Talk:Paper - The origin, growth, and fate of osteoclasts and their relation to bone resorption (1920)
THE ORIGIN, GROWTH AND FATE OF OSTEOCLASTS AND THEIR RELATION TO BONE RESORPTION
LESLIE B. AREY
Anatomical Laboratory of the Northwestern University Medical School
TWENTY-FOUR FIGURES (FOUR PLATES)
Robin ('49) appears to have been the first to distinguish clearly between the giant-cells of bone marrow (megakaryocytes) and those associated, with bone itself (polykaryocytes), although it is probable that F. Bidder ('43) appreciated some such distinction.
Due chiefly to the efforts of Kolliker ('73), the multinucleate cells of developing bone are known as 'osteoclasts' and are regarded commonly as the direct agents of bone resorption.
The present communication comprises a report of certain observations made during the last three years upon the origin, growth, and fate of the osteoclasts, together with a critical
1 Contribution No. 64, January 25, 1918. A grant from the American Association for the Advancement of Science has made possible the publication of the lithographic plate.
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analysis of the evidence upon which their alleged bone-resorptive potentiality rests. 2
The literature concerning polykaryocytes is voluminous, many accounts dealing confusedly with those of normal and pathological occurrence. Observations and speculations on the giant-cells of normal bone development, of bone tumors, and on those of other pathological origins so overlap that a complete literature review would be both tedious and unprofitable. For this reason the following digest of previous work will deal chiefly with the osteoclast associated with normal bone development and resorption.
Origin. Views as to the origin of the osteoclast are not in accord.
Kolliker ('73) observed the presence of osteoclasts on resorption surfaces; these appeared at first discontinuously in the osteoblastic layer and increased in number, size, and multinuclearity concomitantly with the disappearance of the latter. Admitting the absence of direct proof by observation, he nevertheless considered the circumstantial evidence sufficient to render the genetic relation of osteoblasts to osteoclasts highly probable. The latter were supposed to represent single osteoblasts which had undergone repeated nuclear division. For the earliest osteoclasts, and for those found during the resorption of milk teeth, an origin from connective-tissue cells was assumed.
2 Shortly before his death Prof. C. W. Prentiss had been engaged upon a study of the osteoclasts. Except for a brief abstract ('15) of a paper on this topic delivered before a local surgical society and for a number of unlabeled drawings, there were found no notes or other data to indicate the extent or state of completion of the project. For these reasons the perhaps tentative conclusions reached by him are known in outline only. On assuming duties at this laboratory the writer became interested in the same problem and worked over the entire field independently; some of the preparations used have been identified as those upon which Professor Prentiss made observations. It is believed that the scope of the investigation has been extended considerably and that certain interpretations have been profoundly modified. With respect to an osteoblastic origin our conclusions are in accord, but as to the fate and significance of the elements our opinions clearly differ.
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Bassini ('72), Pomraer ('81), and Gegenbaur 3 concurred fully with Kolliker's thesis; Ziegler ('78), Lewis ('13), and others have rejected it. Morrison ('73), working under the inspiration of Kolliker, likewise reported having observed intermediates (in the number of nuclei?) between the osteoblast and osteoclast.
An origin by osteoblastic fusion was inferred by Howell ('90). It is, he argues, "plausible to think that the closely packed cells might become forced to form a polykaryocyte and a number of transitional steps [in size and number of nuclei?] can be seen in sections . . . ."
Bredichin ('67) held the giant-cells of normal and pathological bone resorption to be transitional stages in the transformation of bone tissue into marrow and granulation tissue ; in other words, they are nothing more than liberated bone cells become multinucleate. Murisier (75) and Ziegler (78) expressed a somewhat similar opinion, while Rindfleisch (72) was likewise convinced from the study of giant-cell sarcomata that the polykaryocytes are bone cells 'which have become free and gone over into a peculiar hypertrophic state. The proliferation of bone cells was also noted by Morrison (73). According to Lowe (79), the osteoclastic nuclei arise either from bone cells or from inwandering leucocytes.
Wegner (72) observed a close association between polykaryocytes and blood-vessels in pathological bone resorption. He also maintains that normally the osteoclasts originate as proliferations of the vessel wall. An intimate relation to bloodvessels, although not in every case an actual origin from them, has been emphasized as well by Morrison (73), Brodowski (75), Maas (77), Schaffer ('88), Bidder ('06), and others.
According to Kaczander ('82), giant-cells (osteoclasts?) form from enlarged, liberated cartilage cells by coalescence. These cartilage cells may be multinucleate while within their capsules. This interpretation is modified by Geddes ('13), who considers osteoclasts to be "hybrid syncytial masses composed of fused cartilage cells containing osteoblasts."
3 Cited by A. Bidder ('06).
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Ranvier ('89), Renault ('93), and Duval ('97) trace the origin to lymphoid marrow cells, whereas Mallory ('11) insists that osteoclasts arise unquestionably from fused, large mononuclear leucocytes.
