Aspartic Acid-Assisted Synthesis of Multifunctional Strontium-Substituted Hydroxyapatite Microspheres

Strontium-substituted hydroxyapatite (SrHAP) microspheres with three-dimensional (3D) structures were successfully prepared via hydrothermal method using self-assembled poly­(aspartic acid) (PASP) as a template. By controlling various parameters, including hydrothermal reaction time, amount of l-aspartic acid (l-Asp), and ratio of Sr ions, we were able to investigate the influences of the additive l-Asp on morphology and properties of final products as well as the role of self-assembled PASP template on the formation of HAP microspheres. The change in the amount of Sr substitution significantly affected the particle size, morphology, and concurrent surface area. This difference caused variation in the drug-release properties. In addition, substitution of Sr ions into Ca ion sites affected luminescence of HAP powders. Particularly, multifunctional SrHAP with molar ratios (Sr/[Ca+Sr]) of 0.25 possessed the strongest luminescence as well as superior drug-loading and sustained-releasing properties. These properties were associated with large surface area and large pore size of the SrHAP. This study suggests that the optical and structural properties of the HAP particles can be carefully tuned by controlling the amount of Sr ions doped into HAP particles during synthesis. This work provides new opportunities to synthesize HAP particles suitable for diverse applications including bone regeneration and drug delivery

A comparison of Ki-67 counting methods in luminal Breast Cancer: The Average Method vs. the Hot Spot Method

<div><p>In spite of the usefulness of the Ki-67 labeling index (LI) as a prognostic and predictive marker in breast cancer, its clinical application remains limited due to variability in its measurement and the absence of a standard method of interpretation. This study was designed to compare the two methods of assessing Ki-67 LI: the average method vs. the hot spot method and thus to determine which method is more appropriate in predicting prognosis of luminal/HER2-negative breast cancers. Ki-67 LIs were calculated by direct counting of three representative areas of 493 luminal/HER2-negative breast cancers using the two methods. We calculated the differences in the Ki-67 LIs (ΔKi-67) between the two methods and the ratio of the Ki-67 LIs (H/A ratio) of the two methods. In addition, we compared the performance of the Ki-67 LIs obtained by the two methods as prognostic markers. ΔKi-67 ranged from 0.01% to 33.3% and the H/A ratio ranged from 1.0 to 2.6. Based on the receiver operating characteristic curve method, the predictive powers of the KI-67 LI measured by the two methods were similar (Area under curve: hot spot method, 0.711; average method, 0.700). In multivariate analysis, high Ki-67 LI based on either method was an independent poor prognostic factor, along with high T stage and node metastasis. However, in repeated counts, the hot spot method did not consistently classify tumors into high vs. low Ki-67 LI groups. In conclusion, both the average and hot spot method of evaluating Ki-67 LI have good predictive performances for tumor recurrence in luminal/HER2-negative breast cancers. However, we recommend using the average method for the present because of its greater reproducibility.</p></div

Optimal Ki-67 cutoff values for predicting tumor recurrence.

<p>Optimal Ki-67 cutoff values for predicting tumor recurrence.</p

An example of counting area selection in a tumor with heterogeneous Ki-67 expression.

<p>Three representative areas have been selected including a hot spot (area 1). (Original magnification: x 40 (large photo); x 200 (three small photos)).</p

The distribution of Ki-67 labeling indices in 493 luminal breast cancers.

<p>The X-axis represents the individual cases, arranged in ascending order based on their Ki-67 labeling index (LI) measured by the average method. The Y-axis represents the individual Ki-67 LIs measured by the hot spot method (the edge of the light gray area) and the average method (dotted line), and the values measured at the “cold spot”, the area with the lowest Ki-67 LI (the edge of the dark gray area). The extent of the light gray colored area represents the difference between the Ki-67 LI measured at the hot spot and the cold spot (Δ Ki-67).</p

Kaplan-Meier survival curves of Ki-67 LI based on the average method and hot spot method.

<p>High Ki-67 labeling indices of tumors using a cutoff value of 18% in the average method (A) and 22% in the hot spot method (B) are associated with poor disease-free survival (<i>p</i> = 0.002, <i>p</i> = 0.001, respectively).</p

Correlation between Δ Ki-67, H/A ratio and Ki-67 labeling index measured by the two methods.

<p>(A, B) ΔKi-67 is moderately correlated with the Ki-67 labeling index (LI) based on the average method, and highly correlated with Ki-67 LI measured by the hot spot method (Pearson correlation coefficient, 0.535 and 0.731 respectively). (C, D) The H/A ratio is not correlated with Ki-67 LI measured by either method (Pearson correlation coefficients, -0.298 and -0.119, respectively).</p

Variability of Ki-67 labeling index in repeated counts.

<p>(A) Twenty breast cancers that were initially classified into the low Ki-67 subgroup using the average method (cutoff value: 18%, dotted line) were re-counted by the same method. Only 3 cases were reclassified into the high Ki-67 subgroup in repeated count. (B) Of the 20 cases, 4 cases were initially classified into the low Ki-67 subgroup by the hot spot method (cutoff value: 22%, dot line). One of these was reclassified into the high Ki-67 subgroup, and 8 of the 16 cases originally classified into the high Ki-67 subgroup were reassigned to the low Ki-67 subgroup after a second count using the same method.</p

Disease-free survival according to the degree of tumor-associated macrophage (TAM) infiltration in each compartment.

<p>High levels of infiltration of intratumoral (A), but not stromal (B) and total (C) TAM, are associated with poor disease-free survival of the entire population of patients. However, in the hormone receptor (HR)-positive group, high levels of infiltration of intratumoral (D), stromal (E) and total (F) TAMs are each associated with decreased disease-free survival.</p

The utility of measurements for the detection fetal trisomy 18.

<p>PPV, positive predictive value; NPV, negative predictive value; ACC, accuracy.</p><p>The number in parentheses indicates the 95% confidence interval.</p