A novel quantitative and reference-free ultrasound analysis to discriminate different concentrations of bone mineral content

Abstract

Bone fracture is a continuous process, during which bone mineral matrix evolves leading to an increase in hydroxyapatite and calcium carbonate content. Currently, no gold standard methods are available for a quantitative assessment of bone fracture healing. Moreover, the available tools do not provide information on bone composition. Whereby, there is a need for objective and non-invasive methods to monitor the evolution of bone mineral content. In general, ultrasound can guarantee a quantitative characterization of tissues. However, previous studies required measurements on reference samples. In this paper we propose a novel and reference-free parameter, based on the entropy of the phase signal calculated from the backscattered data in combination with amplitude information, to also consider absorption and scattering phenomena. The proposed metric was effective in discriminating different hydroxyapatite (from 10 to 50% w/v) and calcium carbonate (from 2 to 6% w/v) concentrations in bone-mimicking phantoms without the need for reference measurements, paving the way to their translational use for the diagnosis of tissue healing. To the best of our knowledge this is the first time that the phase entropy of the backscattered ultrasound signals is exploited for monitoring changes in the mineral content of bone-like materials.

Figure 1

Phantom preparation process. The procedure used for phantom preparation is depicted for agarose hydrogels (a), agarose-HA hydrogels (b) and agarose-CaCO₃ hydrogels (c). Agarose (Ag), Hydroxyapatite (HA), Calcium carbonate (CaCO₃).

Figure 2

Experimental setup for US acquisition. (a) Schematic representation of the setup; (b) images of the real experimental setup, including the acquisition system and the pc; (c) structure of the sample holder: in i) the two interlocking parts and the polystyrene membrane are shown, in ii) the assembled system is reported and in iii) the sample holder also containing a hydrogel is shown.

Figure 3

Example of signal envelopes for SoS measurement of Ag 2% samples. The envelopes of the signal in the presence of the sample (black line) and in the absence of the sample (red line) are shown in the figure for the central scanning line.

Figure 4

SEM images and EDX analysis for samples containing different concentrations of CaCO₃ and HA particles. SEM images (left panel) are reported at 700X (first column) and 2400X (second column) magnifications. EDX microanalysis (right panel) is reported at 700X magnification for agarose matrix (2% w/v) and for hydrogels doped with CaCO₃ and HA particles at different concentrations. For EDX microanalysis, the first image (Ag 2% w/v) reports the main elements automatically found by the system, merged with the SEM image channel; the other images show the single elements analyzed. For samples doped with CaCO₃ particles, Ca is reported in green. For samples doped with HA particles, P is reported in green and Ca is reported in red. In the agarose matrix without particles, no traces of Ca and P were found, but only C, O and Au.

Figure 5

B-mode images of hydrogels. Representative B-scan images of agarose gels without particles (Ag 2%), and with particles at different concentrations: CaCO₃ 2%, CaCO₃ 4%, CaCO₃ 6%, HA 10%, HA 20% and HA 50%. Data were acquired using a transmission wave frequency of 15 MHz. For each image, the color bar shows the pixel brightness.

Figure 6

Boxplot statistics for the reflection at the first interface (peak1), the reflection at the second interface (peak2) and SampEn for samples related to different concentrations of CaCO₃ and HA particles. The symbol * indicates p < 0.05 for the comparison of the groups to the control group, the symbol + indicates p < 0.5 for the comparison of the groups to each other.

Figure 7

Boxplot and regression analysis of f(s) for samples containing different concentrations of CaCO₃ and HA particles. The panel (a) shows the trend associated to the model including all the previous parameters (SampEn, peak1 and peak2) for: Ag 2% (control), CaCO₃ at different concentrations (2%,4% and 6% w/v) and HA at different concentrations (10%, 20% and 50%). The statistical comparisons of the closest groups are also presented: the symbol * indicates p < 0.05 for the comparison of the groups to the control group, the symbol + indicates p < 0.5 for the comparison of the groups to each other. In panel (b), the trend of the combined parameter is provided, separating CaCO₃ (left image) and HA (right image). Since agarose values were much higher than HA ones (at least a difference of one order of magnitude), they were excluded from HA representation. Finally, panel (c) shows the regression analysis for both CaCO₃ (left) and HA (right) at different concentrations.