The Chondrification of the Human Ilium' R. D. LAURENSON Department of Anatomy, The University of Alberta, Edmonton, Alberta, Canada ABSTRACT This report on the early human ilium is based on the Streeter collection, Carnegie Institution of Washington, Department of Embryology, Baltimore. The five phases in chondrogenesis, described by Streeter for the humerus ('49) and the horizons (Streeter, '42) are used in the description of the changes observed in the ilium. From horizon XV (7-8 mm, 30-32 days) to horizon XVIII (14-16 mm, 36-38 days) component cells of the ilium are mesoblasts. In horizon XIX (17-20 mm, 38-40 days) young cartilage cells have appeared. Both phase 1 and phase 2 cells appear in horizon XX (21-23 mm, 4 0 4 2 days), the former characterized mainly by the first appearance of intercellular material, the latter by their slender shape and arrangement in tiers. Phase 3 cartilage cells, cuboidal with many vacuoles, appear in horizon XXI (22-24 mm, 4244 days). Increasing vacuolization of the cytoplasm of cells is noted throughout horizons XXII and XXIII until, in horizon XXIII (28-30 mm, 4648 days) total disintegration of cells, a feature of phase 5, is evident. Thus the phases of cartilage differentiation described for the humerus are present also in the ilium. Extensive observations on the pelvis and lower extremity were made by Bardeen and Lewis ('0 1) and Bardeen ('05). They produced from the reconstructions some very informative illustrations, but they did not report the micro-anatomy. Since then, no report on the micro-anatomy of the developing ilium has appeared, except for a brief abstract by Zawitsch ( ' 5 3 ) . Extensive use of the work of Bardeen and Lewis was made by Keibel and Mall ('10). Although they wrote a chapter on the histogenesis of connective tissue and dealt with cartilage and bone, they did not provide any detail concerning the cells of the developing ilium. Some reference to the chondrification of the ilium was made by Strayer ('43), Haines ('47) and by Gardner and Gray ( ' 5 0 ) , while they concentrated on the hipjoint. The present survey of cells from the origin of the ilium in mesenchyme until the cartilage model is well established and primary ossification impending establishes that in the earliest development of the definitive cartilaginous model the ilium and the humerus are alike. The humerus was selected by Streeter ('49) as a representative long bone when he included a chapter on the histogenesis of cartilage and bone in his study of human embryos in the developmental horizon series. He felt that the basic pattern he described underlay other bones of an individualistic type such as the clavicle and the vertebrae; to those bones one may add the ilium. The ilium was selected for further study because of the interest in congenital dysplasia of the hip and of the need to clarify the development of the ilium, if the problem of dysplasia is to be resolved. The basic pattern of chondrification described by Streeter ('49) included five phases of development. Phase one is characterized mainly by the presence of intercellular material enclosing cells emerging from the skeletal blastema, phase two by the slender shape of the cell, phases three and four by their cuboidal shape, the increase in size of the cell and in vacuolization of the cytoplasm, and phase five by the extensive disintegration of the cell. MATERIAL AND METHODS The Streeter collection of human embryos was made available at the Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland, U.S.A. through the courtesy of Dr. J. D. Ebert, director, and the phases of cartilage differentiation (Streeter, '49), and the horizons (Streeter, '42) are adhered to throughout this report. 1This work was supported by a grant from the Canadian Arthritis and Rheumatism Society. 