The results of Jackson ('04), Danchakoff ('09), and Maximow ('10) agree in tracing the origin of the first osteoclasts in the early stages of bone development to enlarged reticular cells of bone marrow. These cells possess at first but two or three nuclei and the cytoplasm is basophilic. Later their cytoplasm appears oxyphilic and the nuclei may become extremely numerous.
Osteoclasts are viewed by Todd ('13) as "masses of preosseus tissue artificially separated from the fully ossified bone during its preparation for histological examination."
The views of Wegner, Kaczander, Todd and Geddes, just presented, are unusual, some of them seemingly even fanciful. In my experience they demand no serious attention. The remaining workers trace or infer an origin from osteoblasts, bone cells, or marrow tissue of some sort. The relation of these opinions to my own observations will be made clear in the pages which follow. Briefly, I recognize all three sources of origin, but the interpretation of the actual mode of genesis and growth of the osteoclast, and the relative importance of each contributory element is novel.
Multinuclearity . A variance of opinion exists also as to the manner in which the osteoclast comes to possess its numerous nuclei.
Kolliker ('73) considered the increase in nuclei to result from nuclear division. Adherents to this view include Bredichin ('67), Wegner (72), Morrison ('73; by amitosis), Bohm and Davidhoff 4 (by mitosis), Jackson ('04 by mitosis); and Jordan ('18; by mitosis, to a limited degree).
Morrison ('73) and Danchakoff ('09) speak of the confluence of mesenchymal cells. Maximow ('10) likewise believes that large osteoclasts arise at the expense of smaller ones; furthermore, he records having never observed nuclear division either by mitosis or amitosis.
4 Cited by A. Bidder ('06).
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Fate. Concerning the ultimate fate of the osteoclast, there is also no general agreement.
These giant-cells were viewed by Bredichin ('67) as transitional stages in the transformation of bone tissue into marrow and granulation tissue.
Wegner ('72), observing some polykaryocytes with cavernous recesses, was led to speculate as to whether new blood-vessels might arise from such (compare p. 327). He also believed in their resolution into connective tissue, or, perhaps, marrow cells.
Kolliker ('73) noted that when bone deposition again succeeds a period of resorption, the osteoclasts disappear from the resorption area' and are superseded by osteoblasts. Furthermore, where resorptive and formative areas join he found intermediate types. The conclusion is drawn that, in such situations at least, the osteoclasts fragment and return to osteoblasts. Kolliker, nevertheless, emphasizes the absence of direct proof and admits (p. 27) that: "Die letzten Schicksale der Ostoklasten sind noch in grosses Dunkel gehlilt." Allowance is also made for the degeneration of some of the giant-cells and for the possibility of a transformation of others into connective-tissue and marrow cells, as Wegner ('72) contended.
Gegenbaur 5 and Bassini ('72) agreed with these views of Kolliker. Pommer ('81) likewise held that osteoclasts not only revert to osteoblasts, but also to cells of a different character and to intercellular material, whereas at the suppression of sufficient nutriment they degenerate.
The removal of the stimulus to absorption was believed by Morrison ('73) to lead to the disappearance of the osteoclasts by 'molecular degeneration.'
Lowe (79) presented a curious and incredible account of the encapsulation of osteoclasts, the fragmentation of their nuclei and cytoplasm into discrete cells, the rupture of the capsular wall, and the dispersal of the individual elements into the marrow. He remarks on the similarity between these stages and those of encystment and spore formation in protozoa.
5 Cited by A. Bidder ('06).
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Jackson ('04) described and figured osteoclasts, which by the enlargement and confluence of cytoplasmic vacuoles formed detached cells; these remain interconnected by processes and are indistinguishable from neighboring reticulum cells.
The view of Maximow ('10) differs from that of Jackson in that some osteoclasts are said to be destroyed through extreme degeneration.
Lewis ('13) holds these polykaryocytes to be degenerating cells produced by those conditions which lead to the dissolution of bone.
Thus, these opinions pertaining to the fate of osteoclasts either uphold their transformation into other cellular elements, their total destruction, or admit both possibilities.
Several provisional communications by the writer on the problem of the origin, growth, fate and significance of the giantcells of bone have appeared previously ('17a; '17b; '08) . 6
The observations recorded in this communication have been made on developing membrane bone of human and pig embryos. A favorable site for study is found about the walls of the dental
6 A publication by Jordan ('18) some time after the present paper had left my hands necessitates supplementary comment. Jordan states (p. 248) that "The osteoclast arises chiefly (at first exclusively) from the marrow reticulum by a fusion process essentially as previously described by Maximow; in the earliest stages the nuclei may multiply slightly by mitosis; their increase, however, is due mainly to exogenous additions either reticular, osteoblastic, or even bone cells. Smaller osteoclasts may fuse to form larger syncytia. These cells finally degenerate, as evidenced chiefly by a vacuolization of their cytoplasm and a karyorrhexis, and eventually they disintegrate. The above-described material gives no evidences of a retransformation into marrow reticulum, as maintained
by certain workers (Jackson, Arey) Osteoclasts may arise to some
extent also from fusing osteoblasts, .... But the osteoblasts involved in this process are not "worn out" as Arey maintains. On the contrary, they are of the less differentiated types and strongly basophilic."
