Angiogenesis in the distal femoral chondroepiphysis of the rabbit during development of the secondary centre of ossification

Abstract

In the developing chondroepiphyses of long bones, the avascular cartilaginous anlage is invaded by numerous blood vessels, through the process of angiogenesis. The objective of this study was to investigate the chronology of this vascular invasion with the spontaneous calcification of the cartilaginous epiphysis during development of the secondary ossification centre in the rabbit distal femur. The time-course of chondroepiphyseal vascular invasion was determined histologically and standardized for eight gestational and four postnatal intervals by plotting kit body mass against crown-rump length. Similarly, microcomputed tomography (micro-CT) helped to visualize calcification at those same gestational and postnatal intervals. To confirm the angiogenic nature of the avascular chondroepiphysis, such samples were assayed on the chick chorio-allantoic membrane (CAM). Neovascular outgrowths from the CAM were apparent 48 h following introduction of an 18-day (gestational) chondroepiphyseal sample. Chondroepiphyseal samples were assayed for the potent developmental angiogenic factors bFGF and VEGF, with the mRNA expression for both these mediators being confirmed using RT-PCR. As angiogenesis and calcification during chondroepiphyseal development occur in a defined tissue environment initially devoid of blood vessels and mineral, those processes provided a unique opportunity to study their progression without complication of injury-related inflammation or extant vasculature and mineral. Furthermore, the discovery of angiogenic, angiostatic or mineral-regulating mediators specific to developing connective tissue may prove useful for analysing the regulation of vascular and mineral pathogenesis in articular tissues.

Fig. 1

Time-course of fetal New Zealand White rabbit growth, from 18 days of gestation, through birth at 30 days, to 11 days after birth. Kit crown–rump length was plotted against mass for each litter of rabbits killed. Litter ages (expressed as days after conception) are shown in the legend below the title.

Fig. 2

Vascular material stained in the distal femoral chondroepiphysis using antithrombomodulin immune-like reactivity in:(a)21 days of gestation metaphyseal cartilage canal erosion,(b)the mineralized diaphysis at 23 days of gestation and (c)the centrally forming secondary centre of ossification (arrow)at 29 days of gestation.Panel (d)illustrates the same frozen section at 29 days of gestation under bright-field transillumination,indicating black regions of calcified mineral (arrow),seen as multiple expanding nidi within the distal femoral chondroepiphysis and in the diaphysis.Scale bars =50 µm.

Fig. 3

Three-dimensional (3D) microcomputed tomography reconstructions of prenatal mineralization. Lateral view of the progression of mineralization in the skeletal structures of the left knee of the fetal New Zealand White rabbit. Gestational days are indicated. Joint integrity was maintained in all samples. All scans are presented at ×10 magnification, and the relative scale of the 3D reconstructions was consistent within the figure.

Fig. 4

Three-dimensional (3D) microcomputed tomography reconstructions of postnatal mineralization. Lateral view of the progression of mineralization in the skeletal structures of the left knee of the newborn (NB) New Zealand White rabbit. Days postnatal are indicated. Joint integrity was maintained in all samples, except in NB17 where it was approximated from two separate scans due to the large sample size. All scans are presented at ×9 magnification, and the relative scale of the 3D reconstructions was consistent within the figure (though scaled 25% smaller than the previous prenatal mineralization panel in Fig. 3).

Fig. 5

Angiogenesis from the chick chorio-allantoic membrane directed toward a 48-h implant (magnifications ×5 and ×15.3 for a and b, respectively), and toward a 72-h implant (magnifications ×5 and ×15.3 for c and d, respectively) of 20 days of gestation fetal rabbit chondroepiphyseal explant. Arrows identify the implanted rabbit chondroepiphysis.

Fig. 6

Gene expression from samples of fetal rabbit chondroepiphyses, as determined by reverse transcription-polymerase chain reaction (RT-PCR). Samples were probed for