197 198 R. D. LAURENSON The youngest embryo is 8.3 mm in length and the oldest 30 mm. Of the 42 embryos examined, 15 are included in this report. They had been fixed in formol, embedded in paraffin or colloidin-paraffin, cut in serial section in a range from 10 v to 50 cc in transverse, sagittal, or frontal planes, and stained by using a variety of stains but mainly hematoxylin-eosin. OBSERVATIONS Horizon XVII, 34-36 days Embryo no. 651 9, 10.8 mm. At the site of the future innominate bone, a core of condensed mesoblast cells lies within a border of more heavily condensed cells that is fairly distinct from the surrounding blastema (fig. 1). Horizon XVlII, 36-38 days Embryo no. 144,16.0 mm. The demarcation of the components of the innominate bone has proceeded further until the triradiate zone is well established (fig. 2). The border of condensed cells that was noted previously is much more distinct, and, over the pelvic surface of the ilium, the cells are in layers typical of perichondrium (fig. 2). Within the ilium there is some evidence of cartilage but, on the whole, the cells are highly differentiated mesoblasts (fig. 3). In embryo no. 991, 17.0 mm, remarked upon by Streeter as being near the older borderline of horizon XVIII, very young cartilage cells have definitely appeared in the ilium (fig. 4). According to these observations, the center of chondrification for the ilium appears in the human embryo at five weeks, and is situated cephalad to the greater sciatic notch. Horizon XX, 40-42 days The outstanding features of this horizon are the rapid increase in the dimensions of the ilium, and the appearance of phase one and phase two cells. In embryo no. 8157, 20.8 mm, for example, cartilage cells throughout the ilium have entered phase one (fig. 5). In the ala of this embryo, however, the elongated shape of an occasional cell and a suggestion of parallel rows of cells suggests that phase two has begun. In slightly older embryo, no. 4059, 21.6 mm, phase two cells are present throughout the ala of the ilium. Their slender shape and their arrangement in tiers is very characteristic (fig. 6 ) . Horizon XXII, 42-44 days Embryo 6832, 25.8 mm. Cartilage cells entering phase three lie in a core cephalad to the greater sciatic notch (fig. 7). Horizon XXIII, 46-48 days Embryo no. 4525, 30.0 mm. The cartilage cells in the vicinity of the greater sciatic notch are passing beyond phase four (fig. 8), and an occasional cell is already in phase five. DISCUSSION During chondrification of the ilium the cartilage model grows as a result of proliferation of fibroblasts in the mesenchyme surrounding the cartilage primordium and their transformation into young cartilage cells. This process of appositional growth probably contributes more to the mass of the model than does interstitial growth (Bloom and Fawcett, '62), except perhaps when the intercellular substance is not yet great in amount and sufficiently malleable (Ham and Leeson, '61) to allow the newly emerged young cartilage cells to divide and grow. Once, however, the intercellular substance is more in amount and less malleable and the perichondrium clearly established interstitial growth will decrease and appositional growth will predominate. As in the humerus (Streeter, '49), so in the ilium, there is no evidence of migration of cartilage cells which retain essentially the same position they occupied when they first appeared. Therefore, growth of the cartilage model is the result of proliferation, not migration, of cartilage cells. Admittedly, there are two additional factors, namely, an increase in size of each cell, and an accumulation of intercellular matrix, but proliferation is considered to be the most important factor in growth of the early cartilage model. The first cartilage cells found in the ilium in horizon XVlII and XIX are so very young that they are really at a stage between frank mesoblast and true cartilage. Cells at this stage, easily recognized, are termed precartilage, procartilage or, more CHONDRIFICATION, HUMAN ILIUM 199 recently, protochondral cells (Bloom and ous model. Nevertheless, it is presumed Fawcett, '62). However, having recognized that an orderly progression of phases does that these cells were in a state of transi- exist. They can be identified, for example, tion, and, I presume, that they defied con- in the acetabular roof. Thus it is concise description, Streeter restricted phase cluded that chondrification of the ilium is one to those cells beginning to acquire an not unusual. Finally, there is no evidence to support environment of increased intercellular substance. Accordingly, phase one cells are the observation that the center of chondrifirst noted in the ilium in horizon XX, 40- fication for the ilium is remote from the 42 days, but their predecessors in horizons center of ossification (Strayer, '43). The XIX and XVIII, 36-38 days, should not centers coincide and it is precisely in the perichondrium over cartilage cells in phase be ignored. Marked change occurs in the shape of five that the first sign of impending ossifithe ilium during horizon XX, when cells in cation is seen in the vicinity of the greater phase two become evident, and they are sciatic notch. particularly conspicuous in the ala of the ACKNOWLEDGMENT ilium, which has increased in length. AcThe author expresses his thanks to Mr. cordingly, with some reservation, phase two cells are concerned with a process of Richard Grill for his excellent photography. rapid lengthening. Probably their elonLITERATURE CITED gated shape and their arrangement in tiers Bardeen, C. R. 1905 Studies of the development is the result of rapid cell division. of the human skeleton. Am. J. Anat., 4: 265In referring to the cells in phases three 302. and four, the increase in the size of each Bardeen, C. R., and W. H. Lewis 1901 Developcell, the number of cytoplasmic vacuoles, ment of the limbs, body-wall and back in man. Am. J. Anat., 1: 1-37. and the amount of intercellular substance, is of note. All three increases are recog- Bloom, W., and D. W. Fawcett 1962 Textbook of Histology. W. B. Saunders Co., Philadelphia. nized features of biochemical activity London. Chap. 6, 148. which leads to calcification of the inter- Gardner, E., and D. J. Gray 1950 Prenatal decellular matrix. Presumably, the activity is velopment of the human hip joint. Am. J. Anat., 87: 163-212. increasing progressively throughout phases three and four. As a result, the cell, shut Haines, R. W. 1947 The development of joints. J. Anat., 81: 33-55. off from its normal source of nutrition Ham, A. W., and T. S. Leeson 1961 Histology. (Ham and Leeson, '61), perishes; as one J. B. Lippincott Co. Philadelphia. Montreal. would expect, the deeply located cells are Chap. 14, 271. first to reach this phase of development. Keibel, F., and F. P. Mall 1910 Manual of human embryology. Lippincott, Philadelphia. Thus, phase five, recognized by the disintegration and disappearance of the affected Strayer, L. M. 1943 The embryology of the human hip joint. Yale J. Biol. Med., 16: 13-26. cell, begins. Streeter, G. L. 1942 Description of age group All the changes just discussed can be XI, 13 to 20 somites, and age group XII, 21 somites to 29 somites. Contributions to Emobserved best in the vicinity of the greater bryology, 30: 211-245. sciatic notch. The earliest cartilage cells 1949 A review of the histogenesis of appear here and are the first to reach cartilage and bone. Contributions to Embryolphase five. The shape of the definitive ogy, 33: 149-167. ilium, however, makes it difficult to follow Zawisch, C. 1953 Die VerknGcherung der knorthe progression of phases of differentiation pelig vorgebildeten platten Knochen. Acta. Anat., 19: 384. during the further growth of the cartilagin- Abbreviations to plates 1 and 2 F, femoral nerve I, innominate bone IL, ilium IS, ischium 0, obturator nerve S, sciatic nerve Photomicrographs of base of limb bud to show innominate bone (figs. 1 and 2) and of cells of ilium (figs. 3-8). PLATE 1 EXPLANATION O F FIGURES Horizon XVII 10.8 mm. Base of limb bud medial to femoral nerve. The anlage of the innominate bone ( I ) is just emerging from the mesoblast. 8 p. Sag. Alum cochineal. x 75. Horizon XVIII 16.0 mm. The triradiate zone separates ilium, ischium and pubis. 40 p. Sag. Alum cochineal x 75. Same embryo as figure 2. The cells of the ilium are well differentiated mesoblast cells. x 375. Horizon XVIII 17.0 mm. An older embryo in the same horizon as the one in figure 3. The first evidence of chondrification. 50 p. Sag. Van Gieson x 375. 200 CHONDRIFICATION, HUMAN ILIUM PLATE 1 R. D. Laurenson 201 CHONDRIFICATION, HUMAN ILIUM PLATE 2 R. D. Laurenson 202 5 Horizon XX 20.8 mm. Cells in ala of ilium intermediate between phase one and phase two. 20 p. Frontal. Hematoxylin-eosin. X 375. 6 Horizon XX. 21.6 mm. Phase two cells in ala of ilium. 15 p. Frontal. Mallory. X 375. 7 Horizon XXII 25.8 mm. Cells cephalad to greater sciatic notch entering phase three. 20 p. Frontal. Hematoxylin-eosin. X 225. 8 Horizon XXIII 30.0 mm. Cells in vicinity of greater sciatic notch i n late phase four. 20 p . Sag. Hematoxylin-eosin. X 225.