As to sources of origin, method of growth, and ultimate fate these conclusions are in harmony with my own, as set forth in former communications ('17 a, '17 b; '18) and in the present contribution. In certain details, however, our opinions diverge widely. The method of osteoclastic origin from marrow reticulum he considers chief in importance, that from osteoblasts secondary; on the contrary,
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alveoli where active bone resorption is preparing for the accommodation of the rapidly growing teeth. Here osteoclasts appear in large numbers.
The material which proved most useful consisted of several series illustrative of tooth development in the pig. In these decalcified preparations the histological preservation was exceptionally good. The jaws of appropriate pig embryos were fixed in Zenker's fluid, decalcified in acid, embedded in celloidin, and stained with hematoxylin and eosin or hematoxylin and orange G. Hematoxylin and Congo red have been employed also, but the hematoxylin-eosin combination was favored for bringing out delicate tinctorial contrasts. Part of the material comprised serial sections; in view of the large size of the osteoclasts, such series are instructive and important.
Typical stages illustrative of the history of these elements were demonstrated before the thirty-third session of the American Association of Anatomists at New York.
The osteoclasts (named 'Ostoclast' by Kolliker) are large, multinucleate cells of irregular shape and without a definite
I have maintained that, except in the youngest stages of bone development, the reverse is true. Furthermore, those giant cells resulting from osteoblastic fusion are traced by Jordan only from young, slightly differentiated cells; in my membrane bone material these stages of osteoclast genesis have never been found, for example, among the active osteoblasts of growing spicule tips, but only farther back amid more or less 'depleted' cells; the term 'depleted' is, of course, relative, and need not necessarily signify 'worn out' — a term not of my using. In the first preliminary announcement of these studies ('17 a) I recorded the abundance of degenerative stages and intimated a probable final disappearance, but further added the tentative observation that "indications of a transformation into marrow reticulum are not lacking." At the time this last statement was penned I had studied the fate of osteoclasts only partially, but had noted the resemblance of fragmenting osteoclasts like figure 18 to those stages held by Jackson ('04) to depict a retransformation into marrow reticulum. In a later publication ('18) and in the present report these stages are believed to represent merely degenerating cells undergoing disintegration ('18, p. 237): "Neither have I seen convincing stages of a fragmentation into reticular cells of the marrow as Jackson and Maximow describe. The entire picture, from the early formation by the fusion of depleted osteoblasts, seems rather to depict a progressive degeneration, culminating in death and removal."
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limiting membrane. Kolliker ('73) records their maximum size in the human new born as 38 m x 91 n and with as many as 50 to 60 nuclei. I have found the measurements in the pig to run as high as 65 ^ x 105 n, with a nuclear count of about 125. These figures undoubtedly are too low, for the entire cell in all probability extended through several sections.
In shape, osteoclasts are rounded or have variably conspicuous processes (figs. 13 and 14). This latter configuration is suggestive of amoeboid motility (compare Maximow, '10), but Kolliker ('73) and Bizzozero failed to confirm this in living osteoclasts examined on a warm stage.
The cytoplasm is typically strongly oxyphilic and contains a variable number of vacuoles; these Jackson ('04) believed not to consist of fat, whereas Dubreuil ('10) is convinced of their lipoid nature. The cytoplasm is granular, sometimes coarsely so, and is notable for the usual absence of debris (compare p. 332). Nuclei tend to be pyknotic, especially in the older, apparently degenerating forms (fig. 16). Some nuclei appear shrunken or folded (fig. 19), but convincing amitotic stages have not been observed by me.
Certain osteoclasts exhibit a brush border along the edge in apposition with the bone. This border stains more intensely than the rest of the cell and may be finely striate or composed of coarse, block-like elements (figs. 8, 9 and 17). Some also have a fringed or toothed appearance. The significance of this condition is obscure.
In regions where bone is actively forming, the osteoblasts are typically separate units, columnar in shape and with basophilic cytoplasm (fig. 2, obi.) ; the nuclei tend to be placed toward the end of the cell farthest from the bone matrix. As development proceeds the cytoplasm diminishes in amount and in older regions the still basophilic osteoblasts flatten out and lose their distinct cell boundaries. There are thus formed syncytial masses of variable size (fig. 1, ocl.). That such do not result from overstaining with basic dyes is proved by the intense oxyphilic reaction of certain other elements in the same preparations. Close to the basophilic syncytium in figure 1, for example, was a brilliant eosinophilic osteoclast.
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Whereas the osteoclasts may arise in the early stages of bone development from the mesenchymal or reticular cells of the marrow, as Jackson ('04), Danchakoff ('09), and Maximow ('10) contend, my observations indicate that in later stages they take origin chiefly from the osteoblastic syncytia just described. There were found all transitional tinctorial stages between these syncytia with basophilic cytoplasm, staining blue with hematoxylin, and typical oxyphilic osteoclasts, staining red with eosin. In figure 2, compare the intermediate purple shade of a very large-sized intermediate with the normal basophilic osteoblasts at the right and with a typical osteoclast such as figure 4. Here again this characteristic coloration does not result from basic overstaining as may be convincingly proved by an inspection of adjacent fields.
