A modular python-based pipeline for quantifying vascular bud density at the chondro-osseous border.

Angiogenesis during endochondral ossification is crucial to skeletal development, bone regeneration, and fracture healing. Vascular buds at the chondro-osseous border represent key markers of angiogenic activity. Conventional quantification approaches, such as global vessel density measurements or manual counting of buds, are time-consuming and often lack reproducibility. Therefore, we developed SACOB (Semi-Automated Counting of Buds), a modular Python-based software pipeline designed for reproducible and anatomically guided quantification of vascular buds in histological sections of chondro-osseous tissues. The SACOB workflow integrates image import, user-defined path annotation along the cartilage-bone interface, extraction and optional smoothing of intensity profiles, and algorithmic peak detection to identify vascular buds. Validation was performed on Endomucin-stained femoral sections from C57BL/6 mice aged 4, 12, and 18 months (n = 18). Four sections per animal, derived from central and peripheral regions, were analyzed to assess both age-related and spatial differences in angiogenesis (n = 6 animals/group; 4 sections/animal (total sections per age group = 24), balanced by sex). The software reliably detected vascular buds and reproduced biological patterns of vascular decline with aging. Bud density was highest in 4-month-old mice (22.23 ± 3.06 mm⁻¹), significantly reduced at 12 months (9.97 ± 1.54 mm⁻¹, p < 0.001), and further declined at 18 months (6.71 ± 2.11 mm⁻¹, p < 0.001). No significant differences were observed between central and peripheral femoral sections, indicating that angiogenesis activity is spatially consistent across the growth plate. Using SACOB workflow enables rapid, reproducible determination of vascular buds at the chondro-osseous border in tissue sections.