THE HISTOGENESIS OF BONE By C. WITHERINGTON STUMP, M.D. From the Laboratory of the Royal College of Physicians, Edinburgh INTRODUCTION FEW processes in histogenesis have been the subject of so much controversy, as that of the development of bone. On reading over the literature, even incompletely, the conviction came with considerable force, that the chief cause of the inability of observers, to find agreement, lay in technical difficulties in obtaining satisfactory preparations for cytological study. The divergent opinions are accounted for by the singular absence of detailed work-a fact which has fostered and sustained a controversy into which there is no need to enter. Microscopic evidence of cell growth and change is yielded by slight morpho- logical variations in different individual cells. Slight differences in structure, and staining reaction, are the only factors by means of which variations can be determined. It is essential, with cells examined in a dead state, to have a standard of fixation which registers detailed and minute structure. Further, it is constantly to be borne in mind, that merely a phase of life is represented, through which the cell was passing at the time of its death. Especially is this so in actively growing, developing tissue, where many structural tendencies are manifested by the act of growth. Thus, in fixed primitive tissue, the ontogeny of any given cell is a matter of conjecture, and it can only be defined with any degree of certainty by an experienced observer. It would be redundant to attempt an historical survey of the contributions to the problem of osteogenesis. Keith(1s), beginning with Duhamel(7) and Haller(12), an hundred and fifty years ago, has traced the main advances in our knowledge of bone growth, from the experimental side. The former asserted the osteogenic function of periosteum, the latter, having observed the deeply placed epiphyseal centres, denied it. Keith writes: "After a century and a half of discussion we still have these two schools." The issue involved in this con- troversy is the origin of the osteoblast, essentially a cytological problem, dependent for its solution on the recognition of the osteoblast as a definite cell entity. As far as has been possible, an attempt has been made to present descrip- tions of the processes studied in developmental sequence. Interpretation in terms of growth has been the object; rather than the mere recording of a series of microscopical phenomena of fixed tissue, a prevalent procedure, bereft of all meaning, inasmuch as living tissue comes to be regarded as though it were dead. The Histogenesis of Bone 137 The results embodied in this research, in a measure find agreement with much that has been done hitherto, but of previous authors, Maximow's (24) is the more completely confirmed. The preparations, though obtained with a dif- ferent technique, render similar cytological values, which in many respects yield a similar interpretation. TECHNIQUE M-aterials and methods. Rabbit, guinea-pig, cat, dog, and human foetal l)ones were examined. No essential differences, in the processes described, were found in these animals. Rabbits and guinea-pigs form more convenient material than other mammals, both because of the ease of breeding, and because of the small bulk of the tissue. The former are pre-eminently more suitable, foetal growth being extremely rapid, a gestation period of thirty days resulting in a full-time foetal animal, approximately the same size and weight as a full- time foetal guiinea-pig, which has a gestation period of seventy days. All types of cartilage bones were used in various stages of development, especially the short and long bones of the fore and hind limbs. Metacarpal and metatarsal bones are exceptionally satisfactory-small, and rapidly penetrated by fixatives: once the integument is removed, they require no further handling. Since the processes described are the same for all bones laid down in cartilage, no reference will be made to specially named bones. For purposes of description and illustration rabbits and giiinea-pigs arc used. The foetal animals were delivered through the abdominal wall, after killing the mother by fractuire-dislocation of the neck. Tissue required was rapidly dissected and transferred to the fixative in as recent a state as possible. FIXATION Bulk tissue, consisting of bones freed from the surrounding skin and muscle, was fixed in a saturated solution of mercuric chloride in saline, at 370 C., to 100 c.c. of which to 1 c.c. of 1 per cent. osmic acid solution was added, immediately after introducing the tissue. To prevent vaporisation of the osmic acid, the specimen bottles were tightly corked. Long bones of more than 5 mm. in diameter were sectioned longitudinally. Ten to seventeen hours' immersion, depending on the size of the tissue, gives adequate fixation. If the quantity of osmic acid solution introduced is sufficient, the tissue becomes dark brown in