A comparison of the nuclei found in osteoclasts and osteoblasts can not be depended upon to furnish very reliable information as to genetic relationship. Not only do the osteoclastic nuclei commonly exhibit pyknosis, but the resemblance between the unchanged vesicular nuclei and those of osteoblasts and connective tissue is close; this is not surprising since the osteoblasts themselves originate from the connective tissue. In some instances, nevertheless, the chromatin disposition and general nuclear structure of the osteoblasts and osteoclasts clearly agree better than do either with the adjacent marrow reticulum. There is evidence that the elongate nuclei in flattened osteoblasts are restored to the spheroidal configuration upon the release of compression.
Not only are basophilic syncytia and syncytial masses of intermediate stainability found, but osteoclasts may be seen frequently continuous at one or both ends with basophilic osteoblasts and particularly with osteoblastic syncytia. This is represented in figure 3, especially at the right; both osteoclast and osteoblasts have been displaced artificially from the bone surface. Figure 4 shows on the right an osteoclast with five nuclei continuous by a bold transition with fused basophilic osteoblasts. Similarly in figure 11, from a human fetus, the osteoclast lapped over the end of a bone spicule, is abruptly
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continuous at both ends, but especially at the right, with osteoblasts.
According to these observations, therefore, the osteoclast arises from depleted osteoblasts which have first formed a syncytium before being transformed into the oxyphilic osteoclast. As bone resorption continues, osteoblasts progressively lose their former relation to the bone, come into association with the advancing osteoclasts and are incorporated into them. If the spicule in figure 11 were resorbed it is believed that the simultaneously advancing (thigmotactic?) osteoclast would take up the osteoblasts in its path. In a similar manner, smaller osteoclasts may, by fusion, merge into larger ones.
But the osteoblasts, as such, are not the only source from which the nuclear and cytoplasmic contents of the osteoclasts are recruited. Bone cells, embedded in the matrix, are laid bare by the resorptive processes and are ingested by the oncoming osteoclasts. All intermediates may be found between the initial and final stages of inclusion. At the left of figure 4 a cytoplasmic process of an osteoclast is in contact with the capsule of a bone cell which is otherwise embedded in the bone matrix. Two succeeding stages appear in figure 5; on the right the area of contact is extensive; at the left the bone cell is half within and half without the osteoclast. Figure 11, from a human fetus, and figure 10 show similar steps, as do text figures A and B. A last stage appears in figure 12, at the right.
Furthermore, bone cells are enclosed normally within a capsule which is known to be resistant, for example, to the action of strong hydrochloric acid. Encapsulated and distinctly stellate cells, which resemble bone cells identically, are occasionally found, embedded in the osteoclastic cytoplasm (fig. 13; compare also figs. 2, 6, and 12). Such cells are interpreted as bone cells whose capsules have resisted cytoplasmic digestion. From the relative infrequency with which such persistent capsules are seen, it is probable that the enclosed bone cells are eventually liberated. Ingested bone cells must contribute in substantial numbers to the formation of osteoclasts. This is perhaps especially true on flat resorption surfaces/
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Hence it appears that the degree of multinuclearity is an index of the number of osteoblasts and bone cells entering into the composition of the osteoclasts. Also, in general, the larger an osteoclast, the more numerous its nuclei and the more extensive its history in relation to bone resorption (compare figs. 1, 21, and 22).
Fig. A The encapsulated bone cell, b.c, is half ingested by an osteoclast, ocl., which lies on a spicule of bone, mix. Only a portion of the entire osteoclast appears in this section. Photograph. X 650.
b . m
Fig. B A stage in osteoclastic phagocytosis similar to that of fig. A. The bone cell is half within and half without the osteoclast. Photograph. X 650.
My observations agree with those of Maximow ('10) in that nuclear division either by mitosis or amitosis has never been observed in these older stages. Shrunken and folded nuclei do appear, especially in cells which show other evidences of degeneration, but convincing stages of amitosis have not been found by me. In much of the material used mitoses were not uncommon in the near-by germinative layer of the epithelium, so the preparations would seem to be favorable for demonstrating nuclear division did it occur.
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Sometimes osteoclasts are seen which are in continuity by fine processes with the marrow connective tissue (figs. 14 and 15). The general appearance of such cells does not indicate advanced degeneration, hence it may be thought that they have arisen by coalescence of connective-tissue elements. A case illustrative of this is seen in the rather thick section shown in figure 15. Such osteoclasts which seemingly lie free in the marrow tissue usually prove to be in contact with the bone when recourse is made to serial sections. It is possible, however, that even in these later stages of bone resorption some osteoclasts are formed from the marrow reticulum, similar to the origin described by Jackson ('04), Danchakoff ('09), and Maximow ('10), and that this is an illustrative stage of such a process. Indeed, from what is known of tooth resorption and of the genesis of foreign-body and other giant-cells it seems reasonable that the osteoclast has no single source of origin. An elaboration of this topic will be found on pages 328 and 329 of the discussion.
