Regenerative biology seeks to uncover the principles enabling organisms to restore complex tissues and organs. The axolotl ( Ambystoma mexicanum ), a salamander with unparalleled limb regenerative capacity, remains a premier model system in this field. However, in vitro studies have been limited by the absence of reliable culture platforms for maintaining blastema cell identity and functionality. Here, we present a robust, gel-free 3D culture protocol that enables the formation of spheroids from axolotl blastema cells under fully defined, serum-free conditions. These spheroids preserve the expression of key regenerative markers such as Prrx1, Msx2 , and CTGF for at least 10 days in culture, and exhibit cellular stability supported by antioxidant and amino acid supplementation. Importantly, spheroids cultured for 10 days were capable of initiating extra digit formation when transplanted into limb bud after amputation. With unique advantage of fully defined incubation conditions, this technique adds a powerful in vitro platform to existing experimental models of axolotl limb regeneration.

The axolotl ( Ambystoma mexicanum ), a neotenic salamander with remarkable regenerative capabilities, serves as a key model for studying nervous system regeneration. Despite its potential, the cellular and molecular mechanisms underlying this regenerative capacity remain poorly understood, partly due to the lack of reliable in vitro models for axolotl neural cells. In this study, we developed a novel protocol for primary cultures of adult axolotl telencephalon/pallium, enabling the maintenance of viable and functionally active neural cells. Using calcium imaging and immunocytochemistry, we demonstrated the presence of neuronal and glial markers, synaptic connections, and spontaneous calcium activity, highlighting the functional integrity of the cultured cells. Our findings reveal that these cultures can be maintained in both serum and serum‐free conditions, with neurons exhibiting robust neurite outgrowth and responsiveness to injury. This protocol addresses a critical gap in axolotl research by providing a controlled in vitro system to study neurogenesis and regeneration. By offering insights into the regenerative mechanisms of axolotl neurons, this work lays the foundation for comparative studies with mammalian systems, potentially informing therapeutic strategies for neurodegenerative diseases and CNS injuries in humans.

Objective: This study aimed to investigate microstructural changes in human dental hard tissues following single-fraction irradiation using high-resolution micro-computed tomography (micro-CT), with particular emphasis on potential differences between flattening filter (FF) and flattening filter-free (FFF) photon beam modalities. Methods: Sixteen extracted, non-carious human third molar teeth were included. All samples underwent baseline micro-CT imaging prior to irradiation. Teeth were then irradiated with 20 Gy in a single fraction using either 6 MV FF (600 cGy/MU) or 6 MV FFF (1400 cGy/MU) photon beams. Micro-CT images following irradiation were obtained with identical imaging conditions. Changes in bone volume (BV), total volume (TV), bone volume fraction (BV/TV), bone surface (BS), specific bone surface (BS/BV), bone mineral density (BMD), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were measured before and after irradiation. Results: Irradiation resulted in a substantial decrease in BV/TV and increases in BS and BS/BV (p < 0.05), suggesting micro-architectural remodeling and enhanced surface irregularity. No significant changes were detected in BV, TV, BMD, Tb.Th, or Tb.Sp. Comparison between FF and FFF beam modalities revealed no statistically significant differences in any micro-CT-derived parameter. Conclusion: Single-fraction 20 Gy irradiation induces early microstructural alterations in dental hard tissues characterized by changes in surface-related parameters rather than bulk mineral loss. Dose-rate differences between FF and FFF photon beams did not result in detectable micro-CT differences, supporting the clinical use of FFF beams at equivalent doses.

The axolotl ( Ambystoma mexicanum ), a neotenic salamander with remarkable regenerative capabilities, serves as a key model for studying nervous system regeneration. Despite its potential, the cellular and molecular mechanisms underlying this regenerative capacity remain poorly understood, partly due to the lack of reliable in vitro models for axolotl neural cells. In this study, we developed a novel protocol for primary cultures of adult axolotl telencephalon/pallium, enabling the maintenance of viable and functionally active neural cells. Using calcium imaging and immunocytochemistry, we demonstrated the presence of neuronal and glial markers, synaptic connections, and spontaneous calcium activity, highlighting the functional integrity of the cultured cells. Our findings reveal that these cultures can be maintained in both serum and serum-free conditions, with neurons exhibiting robust neurite outgrowth and responsiveness to injury. This protocol addresses a critical gap in axolotl research by providing a controlled in vitro system to study neurogenesis and regeneration. By offering insights into the regenerative mechanisms of axolotl neurons, this work lays the foundation for comparative studies with mammalian systems, potentially informing therapeutic strategies for neurodegenerative diseases and CNS injuries in humans.

Gold nanoparticles (AuNPs) have attracted considerable interest as emerging CT contrast agents; however, the influence of reconstruction parameters on their quantitative detectability remains insufficiently characterized, limiting the optimization of quantitative CT imaging. This study quantitatively compared the CT imaging performance of citrate-stabilized AuNPs, PEGylated AuNPs (PEG-AuNPs), and a conventional iodinated contrast agent (BMX) under different reconstruction conditions. AuNPs were synthesized by citrate reduction and PEGylated with SH-PEG5K-COOH, followed by physicochemical characterization using dynamic light scattering, zeta potential analysis, and ICP-OES. Contrast samples (≤10 mg/mL) were imaged using a micro-CT system (50 kV, 0.43 mA) with two angular sampling intervals (0.750° and 0.500°), Gaussian and Hann reconstruction kernels, and voxel sizes of 100, 160, and 200 µm. Quantitative performance was evaluated using sensitivity, coefficient of determination (R²), background noise, limit of detection (LOD), and limit of quantification (LOQ). PEG-AuNPs exhibited the highest sensitivity (80.55 HU/mg/mL) and the lowest detection limits (LOD: 0.53 mg/mL; LOQ: 1.61 mg/mL), outperforming bare AuNPs and BMX. Voxel size was the dominant reconstruction parameter, with optimal detectability achieved at 160 µm due to an optimal balance between spatial resolution and image noise, whereas reconstruction kernel and angular sampling had comparatively minor effects. These findings provide a quantitative framework for optimizing reconstruction parameters in nanoparticle-enhanced quantitative CT imaging.