colour. If left for a longer period, the mercuric chloride tends to make the tissue very brittle. DECALCIFICATION Foetal bones; rabbits, lip to the termination of the gestation period, were transferred after fixation in Zenker's fluid (to which acetic acid was not added) or Midler's fluid, for 12 to 24 hours. This treatment serves a double purpose, b)oth decalcifying young bone, and acting as a satisfactory mordant, especially if azur stains are subsequently used. 138 C. Witherington Stump Perenyi's solution was found to be the most suitable reagent for the decalcification of more mature bone. If not used too long, the cell preservation is not affected; a small amount of shrinking obscures the finer detail, but does not interfere with the identification of the different cell elements. TEASED AND SMEAR PREPARATIONS These were treated with the above fixative for 10 minutes, and then transferred to Zenker's fluid for a further 10 minutes. Osmic acid vapour was also used for smear preparations, in which case Mallory's triple connective tissue stain was found to give very satisfactory results. EMBEDDING Paraffin and celloidin were both employed. For paraffin embedding, the tissue was passed through graduated spirits, beginning at 30 per cent., and rapidly ascending through 50, 60, and 70 to 80 per cent., hardening in the latter for 8 to 12 hours, finally ascending to absolute through 90 per cent. alcohol, using two changes of the former over a period of 4 hours. Cedar wood oil was used in two changes over 72 hours, for removing the alcohol. The tissue was then transferred direct to paraffin with a melting point either of 490 or 520 C. Indifferent results may be anticipated if the paraffin is permitted to pass above 560. Four baths of paraffin over 12 hours remove the cedar wood oil. The thin sections obtained with the paraffin afford a considerable advantage over the celloidin method. STAINING Eosin and azur, or Wright's (23) polychrome methylene blue, and eosin, give good results. The least capricious staining was obtained with 5 per cent. aqueous eosin and Grubler's polychrome methylene blue i per cent. differen- tiated in 3 per cent. colophonium in 95 per cent. alcohol (after Wright). This stain is especially good for cytological work when employed after the fixation described above. THE FORMATION OF THE CARTILAGE MODEL The primitive connective tissue of the chondrogenetic areas originally forms a syncytium resembling Wharton's jelly. The cellular elements increase and eventually form a dense packed mass. Geddes(1o) and Mall(22) have separately described the process of this condensation. At this stage it is not possible to differentiate between the cells which ultimately form the muscle and perimysium, and those which form the cartilage model and perichondrium. The varying growth potentials inherent in the cells express the resultant tissues by group change. In the chondrogenetic area, there appears a condensation, followed, first by an enlargement, then by a separation of the cells, resulting from the deposition of chondromucin as an exoplasm (Fig. 1). The Histogenesis of Bone 139 Growth of the cartilage area, thus formed, ensues by peripheral increments, the adjacent close-packed mesenchymal cells undergoing transformation by secreting chondromucin, and thereby collectively contributing to the develop- ment of the cartilage model. Apart from enlargement, this cytomorphosis is not marked by any special change in the primitive connective tissue cells. The appearance of the chondromucin is the essential feature of the transition. In its own increase, the mesenchyme is exceedingly active. Cell division in the new-formed cartilage is also a frequent appearance. Free movement fol- lowing division is apparently not impeded to a greater extent than in less dense tissue. Later, it will be seen that the capacity of the cartilage cell, to move freely in its matrix, is the causal factor in effecting the growth of diaphyseal bone longitudinally. Finally, a cartilage mass arises, assuming, in broad outline, the shape of the bone which eventually replaces it. Concurrently with the growth of the cartilage, muscle and perimysium appear, arising from the same type of connective tissue energid. As the cartilage model nears completion, part of the surrounding mesen- chyme immediately subjacent to the perimysium and muscle undergoes a transition into loose cellular fibrous tissue. In this way the zone of the primitive connective tissue becomes delimited forming cartilage centrally and fibrous tissue peripherally. Both the mesenchyme and the fibrous tissue which arises from it are usually grouped together under the one term-perichondrium. The confusion arising from this use of the term, will be considered later. Joint cavities arise by cleavage of the mesenchyme (Fig. 2), and by isolation fol- lowing cleavage, the joint menisci are formed. Capsular tissue appears in continuity with the loose fibrous tissue previously described.