Finally, the fate of the osteoclasts demands attention. That these cells may be resolved ultimately into osteoblasts and again resume bone formation seems improbable. Kolliker, who championed this view, was unable to produce direct evidence in its support. My preparations show nothing in favor of such a cycle, whereas pictures of degeneration in varying degrees are abundant.
A vacuolated cytoplasm is a common characteristic of many osteoclasts. The degree of vacuolization appears to be fairly closely correlated with the extent of degeneration (figs. 16, 17, 18, 19, and 6). In other cells granular degeneration of the cytoplasm occurs (figs. 7 and 20) . Nuclei may show varying degrees of pyknosis while the cytoplasm still appears to be in good condition (figs. 2 and 5) ; on the other hand, the nuclei of cells otherwise exhibiting extreme degenerative changes appear to be constantly pyknotic. Through stages of increasing vacuolization and pyknosis conditions are reached such as are depicted in figures 6, 17 and 19. In an osteoclast like figure 6 both nuclei and cytoplasm are far from normal; the nuclei are highly pyk
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notic while the cytoplasm takes the eosin poorly and is riddled with vacuoles which at the edges produce a ragged, laced appearance. In short, the optical appearance is such as one normally associates with extreme degeneration.
As the resorption of local areas of bone is completed, the accompanying osteoclasts become left behind, stranded in the marrow tissue. Thus, one occasionally finds a small portion of bone enclosed by an osteoclastic mass (fig. 17). Only when serial sections are available is one certain that a paratangential section of a spicule-tip had not produced a deceptive appearance of an isolated bone fragment and enclosing osteoclast.
Left behind in regions where resorption has apparently finished its course are sometimes found also nests of large osteoclasts (figs. 19 and 22). Such a stage as figure 21 apparently shows in its inception how these masses become thus isolated, for this particular giant-cell is at the rear of an area of bone dissolution that is almost completed locally. Some of these stranded elements show excessive degenerative changes. Illustrations show but poorly the pale, reticulate or fragmented cytoplasm and the shrunken and distorted, pyknotic nuclei.
Jackson ('04) and Maximow ('10), in particular, have upheld the fragmentation of osteoclasts into detached cells which become indistinguishable from the reticulum of the marrow (compare p. 327). I have seen several stages which show stellate portions of the osteoclast being cut off by vacuolization (fig. 18). That these moieties, some of which may even lack nuclei, persist as elements indistinguishable from the reticulum is doubtful; on the contrary, the general appearance of such osteoclasts seems rather to point to ultimate degeneration. If my interpretation of the osteoclasts be correct, the entire course from the time of osteoblastic coalescence is one of progressive decline (see foot-note 6, p. 320). To return to a healthy, active state, the fragmentation products of such osteoclasts would seemingly have to undergo extensive rejuvenation both as regards nucleus and cytoplasm.
Large osteoclasts were observed within the blood-vessels of the marrow (figs. 7 and 20) . That such gain admittance and
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do not arise in situ from the endothelium is supported by their usual retrograde appearance; vacuolization, granulation and loss of stainability of the cytoplasm, and pyknosis of the nuclei occur. No stages have been observed which could be interpreted as illustrative of an origin from the blood-vessel itself; on the contrary, the condition appears to indicate a method of final removal. This admission into embryonic vessels does not of itself prove or imply an amoeboid activity by the osteoclast; Meyer ('18) is wrong in assuming I hold such a belief.
The conclusions of Kolliker regarding the history and significance of the osteoclasts have gained great prominence. It should be kept clearly in mind, however, that his opinions were almost wholly inferential. He neither offered direct proof of the origin of osteoclasts nor of their fate, as has been pointed out in the historical section. The apparent reasonableness of these deductions, and the prestige of their originator, doubtless account for their acceptance by numerous later investigators and for their widespread inclusion in texts.
Concerning the validity of many of the claims advanced by other workers the writer can offer little except the negative evidence of not having seen corroborative stages. Unfortunately, in the past, too many dogmatic statements have been made wholly unsupported by appropriate evidence. Only a few have presented their claims adequately described and illustrated. Under these conditions it is not always easy to distinguish between surmises and conclusions drawn from actual observations.
Moreover, it seems reasonable to believe that several morphologically similar but developmentally distinct elements masquerade under the generic term osteoclast, so that, historically, the controversy has not always been over identical structures. The careful studies of Jackson ('04), Danchakoff ('09, and Maximow ('10) appear to show convincingly that, at least in the early stages of bone development, the osteoclasts arise from the primitive connective tissue of the marrow. At
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the time of resorption of the milk teeth an osteoblastic historyis likewise excluded. In the present communication are recorded observations of osteoclasts in continuity with the marrow reticulum; it is entirely probable that this is not a secondary union, but primary, and of developmental significance. My chief results, nevertheless, point to a widespread formation of osteoclasts in the later stages of development from osteoblasts and bone cells.
