COPYRIGHT © 2002 BY THE JOURNAL OF BONE AND JOINT SURGERY, INCORPORATED Structural Stages in the Development of the Long Bones and Epiphyses A STUDY IN THE NEW ZEALAND WHITE RABBIT BY ROBERTO RIVAS, MD, AND FREDERIC SHAPIRO, MD Investigation performed at the Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Children’s Hospital, Harvard Medical School, Boston, Massachusetts Background: Histologic delineation of the events involved in the development of long bones and the develop- mental age at which these events occur is needed to elucidate the genetic and molecular mechanisms associ- ated 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. Methods: 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. Results: 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 begin- ning 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 ca- nals (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); full- est 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). Clinical Relevance: The detailed classification system presented here will allow for correlations between genetic and molecular mechanisms and histologic events in normal and abnormal development of long bones and their epiphyses. Many of the nonosseous structures formed during long-bone and epiphyseal development in the fetus, infant, and child are amenable to assessment with sonography and magnetic resonance imaging. An understand- ing of the histopathological features of developmental abnormalities of the long bones and their epiphyses re- vealed with newer imaging techniques should greatly improve management by allowing earlier diagnosis. cientific studies have revealed that bones grow in length edge of the precise sequence of these histologic events and the by increments at their cartilaginous ends and in width by times at which they occur. periosteal apposition and that they remodel by resorp- The purposes of the present study were to determine the S 1-3 tion at the metaphyseal and inner cortical regions . In the last sequence of histologic events involved in the formation of several years, much has been learned about the molecular long bones and their epiphyses from the embryonic limb-bud mechanisms involved in limb morphogenesis4-10. Correlation stage to skeletal maturity, to classify the various stages, and to of the continuum of histologic events with molecular mecha- define the time at which each event occurs in the New Zealand nisms during the development of a long bone requires knowl- White rabbit. THE JOURNAL OF BONE & JOINT SURGERY · JBJS.ORG STRUCTURAL STAGES IN THE DEVELOPMENT OF THE VOLUME 84-A · NUMBER 1 · JANUARY 2002 LONG BONES AND EPIPHYSES TABLE I Histologic Stages in Long-Bone and Epiphyseal Development Stage* Histologic Events 1 Limb-bud formation, uniform distribution of mesenchymal cells, and formation of apical ectodermal ridge 2 Mesenchymal condensation 3 Cartilage differentiation 3a Interzone formation 3b Chondrocyte hypertrophy in middle part of long-bone cartilage model 4 Epiphyseal shaping 4a Formation of intramembranous periosteal bone at mid-diaphysis (primary center of ossification) 5 Resorption of joint interzone and formation of smooth articular cartilage surface 5a Vascular invasion of hypertrophic chondrocyte area, endochondral bone formation (mid-diaphysis), and completion of formation of primary center of ossification 6 Formation of the physis and of peripheral perichondrial groove tissue 6a Farthest relative extent of epiphyseal/physeal position 7 Vascularization of epiphyseal cartilage with formation of cartilage canals 8 Central chondrocyte hypertrophy to form spherical mass, development of growth plate completely surrounding secondary ossification center 9 Vascular invasion of developing secondary ossification center into hypertrophic chondrocytes adjacent to mineralized cartilage matrix 10 Bone formation and marrow cavitation in secondary ossification center, formation of hematopoietic marrow 11 Increase in size of secondary ossification center, decrease in size of epiphyseal cartilage 12 Central chondrocyte hypertrophy and secondary-ossification-center growth-plate change from spherical to hemi- spherical orientation 13 Fat in marrow, hematopoietic marrow adjacent to secondary-ossification-center