Solute transport in growth plate cartilage: in vitro and in vivo

Abstract

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. Because cartilage is generally avascular, all nutrients, oxygen, signaling molecules, and waste must be transported relatively long distances through the tissue for it to survive and function. Here we examine the transport properties of growth plate cartilage. Ex vivo, fluorescence photobleaching recovery methods are used in tissue explants. In vivo, multiphoton microscopy is used to image through an intact perichondrium and into the cartilage of anesthetized mice. Systemically introduced fluorescent tracers are monitored directly as they move from the vasculature into the cartilage. We demonstrate the existence of a relatively permissive region at the midplane of the growth plate, where chondrocytes transition from late proliferative to early hypertrophic stages and where paracrine communication is known to occur between chondrocytes and cells in the surrounding perichondrium. Transport in the living mouse is also significantly affected by fluid flow from the two chondro-osseus junctions, presumably resulting from a pressure difference between the bone vasculature and the cartilage.

FIGURE 1

Diagram of a juvenile bone, showing the three major vascular sources to the avascular growth plate. Chondrocytes align in axial rows and proliferate extensively. Differentiation and hypertrophy of these cells is thought to be involved in the pushing of the epiphysis away from the elongating metaphysis. Secreted matrix acts as the scaffolding for bone formation at the metaphyseal end. Spatially varying transport coefficients are described in terms of the normalized axial position from 0.0 at the epiphyseal COJ to 1.0 at the metaphyseal COJ. Note that the proliferative-to-hypertrophic transition occurs roughly at the axial center of the growth plate.

FIGURE 2

Verification of our image-based method for measuring diffusion. Images of (a) FL and (b) 40k-FL diffusing in a 1-mm-wide channel with flow. Images are acquired at 10-s intervals but displayed at 200 and 100 s intervals for FL and 40k-FL, respectively. To avoid edge effects, only data from the center of the channel were used. Fluorescence data from the FL and 40k-FL time series were averaged laterally across the channel and displayed in curves that are color-coded to indicate time (c and d, respectively). Note that at these temporal and spatial scales, FL transport is dominated by diffusion, which tends to equilibrate concentration gradients, but 40k-FL transport is dominated by flow, which causes a translocation of concentration gradients. Fits to the curves yielded average diffusion (flow) values of 223 μm²/s (2.3 μm/s) and 56 μm²/s (4.2 μm/s) for FL and 40k-FL, respectively. Solution diffusion coefficients were within 30% of those measured using standard FPR measurements described subsequently and those reported in the literature (51). Multiphoton images were acquired as described in Materials and Methods, except that a low magnification 4×/0.28 NA Olympus macro objective was used for obtaining the necessary large field-of-view.

FIGURE 3

Multiphoton fluorescence photobleaching recovery diffusion measurements. Proximal tibial sections are isolated, sectioned once midsagitally, and incubated in either FL, 10k-FL or Ova-FL. (a–c) Representative data from an Ova-FL incubated growth plate. Scale bar = 25 μm. The laser is parked at the locations indicated (squares) and a 50-μs pulse bleaches out ∼20% of the fluorescence in the focal spot; the timescale of fluorescence recovery is fit to determine diffusion coefficients for the species in that location. Example FPR traces from the red and orange squares are shown in panel b with their respective fits. (c) Diffusion coefficients for the locations indicated in panel a are plotted against the normalized axial position along the growth plate. The curve shows a polynomial fit to the data. (d) Cumulative plot of data such as those in panel c from multiple experiments and multiple tracers. These data represent measurements from n = 7 (FL, blue), n = 6 (10k-FL, green), and n = 6 (Ova-FL, yellow) similar experiments.

FIGURE 4

Multiphoton images of the proximal tibial growth plate 165-μm deep from the superficial surface of the perichondrium in a 32-day-old mouse (a) 18, (b) 54, and (c) 102 s after an IC injection of FL. The tracer is distributed to the epiphyseal and metaphyseal vasculature (EV and MV) within seconds and reaches the axial center of the growth plate within minutes (scale bar = 50 μm). Such images are recorded every 5.8 s and the intensity values are averaged laterally and displayed color-coded for time in panel d. Polynomial fits to these curves (e) provide functions that are smoothed and easily differentiable. Local flow rates and diffusion coefficients are calculated by analyzing the rate-of-change of the fluorescence and comparing it to the first and second spatial derivatives at every point. For comparison among growth plates, the axial position along the growth plate is normalized such that the E and M COJs are at 0.0 and 1.0, respectively; positions are demarcated by vertical dotted lines throughout the figure. (f) The blue and orange curves represent the local D and v values for the image series in panels a–c. The error bars show average mean ± SE for n = 7 similar experiments.

FIGURE 5

Arrival patterns of 10k-FL and FL at the same location. Multiphoton images of the growth plate edge, the “groove of Ranvier”, acquired (a) 63, (b) 168, and (c) 290 s after an IC 10k-FL injection and (d) 12, (e) 46, and (f) 93 s after a second injection, this time with FL. Note that the 10k-FL exhibits a more circumferential arrival whereas the FL clearly enters from both bone vasculatures. Scale bar = 50 μm.

FIGURE 6

10k-FL transport is effectively blocked at the metaphaseal COJ. (a) Time series of images at 90 s intervals showing that the IC-injected 10k-FL leaks from the metaphyseal vasculature into the primary spongiosa, but not to an appreciable degree into the cartilage. (b) A similar series of images (12 s intervals) after a FL injection showing that FL immediately enters the cartilage as well. Scale bar = 50 μm. (c) Average intensity plotted as a function of time for the 10k-FL series in panel a. Different regions (the vasculature, the primary spongiosa, and the cartilage just adjacent to the metaphyseal COJ) are color-coded in red, blue, and green, respectively (see inset). Note that the fluorescence peaks at 90 s in the vasculature and in the spongiosa a few minutes later. The tracer does not significantly enter the cartilage. (d) A similar analysis of the FL-injected images shows that all regions peak within less than a minute.

FIGURE 7

The illustration to the left displays transport routes critical to chondrocyte development and ultimately bone growth (4,6,9,12). To the right, hypothesized transport patterns within the growth plate are diagramed. Cartilage permissivity is coded by shading around the chondrocytes, with hindered diffusion near the two COJs. Axial flow measurements (solid lines) reveal flow that is directed away from the COJs. Draining toward the perichondrium is presumed (dotted lines). Note that due to both the heterogeneous diffusion characteristics and the overall flow profile, perichondrium-produced molecules will be enriched in the proliferative and early hypertrophic zones.