Gold is for the mistress, silver for the maid: Enhanced mechanical properties, osteoinduction and antibacterial activity due to iron doping of tricalcium phosphate bone cements

Abstract

Self-hardening calcium phosphate cements present ideal bone tissue substitutes from the standpoints of bioactivity and biocompatibility, yet they suffer from (a) weak mechanical properties, (b) negligible osteoinduction without the use of exogenous growth factors, and (c) a lack of intrinsic antibacterial activity. Here we attempt to improve on these deficiencies by studying the properties of self-setting Fe-doped bone-integrative cements containing two different concentrations of the dopant: 0.49 and 1.09 wt% Fe. The hardening process, which involved the transformation of Fe-doped β-tricalcium phosphate (Fe-TCP) to nanocrystalline brushite, was investigated in situ by continuously monitoring the cements using the Energy Dispersive X-Ray Diffraction technique. The setting time was 20 min and the hardening time 2 h, but it took 50 h for the cement to completely stabilize compositionally and mechanically. Still, compared to other similar systems, the phase transformation during hardening was relatively fast and it also followed a relatively simple reaction path, virtually free of complex intermediates and noisy background. Mössbauer spectrometry demonstrated that ⁵⁷Fe atoms in Fe-TCP were located in two non-equivalent crystallographic sites and distributed over positions with a strong crystal distortion. The pronounced presence of ultrafine crystals in the final, brushite phase contributed to the reduction of the porosity and thereby to the enhancement of the mechanical properties. The compressive strength of the hardened TCP cements increased by more than twofold when Fe was added as a dopant, i.e., from 11.5 ± 0.5 to 24.5 ± 2.0 MPa. The amount of iron released from the cements in physiological media steadied after 10 days and was by an order of magnitude lower than the clinical threshold that triggers the toxic response. The cements exhibited osteoinductive activity, as observed from the elevated levels of expression of genes encoding for osteocalcin and Runx2 in both undifferentiated and differentiated MC3T3-E1 cells challenged with the cements. The osteoinductive effect was inversely proportional to the content of Fe ions in the cements, indicating that an excessive amount of iron can have a detrimental effect on the induction of bone growth by osteoblasts in contact with the cement. In contrast, the antibacterial activity of the cement in the agar assay increased against all four bacterial species analysed (E. coli, S. enteritidis, P. aeruginosa, S. aureus) in direct proportion with the concentration of Fe ions in it, indicating their key effect on the promotion of the antibacterial effect in this material. This effect was less pronounced in broth assays. Experiments involving co-incubation of cements with cells in an alternate magnetic radiofrequency field for 30 min demonstrated a good potential for the use of these magnetic cements in hyperthermia cancer therapies. Specifically, the population of human glioblastoma cells decreased six-fold at the 24 h time point following the end of the magnetic field treatment, while the population of the bone cancer cells dropped approximately twofold. The analysis of the MC3T3-E1 cell/cement interaction reiterated the effects of iron in the cement on the bone growth marker expression by showing signs of adverse effects on the cell morphology and proliferation only for the cement containing the higher concentration of Fe ions (1.09 wt%). Biological testing concluded that the effects of iron are beneficial from the perspective of a magnetic hyperthermia therapy and antibacterial prophylaxis, but its concentration in the material must be carefully optimized to avoid the adverse effects induced above a certain level of iron concentrations.

Keywords: Bone cement; Escherichia coli; Hardening behaviour; Iron-doped tricalcium phosphate; Osteoblastic MC3T3-E1; Pseudomonas aeruginosa; Salmonella enteritidis; Staphylococcus aureus.

Fig. 1.

EDXRD of the 0.05Fe-TCP powder.

Fig. 2.

Raman spectra collected on 0.05Fe-TCP, 0.1Fe-TCP and red grains.

Fig. 3.

⁵⁷Fe Mössbauer spectra (experimental hollow dots) of (A) 0.1Fe-TCP powder and (B) 0.05Fe-TCP powder recorded at T = 298 K

Fig. 4.

Initial (60 s) and final (100 h) EDXRD spectra of the Fe-doped cement. CaFePh refers to calcium iron phosphate; DCPD refers to brushite; Fe₂O₃ refers to hematite.

Fig. 5.

3D diffraction map collected during the setting-hardening of the cement: (a) perspective view and (b) frontal view.