growth plate 13a Epiphyseal bone-plate formation 14 Fullest relative extent of secondary-ossification-center development in epiphyseal cartilage 15 Thinning of physis 15a Involution of secondary-ossification-center growth plate 15b Subchondral bone-plate formation 16 Resorption of physis with linkage of epiphyseal and metaphyseal circulations 16a Calcification of lowest zone of articular cartilage, tidemark formation, and transformation of all marrow to fat *The substages, labeled a and b, refer to events occurring at the same time as a particular stage in different parts of the same bone or to a structurally important continuation of the same process at a slightly later time. Fig. 1-A The components of the developing end of a long bone. The epiphysis is composed of the articular cartilage (AC), the epiphyseal cartilage (EC), and the physis (growth plate). The secondary ossification center (SOC) forms by the endochondral mechanism within the epiphyseal cartilage. It is completely surrounded in the earlier phases of development by another growth plate, the growth plate of the secondary ossification center (GP-SOC), which is responsible for the circumferential growth of the secondary center. THE JOURNAL OF BONE & JOINT SURGERY · JBJS.ORG STRUCTURAL STAGES IN THE DEVELOPMENT OF THE VOLUME 84-A · NUMBER 1 · JANUARY 2002 LONG BONES AND EPIPHYSES Materials and Methods animals. The embryos were obtained from Pel-Freez Biologi- Study Group cals (Rogers, Arkansas). The embryos were staged according he formation and development of the long bones and epi- to the external criteria described by Edwards11 and in previous Tphyses in the New Zealand White rabbit were studied studies of rabbit embryos12,13. Twelve, fourteen, and fifteen- prenatally in twelve, fourteen, fifteen, sixteen, eighteen, twenty- day-old embryos were examined and photographed with use one, twenty-four, and twenty-seven-day-old embryos and post- of a dissecting photomicroscope for limb-bud definition. Pre- natally in newborn; three-to-four-day-old; one, two, four, and natal studies were performed on serial sections of developing six-week-old; and two, three, four, six, and eight-month-old upper and lower limbs. Twelve, fourteen, and fifteen-day-old Fig. 1-B The major stages in the formation and development of long bones and epiphyses (defined in Table I), from stage 1 to stage 16a. THE JOURNAL OF BONE & JOINT SURGERY · JBJS.ORG STRUCTURAL STAGES IN THE DEVELOPMENT OF THE VOLUME 84-A · NUMBER 1 · JANUARY 2002 LONG BONES AND EPIPHYSES TABLE II Relationship Between Age and Developmental Stage at Major Long Bones and Their Epiphyses ➤ Humerus Proximal Parts of Age Proximal* Middle Distal Radius and Ulna Prenatal 12 d 1 1 1 14 d 2 3 2 2 15 d§ 3, 3a, 4 3b 3, 3a, 4 3, 3a, 4 16 d 4 4a 4 4 18 d 6a 5a 5 5 21 d 7 6 6 24 d 8 8 6a 27 d 8/8 8 8 Postnatal Newborn 9/9 9 3-4 d 10/9 9 1 wk 10/10 10 2 wk 12/11 12 12 4 wk§ 13/13, 13a/13a 13, 13a 13a 6 wk 2 mo 15/15 15 3 mo§ 15, 15a, 15b 15, 15a, 15b 4 mo 6 mo 16 16a 8 mo 16 16 16 *Two separate secondary ossification centers initially form in the proximal part of the humerus. From stages 8 to 15, the developmental stages for the medial center and the lateral center are separated by a slash. Differentiation is not made after fusion into one osseous mass. †Two separate secondary ossification centers initially form in the proximal part of the femur. The developmental stages for the femoral head center and the greater trochanter center are separated by a slash. Differentiation is not made after fusion into one osseous mass. ‡Two sep- arate secondary ossification centers initially form in the proximal part of the tibia. The developmental stages for the main proximal center and the tibial tubercle center are separated by a slash. Differentiation is not made after fusion into one osseous mass. §When two or more stages are identified in the same epiphysis at the same time, the stages are separated by commas. embryos were serially sectioned intact, whereas sixteen, eigh- femora of newborn, three-to-four-day-old, and one and two- teen, twenty-one, twenty-four, and twenty-seven-day-old em- week-old animals and from the humeri of newborn and one- bryos were studied with use of serial sections of the upper and week-old animals.