demonstrated that while some calcium ion channels may be non-selective to some cations, Mg²⁺ efflux remained low through these same channels [49]. While dietary deficiencies have been linked to osteoporosis, this study provides no evidence that an abundance of Mg²⁺ will help in the maturation and differentiation of osteoblastic cells. On the other hand, significant results were seen in cellular proliferation at days 7 and 11, confirming Mg²⁺ as a valuable mitogenic element. SrO, ZnO and SiO₂ all seemed to have positive effects on Runx2 regulation, increasing early expression and decreasing at the latter stages of the cell life cycle indicating the ability for accelerated differentiation when compared to pure TCP samples. The mineralization capabilities of the osteoblast cells were also positively affected by the ZnO and SrO dopants, demonstrated by ALP activity at day 11. Overall results suggest that ZnO, SrO and SiO₂ dopants can have profound effects on the differentiation capabilities of osteoblast cells in vitro and likely act through the intracellular Ca²⁺ signaling pathway as a competitive agonist with calcium to activate key proteins in this pathway. While Si⁴⁺ was found to cause functional destabilization of ALP, a key enzyme in the mineralization process of osteoblasts, it has also been shown that it is an important trace element in bone and connective tissue formation and can stimulate biological activity by increasing the solubility of the CaP material, generating a more electronegative surface and it can also create a finer microstructure resulting in a nucleation site that is favorable for apatite formation [22,59].

5. Conclusions

CaP materials have been widely studied for use as an osteogenic bone replacement material and are effectively used in several medical and dental applications today. In this study, by incorporating the dopants SiO₂, ZnO, and SrO into TCP, the important transcription factor Runx2 was able to be modulated in a beneficial manner as to induce accelerated bone cell differentiation, with high expression in the early stages of cell maturation and low expression in the terminal stage. ALP activity was increased with the introduction of ZnO and SrO into the samples when compared to pure TCP indicating increased affinity for matrix mineralization. ZnO and MgO dopants both demonstrated favorable mitogenic properties, but Mg did not have any significant effect on the differentiation markers. In addition to these results a signaling mechanism was proposed as to the action of these dopants for consideration of further study and understanding of the system.