In vivo delivery of fluoresceinated dextrans to the murine growth plate: imaging of three vascular routes by multiphoton microscopy

Abstract

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.

Figure 1

Figure 1A depicts the proximal tibia in a young mouse showing the three potential routes of vascular access to the growth plate: vessels of the epiphysis, vessels of the metaphysis, and vessels associated with a ring vessel and plexus running just deep to the periosteum. The latter vessels would not be visible on a sagittal section, since they are associated with the periosteum. Figure 1B is a one-micron-thick histological section of the proximal tibia from a four-week-old mouse, fixed in 2% paraformaldehyde/ 2% glutaraldehyde with 0.7% ruthenium hexamine trichloride, embedded in Epon-Araldite (Hunziker and Schenck, 1989), and stained with methylene blue/azure II/basic fuchsin. Note the articular cartilage (a), trabecular bone of the secondary center of ossification (soc), the perichondrium (p), and the longitudinally oriented bone of the elongating metaphysis (m). As shown in C, the reserve cell zone (rz) is limited to one or two cells adjacent to the secondary center. Proliferative zone cells (pz) are arranged in columns with their long axis perpendicular to the direction of growth; hypertrophic zone cells (hz) have their long axis parallel to the direction of elongation. Figure 1D is a multiphoton image of a sagittally sectioned, freshly isolated growth plate, non-stained, showing the same cellular morphology. Cellular autofluorescence (grey pseudocolor) and collagen second harmonic generation (blue pseudocolor) can be seen when the section is illuminated at 770 nm.

Figure 2

For surgery and imaging the mouse was positioned in dorsal recumbency, on a heating pad under isoflurane anaesthesia (A). A surgical approach was made to the medial side of the left tibia using the patellar ligament (p), the medial collateral ligament (m) and the saphenous vessels and nerve (s) as landmarks (B). An asterisk (*) indicates the position of the growth plate with the perichondrium intact. The surgical field is illuminated to demonstrate the yellow-green fluorescence of OTC in the epiphyseal (e) and metaphyseal (m) bone (C). The growth plate appears as a non-fluorescent band between the two areas of fluorescing bone. Figure 2D is the identical surgical field, illuminated to show the green fluorescence of the GFP-positive chondrocytes of the growth plate, between the arrowheads. Asterisks mark the medial collateral ligament.

Figure 3

Figures A-D are four frames of a z-series, from superficial to the periosteum (A), through the periosteum (B,C), and to the growth plate (D) to verify orientation and positioning. Separation of emission colors allows simultaneous imaging of collagen second harmonic generation (blue), and OTC and GFP-fluorescence (green). GFP-positive proliferative cells are adjacent to the asterisks. OTC-fluorescent epiphyseal and metaphyseal COJs are indicated by double and single arrowheads respectively. The growth plate as imaged in Figure D is 70μm deep to the perichondrium. e=epiphyseal bone; m=metaphyseal bone Figures 3. E-H demonstrate the network of the vascular plexus, visualized after an IP injection of fluorescein so that the plasma is fluorescent and blood cells can be seen as dark shadows within the vessels. Frames E and F (taken 5 μm deeper than E), demonstrate that the network of the plexus is on the deep side of the collagen of the perichondrium, and that the ring vessel is in a plane slightly deeper than the plexus. In frames G (another 20 μm deeper) and H (another 10 μm deeper) asterisks are positioned at the metaphyseal COJ. The ring vessel encircles the growth plate at approximately the P/H junction, and the plexus extends entirely on the hypertrophic zone side.

Figure 4

Each row shows four frames from a timed series after injection of fluorescein (vascular concentration ∼2 mM), IP, pseudo-colored in red. The pattern of arrival of fluorescein into the growth plate reflects the depth of the imaging plane relative to ring vessel and plexus, more superficial in frames E-H than in frames A-D. The epiphyseal COJ is indicated by double arrowheads at the left in A and E, the metaphyseal COJ by a single arrowhead, and OTC-labeling of the bone is green. e=epiphyseal bone; m=metaphsyeal bone. In series A-D from a GFP-/- mouse, fluorescein arrives essentially simultaneously into the growth plate from both the epiphyseal and the metaphyseal sides (C, D are 336 and 590 seconds after injection, respectively), and the last area of the growth plate to be reached is the P/H junction (asterisk). By contrast, in a more superficial focal plane shown in E-H from a GFP +/- mouse, the P/H junction is the first region to receive fluorescein (F and continuing into G and H). Times after injection for F,G,H are 94, 242, and 401 seconds, respectively. The shadow indicated by three arrows in G and H is that of the ring vessel, with intense fluorescein on either side.

Figure 5

Graphs show tracer arrival half-time (vertical axis) relative to the tracer's position-specific fluorescence at five minutes. The epiphyseal bone is on the left and the metaphyseal bone on the right, with the respective COJs represented as vertical lines. Figures A,B plot the entrance of fluorescein (∼2 mM vascular concentration), in a relatively deep (A, 90 μm deep to the periosteum) versus a relatively superficial (B, 45 μm deep to the periosteum) position. In the deep location (A), tracer arrives first from both COJ vasculatures, whereas in the superficial position arrival occurs just as early at the P/H junction, presumably from the ring vessel and plexus (arrow in B). Figures C,D and E,F are analogous graphs for the entrance of 3 kDa and 10 kDa dextrans, respectively, after IC injection of 50μl, 0.1% solution. Patterns of arrival are identical to those for fluorescein. Figure G demonstrates that the 40 kDa dextran, injected IC, does not enter the growth plate. Figure H demonstrates an IP injection of fluorescein (200μl, 0.5% solution) in a superficial position. The form of the graph indicates the arrival of fluorescein to the growth plate is significantly slower than when injected IC (B), and also that in this image plane a significant amount is reaching the hypertrophic zone from the plexus, indicated by the relatively long straight line in the graph covering most of the hypertrophic cell zone.

Figure 6

This bar graph presents data for estimating the relative amount of tracer that leaves the vasculature and enters the growth plate. Data are calculated as the ratio of fluorescence intensity in the growth plate relative to the maximal vascular fluorescence. Both IC and IP fluorescein enter the growth plate readily from the vasculature and to the same extents. By contrast, entrance of the 3 kDa dextran is half that compared to fluorescein, and the 10 kDa dextran only one tenth.

Figure 7

These graphs are presented with the same conventions as Figure 5. Figures A,B demonstrate that IC-injected fluorescein (50μl, 1% solution) can enter entirely from the epiphyseal vasculature (A), or almost entirely from the metaphyseal vasculature (B), if the experimental situation is such that the vasculature of one side of the growth plate fills more rapidly than that of the other. In this situation, the fluorescein that enters has the potential to move throughout the growth plate, reaching the opposite COJ (7A). Figure 7C demonstrates that dextrans also behave this way. The specific graph demonstrates entrance of a 10 kDa dextran, primarily from the metaphyseal side and diffusing from the metaphyseal COJ well into the proliferative cell zone. Figure 7D is an IC injection of fluorescein, following the 10 kDa injection, demonstrating that directionality of entrance of fluorescein mirrors that of the 10 kDa dextran.