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Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4-to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. Key words: growth plate; vasculature; multiphoton microscopy; endochondral ossificationBone elongation in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. Chondrocytic differentiation in the growth plate begins with clonal expansion of stem cells, continues during cellular proliferation, and ends with cellular enlargement, followed by death and replacement by bone. The kinetics of chondrocytic activity in each stage and of the regulatory transitions between them determine the rate of bone elongation achieved. As in any cellular growth system, nutrients are required to sustain the process, and multiple endocrine and paracrine inputs regulate at each stage. For growth plate cartilage, the role of the extracellular matrix also must be considered, not only as a substantive contributor to elongation per se, but through its potential role as a communication link among chondrocytes of the growth plate, and between growth plate chondrocytes and cells of surrounding tissues such as the peri-

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