Polykaryocytes which resemble osteoclasts morphologically are the so-called foreign-body giant-cells produced within the body about fat (Mallory, '11) as a center, or developed experimentally about introduced foreign bodies, such as agar, paraffin, lycopodium spores, or bone dust (Maximow, '02; Lambert, '12; Mallory, '11, et al). These are said to form from fused, wandering leucocytes, and Mallory ('11) even goes so far as to derive all osteoclasts (but on insufficient evidence) from a like source. The foreign body origin is, nevertheless, an important concept. It seems reasonable that on the cessation of active growth a spicule of bone, or any quiescent portion of a spicule, becomes essentially a foreign body and the cells in contact with it, not necessarily all of one origin, may respond to whatever stimulus it offers and fuse into syncytial masses. Kolliker (73) found that ivory pegs driven into bone became eroded, the lacunae thus formed containing polykaryocytes. Rustizky (74) similarly produced giant-cells, but no lacunae, by introducing pieces of bone in the dorsal lymph sacs of frogs.
The causative factors of giant-cell formation are necessarily obscure. Kolliker considered it due to a pressure by the soft parts underlying bone (compare also Wegner). Pommer ('81), on the contrary, held as responsible a locally increased blood pressure. On spongy bone, at least, it may be due to the augmented mutual pressure resulting from the decreased size of the spicules and to a concomitant loss of cell individuality as a result of general osteoblastic degeneracy. The formation of ordinary foreign-body giant-cells would demand a different explanation. Presumably a definite thigmotaxis is operative in all giant-cell formation.
THE AMERICAN JOURNAL OF ANATOMY, VOL. 20, NO. 3
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Finally comes the vexed question as to whether the osteoclasts actually are bone destroyers. Kolliker ('73) was the first and most persistent exponent of their bone-destroying function. He believed that their presence in the Howship's lacunae of resorption surfaces, on ivory pegs, and on milk teeth during resorption, demonstrated his thesis beyond controversy. Among the many who have subscribed to these views are Wegner ('72), Morrison (73), Jackson ('04), and Maximow ('10). Mallory ('11) has even suggested that the erosion of bone may be accomplished mechanically by the brush border. Danchakoff ('09) refers to the dissolving action on bone, but calls attention to the relative infrequency of osteoclasts during the erosion of calcified cartilage and the irrationality of attributing the destruction of cartilage solely to them. Shaffer ('81) likewise held that they have a bone-destroying action, which, however, blood-vessels chiefly perform.
Howell ('90; p. 119) took a decided stand against the probability of the commonly accepted osteolytic function:
The function of these cells is unknown. The common view that they are concerned in the absorption of bone (osteoclasts) seems to me to rest upon very slight evidence. If we find them in developing bone lying upon the cartilage trabeculae which are being absorbed, we find them also on the partitions of sponge or pith, introduced into serous cavities where no absorption is taking place; and the conclusion in the first case that the absorption which is going on is due to the giant cells (osteoclasts) is illogical. Absorption of tissues is an occurrence common enough in the body, and it is difficult to understand why the absorption of bone or cartilage should require the activity of a special cell, when the absorption of other tissues does not. It would seem more probable that this form of cell has no specific function, and that its formation is, in fact, accidental, or, in a certain sense, pathological: that the presence of a solid substratum leads to an abnormally rapid growth of lymphoid cells, leucocytes, osteoblasts, as the case may be, and the fusion of some of these to produce multinucleated giant cells.
The evidence in favor of a dissolving activity by the osteoclasts rests upon the following relations: these elements appear at the onset of resorption and disappear at its cessation; they are closely applied to bone, sometimes being wrapped around eroded spicules (fig. 10), and sometimes occupying the so-called How
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ship's lacunae; 7 their coloration with acid dyes often resembles that of bone. The occasional presence of a striate border (figs. 8 and 17) on the side in contact with bone might be thought to indicate cellular activity (compare the brush order of the epithelial cells of the intestine or kidney) , but this phenomenon is open equally well to other interpretation.
Against the view of the osteoclast as a causative resorptive agent may be presented several objections. They are notably scarce during the resorption of calcified cartilage. Howship's lacunae not infrequently are seen without associated osteoclasts (compare Rustizky, '74) ; this may represent a distinct method— "lakunare Resorption ohne Riesenzellen" (Kaufmann, '07). A 'smooth resorption' of bone occurs where Howship's lacunae are entirely lacking (Busch, '77), and they are often infrequent where osteolysis is active. In osteomalacia, leprosy, and in tumors and many inflammations (Ziegler, '78), the lime salts are removed in the absence of osteoclasts — it is significant that in these cases typical Howship's lacunae usually occur (Ziegler, '78, et al). The absorption of various other tissues in the body is accomplished without the intervention of special cells. It is not impossible that the relation of osteoclast to Howship's lacunae may be given an inverse interpretation; if the lacunae represent softer regions in the bone, or regions for some reason more directly subjected to the resorbing principle, perhaps these depressions serve merely as traps in which the giant-cells collect.
If the osteoclasts arise largely from osteoblasts and bone cells, as set forth in this communication, and if, furthermore, the subsequent course is one of progressive degeneration, it seems unlikely that such cells, which a short time before had been active bone formers, now T would take over the diametrically opposed function of destruction. On the contrary, the concept of progressive degeneration seems to militate against such a view.
