Structural stages in the development of the long bones and epiphyses: a study in the New Zealand white rabbit

Histologic delineation of the events involved in the development of long bones and the developmental age at which these events occur is needed to elucidate the genetic and molecular mechanisms associated with these events. This report describes the sequence of histologic events involved in the formation of long bones and their epiphyses in the New Zealand White rabbit.

Prenatal studies were performed on twelve, fourteen, fifteen, sixteen, eighteen, twenty-one, twenty-four, and twenty-seven-day-old rabbit embryos, and postnatal studies were performed on newborn rabbits and on three-to-four-day-old; one, two, four, and six-week-old; and two, three, four, six, and eight-month-old rabbits. Histologic specimens from embryos were embedded in plastic and stained with toluidine blue or safranin O-fast green, and specimens from postnatal rabbits were embedded in paraffin and stained with hematoxylin and eosin or safranin O-fast green.

Studies of twelve-day-old embryos demonstrated upper and lower limb buds filled with undifferentiated mesenchymal cells, and studies of fourteen-day-old embryos showed mesenchymal condensation and beginning cartilage formation outlining major long bones. Long-bone and epiphyseal development progressed through sixteen structural stages, and the developmental age at which these stages occurred was determined. These stages included limb-bud formation with uniform distribution of mesenchymal cells and formation of an apical ectodermal ridge (stage 1); mesenchymal condensation (stage 2); cartilage differentiation (stage 3); formation of a primary center of ossification (stage 4a); epiphyseal cartilage vascularization with formation of cartilage canals (stage 7); vascular invasion of the developing secondary ossification center (stage 9); bone formation and marrow cavitation in the secondary ossification center with formation of hematopoietic marrow (stage 10); fullest relative extent of secondary-ossification-center development in epiphyseal cartilage (stage 14); thinning of the physis (stage 15); and resorption of the physis with establishment of continuity between epiphyseal and metaphyseal circulations (stage 16).