Fig. 6.

Kinetic curve obtained on the peak (041) of brushite, fitted by the Boltzmann fit curve.

Fig. 7.

SEM micrographs of the cement showing the medium-to-small sized plate-shaped crystals (light blue dotted rectangles), larger plate-shaped crystals (red dotted rectangles) and the cauliflower-like structures (magnification 1,200 ×) (a); medium sized and fine sized crystals (magnification 6,000 ×) (b); unreacted residuals of doped-TCP (magnification 3,000 ×) (c).

Fig. 8.

Histogram of the frequencies of Feret’s diameter values of the pores obtained from the SEM micrograph analysis. Log-normal fit curve was used to analytically fit the count distribution.

Fig. 9.

(a) Cumulative iron release curve obtained on 0.05FeTCP-derived cement. Relative uncertainty for all the measures is 1 %. Reduced chi squared for the estimated fit is ${\tilde{\chi }}^2=0.001$. (b) Comparison between compressive strength of hardened doped and non-doped TCP-derived cements. *** = p<0.001

Fig. 10.

mRNA expression of osteocalcin (BGLAP) (a) and the transcription factor Runx2 (b) in MC3T3-E1 cells incubated for 7 days with 5 mg/ml of 0.49 wt.% or 1.09 wt.% Fe-TCP cement. mRNA expression was detected by quantitative RT-polymerase chain reaction and normalized to the expression of the housekeeping gene β-actin (ACTB). C− and C+ represent the expressions in undifferentiated and differentiated (50 μg/ml ascorbic acid and 10 mM β-glycerophosphate) negative, untreated control cells, respectively. Data normalized to the expression of ACTB are shown as averages with error bars representing standard deviation (n = 3 × 3). Statistically significant difference between sample groups is represented with * (p < 0.05), ** (p < 0.005), and *** (p < 0.0001).

Fig. 11.

Temperature increase of the aqueous medium (a) and the viability of human E297 glioblastoma and mouse K7M2 osteosarcoma cells (b) challenged with 10 mg/cm² of 1.09 wt.% Fe-TCP cement for 30 minutes in an alternate magnetic field (300 kHz, 1.16 μT) compared to the negative, untreated control. All the cells were allowed 24 h to recover after the 30 min treatment in the magnetic field. Treatments of the cells in the magnetic field (Fe-TCP 1.09 wt.% + H) were compared against the no-cement controls and against the treatments with the cements in the absence of the magnetic field (Fe-TCP 1.09 wt.%). Data points are shown as averages with error bars representing the standard deviation (n = 2). Statistically significant difference between the sample groups is represented with either * (p < 0.05) or ** (p < 0.005).

Fig. 12.

Optical densities of liquid broths, directly indicative of the number of colony forming units, following a 24 h inoculation with Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa or Staphylococcus aureus without (control) or with either 20 mg/ml of a precursor HAp component of the cement formulation (carbonated HAp) or Fe-TCP cements with different concentrations of Fe (low Fe = 0.49 wt.%, high Fe = 1.09 wt.%). Data are shown as averages with error bars representing standard deviation. Data points significantly lower than the untreated control (p < 0.05) are topped with an asterisk.

Fig. 13.

(a) The ratio of the diameter of the inhibition zone (d_z) to the diameter of the spherical cement sample deposit (d_s) on an agar plate following a 24 h inoculation with Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa or Staphylococcus aureus without (control) or with a precursor HAp component of the cement formulation (carbonated HAp) or Fe-TCP cements with different concentrations of Fe (low Fe = 0.49 wt.%, high Fe = 1.09 wt.%). Data are shown as averages with error bars representing standard deviation. Data points significantly different from the untreated control (p < 0.05) are topped with an asterisk. (b) Visual appearance of either homogeneous (E. coli, S. aureus) or concentrically circled (S. enteritidis, P. aeruginosa) inhibition zones around Fe-TCP cements for different bacterial cultures.

Fig. 14.

Single-plane confocal optical micrographs of fluorescently stained osteoblastic MC3T3-E1 cells (cytoskeletal f-actin - red; nucleus – blue; TCP cement - green) in interaction with 5 mg/ml of either (a) the control HAp or (b, c) Fe-TCP cements with different concentrations of Fe (low Fe = 0.49 wt.%, high Fe = 1.09 wt.%) following a 72 h incubation with the particles.