Image analysis software

Fluorescent calcein accumulation and trabecular bone histology in methyl acrylate-embedded tibiae were probed using fluorescence microscopy and von Kossa staining, respectively. In some experiments, osteoclasts and osteoblasts in sections of decalcified specimens were probed using tartrate-resistant acid phosphatase and alkaline phosphatase staining, respectively. Mineral acquisition rate (μm/day), trabecular bone histology (BV/TV, %), osteoclast number (Oc.N/mm), and osteoblast number (Ob.N/mm) were measured using Zeiss image analysis software. Three random fields in each section, three sections of each mouse, and six animals were randomly selected for analysis.

Primary osteoclast precursor cells were isolated from mouse femurs to explore the impact of DKK1-AS or DKK1-S treatment on the osteoclastogenic potential of the harvested precursors. Nucleated cells were obtained from the bone marrow using RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) and incubated in α-minimum essential medium (MEM) supplemented with 10% FBS and 20 ng/mL macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) for 24 h. Macrophages typically exhibit non-adherence to surfaces; thus, non-adherent cells were collected following incubating nucleated cells in M-CSF treatment. Subsequently, 10⁵ collected non-adherent cells per well were seeded into 24-well plates and cultured in osteoclastogenic medium consisting of α-MEM, 10% FBS, 20 ng/mL M-CSF, and 20 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL) (R&D Systems, Minneapolis, MN, USA) for 7 days. Osteoclasts were identified using TRAP staining, and six × 125 magnification fields were randomly selected from each well, with a total of six wells for each treatment, to quantify osteoclasts using image analysis software (Carl Zeiss) (Wang et al. 2013 (link); Zhang et al. 2024 (link)).

CoPA neurons were scored for the anterior or posterior direction of post-midline crossing axons. Only CoPA axons caudal to the 9th somite were scored for reproducibility. Analysis of pre-crossing axons was achieved by drawing a line perpendicular to the A–P axis at the axon hillock of each CoPA cell. A second line was drawn from the axon hillock to the point of entry at the floorplate. If the angle of the two lines was greater than 3°, the axon fiber was considered to have an anterior or posterior direction bias. Pearson’s Chi square test was utilized to test percentages for statistical significance. We measured the distance travelled by CoPA axons within the floorplate along the A–P axis from lateral view confocal stacks using image analysis software (Zeiss). The commissure can be defined as the area of the midline occupied by CoPA axons along the longitudinal axis, which is devoid of ventrally-projecting ipsilateral axon, or the dorsally projecting contralateral axon. Statistical significance for commissure length was determined using a two-tailed Student’s t test. All statistical tests were conducted with JMP11 statistical software provided by Virginia Commonwealth University.

Animal studies were approved by the Committee for Animal Care and Use of The First Faculty of Medicine in Prague. In order to study the tissue reaction, the hydrogel expanders were implanted intramuscularly into the dorsal muscles of BALB/c mice (Velaz, Prague, Czech Republic). The experimental animals were anesthetized with isoflurane and the skin, subcutaneous tissue and muscle was cut. The implants (of approx. size of 8 mm × 4 mm × 2 mm) were carefully inserted into the dorsal muscle and the wound was sutured layer by layer. The animals were given analgesics and antibiotics after surgery. 3, 5, 7, 18 and 31 days after implantation the mice were sacrificed (3 animals in each group, each animal having implant in right and left leg), the implant was removed, measured and weighed. Surrounding tissue was fixated with paraformaldehyde and stained for Hematoxylin & Eosin (Sigma-Aldrich, Prague, Czech Republic). The thickness of connective tissue capsule around the implant was measured by the image analysis software (Zeiss, Prague, Czech Republic).