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In vivo visualization of Long Bone Growth

Bone elongation originates from cartilaginous discs (growth plates) at both ends of a growing bone. Here chondrocytes proliferate and subsequently enlarge (hypertrophy), laying down a matrix that serves as the scaffolding for subsequent bone matrix deposition (Figure 1a). To accomplish this feat, chondrocytes in endochondral cartilage align in an axial differentiation cascade directed towards the metaphysis or shaft of the bone. The entire process is orchestrated by at least five different cell types and countless signaling molecules. Because growth plate cartilage is avascular, all nutrients, oxygen, signaling mediators and waste must be transported relatively long distances through the tissue in order for it to survive and function. This project grew out of initial experiments designed to test hypotheses of unidirectional sourcing of nutrients and oxygen to growth plate Chondrocytes. Our In vivo imaging experiments revealed that small solutes experienced rapid equilibration (~ 1 min) through mouse growth plate cartilage from all vascular sources, differing from what was previously hypothesized. This early work at DRBIO resulted in an R01 and an R21 for our collaborator and spawned several new avenues of research. The overall goal is to gain a better understanding of the mechanisms of long bone growth by direct imaging of the dynamic processes in growth plate cartilage.

Multiphoton microscopy enables us to visualize transport directly in this region without disturbing the surrounding (sourcing) vasculature in the bone and perichondrium. To image transport in vivo, a bolus injection of (biologically inert) fluorescent tracer is monitored as it enters the growth plate (Figure 1b) from the vasculature, and the resulting time dependent fluorescence profiles are fit to determine spatially heterogeneous diffusion and flow coefficients. We demonstrated the existence of a highly-diffusing cartilage channel at the proliferative and early hypertrophic regions of the growth plate, where paracrine communication occurs between chondrocytes and cells in the surrounding perichondrium. These results are corroborated ex vivo using two-photon photobleaching recovery techniques in isolated growth plate slabs. Transport of small molecules in the living mouse is also significantly influenced by fluid flow from both the epiphyseal and metaphyseal chondroosseous junctions (Figure 1b), presumably resulting from a pressure difference between the bone vasculature and the cartilage. We suggest that due to these transport properties, small signaling and nutrient molecules originating in the bone will be distributed throughout the growth plate, whereas those originating in the perichondrium or cartilage will tend to be concentrated in the proliferative and early hypertrophic zones. These experiments have resulted in two recent publications (Farnum et al., 2006; Williams et al., 2007).

We are now concentrating more on specific questions concerning the nature of signaling within the growth plate. We have worked out imaging conditions so that individual chondrocytes within the growth plate can be monitored (with subcellular resolution) over many hours (Fig. 1c). General tissue movement and drift are eliminated using image analysis methods post acquisition. Our current research is focused specifically at the metaphyseal chondroosseous junction (COJ). Several decades ago, it was shown that growth plate chondrocytes at all stages of differentiation die after compromising the epiphyseal vasculature, but a compromise to the metaphyseal vasculature does not result in chondrocytic death, rather only a failure of chondrocytic apoptosis and bone formation on the metaphyseal side. These findings have led to a hypothesized unidirectional entrance of nutrients via the epiphyseal vasculature. The hypertrophic chondrocytes appear to be hypoxic, evidenced by the fact that angiogenesis in the hypertrophic zone is regulated by hypoxia inducible transcription factor 1a (HIF-1a). These ideas suggest that chondrocytic access to oxygen and nutrients from the metaphyseal vasculature is somewhat limited, even though the vessels at this location are known to be leaky as is characteristic of actively growing and remodeling vasculature. Our results showed that tracer entry to this region was just as rapid as that from other directions (Fig. 1b).

However, levels of nutrients and oxygen in the metaphyseal blood supply are still unknown. The accepted hypothesis is that in response to hypoxic conditions, hypertrophic chondrocytes express VEGF that promotes angiogenesis from the metaphyseal bone (Ortega et al., 2004); however, this model has recently come into question. Our own preliminary results correlate more with the latter. We also plan to image extracellular pH, which should decrease with anaerobic metabolism (preliminary results indicate no gradient) and are waiting for oxygen probes under development in Core R&D project 1, Aim 3. DRBIO has been essential in these imaging experiments. The Resource has developed an instrument and protocols for live mouse imaging, with excellent emission sensitivity. Which events, processes or environments that can be imaged successfully within this region will also depend on the probe development and screening project that is currently underway for in vivo use. We are particularly interested in the ability for imaging of apoptosis, MMP activity and/or oxygen concentrations near the metaphyseal COJ.  In the long-term studies, we often miss critical data due to breathing artifacts.  A scanner that was synchronized to the mouse would alleviate these problems.  Core R&D project II is expected to facilitate imaging of NIR-indicators such as MMPsense (Visen).  The ability to image deeper into the growth plate would better alleviate the effects of surface vasculature. 


Farnum, C.E., M. Lenox, W. Zipfel, W. Horton, and R. Williams (2006) In vivo delivery of fluoresceinated dextrans to the murine growth plate: imaging of three vascular routes by multiphoton microscopy. Anat Rec A Discov Mol Cell Evol Biol 288(1):91-103.

Williams, R.M., W.R. Zipfel, M.L. Tinsley, and C.E. Farnum (2007) Solute transport in growth plate cartilage: in vitro and in vivo. Biophys J 93(3):1039-1050.

Ortega, N., D.J. Behonick, and Z. Werb (2004) Matrix remodeling during endochondral ossification. Trends Cell Biol 14(2):86-93.



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