7 The relation of polykaryocytes to such lacunae is demonstrated strikingly during the resolution of deciduous teeth. At this time, the root may be completely scalloped with adjacent pits, each harboring a giant-cell. It is hoped that a subsequent report will clear up the puzzling questions of the origin and significance of these elements.
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In working over this field and evaluating the total evidence the writer has become highly skeptical concerning the potency of the osteoblast in bone resorption. With Lewis ('13, p. 86) he would take the position that 'There seems to be no satisfactory evidence that the osteoclasts are the active causes of bone destruction. On the contrary, they appear to be degenerating cells, . . . ."
Attention has been directed in another publication (Arey, '17 b) to the uncritical nature of the assumption that the failure of a cell to drink in vital dyes warrants the denying to it of phagocytic potentiality. The' osteoclast refuses to 'stain' with trypan blue (Shipley and Macklin, '16), yet bone cells, encapsulated or naked, laid bare by the resorptive processes, are demonstrably engulfed by it; 8 (compare also the observations of Wegner 9 and of Rustizky ('14) on 'Kalkkorner' within polykaryocytes, and note the red blood corpuscles within the body of the osteoclast shown in figure 7 of the present communication.) Furthermore, the mere presence of cytoplasmic inclusions within an osteoclast by no means indicates that the latter was responsible for the dissolution of the material ingested; to imply such a causal relation is to exceed the limits of legitimate deduction. When Jordan ('18, p. 251) writes that "The osteolytic function of the giant-cells of reticular and osteoblastic origin is proved by the presence of globules of resorbed osseus substance [p. 262, globules of absorbed bone] within the cytoplasm," he perhaps is using 'osteolytic' as a term interchangeable with 'phagocytic;' this is supported by a further reference (p. 255) to the ' ' 'phagocytic' (osteolytic) function" of osteoclasts. Yet in another place (p. 246) we read:
8 The presentation of these facts ('17 b) has led Meyer ('18 t p. 100) to conclude that it involved "apparently implying that fusion products never observed to undergo mitosis, nevertheless may be physiologically active and continue a progressive evolution." That these 1 giant -cells are active enough to engulf bone cells, osteoblasts, or even fragments of bone matrix is unquestionably true, but that the osteoclasts enjoy a progressive evolution is not supported by my observations. On the contrary, both in my 1918 communication and in the present contribution the h'story of these elements is held to be one of advancing degeneration, culminating in death and removal.
9 Cited by Rustizky (74).
ORIGIN AND FATE OF OSTEOCLASTS 333
"the osteoblasts are constructive osseus elements; the osteoclasts are destructive elements. The former elaborate bone; the latter resorb it."
There is no direct evidence as to how bone matrix (inorganic and organic) is resorbed. One might assume it is essentially a double process of decalcification and of digestion of the organic substrate; conditions such as osteomalacia, in which the lime salts are removed leaving the organic framework, perhaps support such a view. In the absence of more appropriate evidence, therefore, the activity of an acid (carbon dioxide or lactic acid?) and an enzyme can be tentatively suggested.
According to Morpurgo and Satta ('08 a, '08 b), there is associated with bone a thermolabile entity, an enzyme they believe, responsible for calcium removal during experimental autolysis of bone. The provisional nature of these two communications renders their evaluation difficult.
In his Harvey Lecture, Wells ('10-' 11) marshals numerous facts to support the view that bone resorption is accomplished through the agency of carbon dioxide. It is demonstrable that calcium is normally contained in the blood in amounts approximating saturation and that this content is from two to four times that soluble in water. This amount of calcium in the blood is held in solution by the colloids and the carbon dioxide. "In normal ossification, and in most instances of pathological calcification, the deposition is probably initiated by a process of colloidal adsorption .... Reduction in the amount of carbon dioxide in such areas, or some unknown agency, causes a precipitation in this colloid matrix, . . . ."It is well known "that, no matter how sclerotic the walls of the veins may become, they rarely, if ever, calcify so long as there is venous blood rich in C0 2 flowing through them. As soon as they are occluded, however, calcification occurs readily enough (e.g., phleboliths)." It has been long established that carbon dioxide in solution will dissolve calcium from bone but that NaHC0 3 cannot so act. Furthermore, studies on this solubility in vitro and in vivo show "that pieces of ivory are absorbed most rapidly in tissues whose metabolism is the most active, and where, by
334 LESLIE B. AREY
inference, there is the most carbon dioxide." From these and other considerations Wells concludes: "It is indeed probable that it is the C0 2 which accomplishes the resorption of dead bone in the living body, and perhaps also the normal resorption of bone in the various conditions in which this process takes place." It is, perhaps, difficult to imagine the mechanism of the localization of carbon dioxide ( or the stronger lactic acid?) in sufficient concentrations to effect the selective erosion of small areas, or to account for the frequent directional polarization of the resorptive wave. However this may be, there is, of course, no basis for suspecting the osteoclast of special carbon dioxide production. On the contrary, if it is indeed a degenerating cell, its carbon dioxide output is presumably low. The conception of Wells adds to the objections already enumerated against the osteoclast as a specific osteolytic agent.
