Imaging IGF-I uptake in growth plate cartilage using in vivo multiphoton microscopy

Bones elongate through endochondral ossification in cartilaginous growth plates located at ends of primary long bones. Linear growth ensues from a cascade of biochemical signals initiated by actions of systemic and local regulators on growth plate chondrocytes. Although cellular processes are well defined, there is a fundamental gap in understanding how growth regulators are physically transported from surrounding blood vessels into and through dense, avascular cartilage matrix. Intravital imaging using in vivo multiphoton microscopy is one promising strategy to overcome this barrier by quantitatively tracking molecular delivery to cartilage from the vasculature in real time. We previously used in vivo multiphoton imaging to show that hindlimb heating increases vascular access of large molecules to growth plates using 10-, 40-, and 70 kDa dextran tracers. In order to comparatively evaluate transport of similarly sized physiological regulators, we developed and validated methods for measuring uptake of biologically active IGF-I into proximal tibial growth plates of live 5-week old mice. We demonstrate that fluorescently labeled IGF-I (8.2 kDa) is readily taken up in the growth plate and localizes to chondrocytes. Bioactivity tests performed on cultured metatarsal bones confirmed that the labeled protein is functional, assessed by phosphorylation of its signaling kinase Akt. This methodology, which can be broadly applied to many different proteins and tissues, is relevant for understanding factors that affect delivery of biologically relevant molecules to the skeleton in real time. Results may lead to the development of drug-targeting strategies to treat a wide range of bone and cartilage pathologies.

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