The multinucleate giant-cells known as 'osteoclasts' probably include several morphologically similar but development ally distinct elements.
In the earliest stages of bone development, and to a certain extent in later stages, osteoclasts apparently arise from the confluence of the mesenchymal cells and connective tissue of the marrow.
The chief source of osteoclasts, however, is from old osteoblasts and bone cells.
Depleted, basophilic osteoblasts coalesce to form multinucleate masses. These syncytial elements become typical osteoclasts when their cytoplasm assumes an oxyphilic stainability. All intermediate tinctorial stages are demonstrable.
True oxyphilic osteoclasts also exist in cytoplasmic continuity with basophilic osteoblasts. Increase in size and nuclear content results from the engulfing of osteoblasts met in the path of resorption and from bone cells which are ingested as the bone matrix is resorbed.
Osteoclasts undergo retrograde changes and ultimately disappear through extreme degeneration.
ORIGIN AND FATE OF OSTEOCLASTS 335
Only indirect and insufficient evidence points to the osteoclasts as the active, specific agents of bone resorption. That they are merely degenerating, fused osteoblasts, accords better with the known facts.
Arey, L. B. 1917 a On the origin and fate of the osteoclasts. Anat. Rec, vol. 11, no. 6, pp. 319-322.
1917 b Phagocytosis by osteoclasts. Anat. Rec, vol. 13, no. 5, pp. 269-272, 4 figs.
1918 The origin, fate and significance of osteoclasts. Trans. Chicago Pathol. Soc, vol. 10, no. 7, pp. 231-234.
Bassini, E. 1872 Sul processo istologico di riassorbimento del tessuto osseo.
Nota preventica, 1872. Bidder, A. 1906 Osteobiologie. Arch. f. mikr. Anat., Bd. 68, S. 137-214, 33
Fig. Bidder, F. 1843 Zur Histogenese der Knochen. Arch. f. Anat., Physiol, u.
wissensch. Med., Jahrg., S. 372-395, 1 Fig. Bredichin, J. 1867 IJber die Bedeutung der Riesenzellen in den Knochen.
Centralbl. f. d. med. Wissensch., Jahrg. 5, S. 563-564. Brodowski, W. 1875 tJber den Ursprung sogenannter Riesenzellen und liber
Tuberkeln im allgemeinen. Arch. f. path. Anat. u. Physiol., Bd. 63,
S. 113-135, 8 Fig. Btjsch, F. 1877 Die Knochenbildung und Resorption beim wachsenden und
entziindeten Knochen. Arch. f. klin. Chirurgie, Bd. 21, S. 150-181. Danchakoff, W. 1909 tJber die Entwicklung des Knochenmarks bei den
Vogeln und fiber dessen Veranderungen bei Blutentziehungen und
Ernahrungsstorungen. Arch. f. mikr. Anat., Bd. 74, S. 855-926, 18
Fig. Dtjbrettil, G. 1910 Vacuoles a lipoides des osteoblastes, des cellules osseuses
et des osteoclasts. Compt. rend. d. 1. Soc. Biol., T. 69, pp. 189-190. Duval, M. 1897 Precis, d.' histologic Masson et Cie, Paris, 31 + 956 pp. Geddes, A. C. 1913 The origin of the osteoblast and of the osteoclast. Jour.
Anat. and Physiol., vol. 47, pp. 157-176, 12 figs. Howell, W. H. 1890 Observation upon the occurrence, structure, and function
of the giant cells of the marrow. Jour. Morph., vol. 4, no. 1, pp. 117—
130, 15 figs. Jackson, C. M. 1904 Zur Histologic und Histogenese des Knochenmarkes.
Arch. f. Anat. u. Entw., Jahrg., S. 33-70, 16 Fig. Jordan, H. E. 1918 A contribution to the problems concerning the origin,
structure, genetic relationship and function of the giant-cells of hemopoietic and osteolytic foci. Am. Jour. Anat., vol. 24, no. 2, pp. 225 269, 70 figs. Kaczander, J. 1882 tJber den Ossificationsprozess. Arch. f. path. Anat. u.
Physiol., Bd. 87, Heft 1, S. 189-193, 7 Fig. Kaufmann, E. 1907 Lehrbuch der speziellen pathologischen Anatomie. Rei mer, Berlin, 10 4- 1363 S., 693 Fig.
336 LESLIE B. AREY
Kollikeh, A. 1S73 Die normale Resorption des Knochengewebes und ihre
Bedeutung fur die Entstehung der typischen Knochenformen. Vogel,
Leipzig, 6 + 86 S., 95 Fig. Lambert, R. A. 1912 The production of foreign body giant cells in vitro.
Jour. Exp. Med., vol. 15, no. 5, pp. 510-515, 7 figs. Lewis, F. T. 1913 A text-book of histology. Blakiston, Phila., 11 + 539 pp.,
495 figs. Lowe, L. 1879 Kleinere histologische Mittheilungen. C. Uber die Umwand lungen der Osteoklasten im Knochenmark nebst Bemerkungen iiber
Knochenwachstum. Arch. f. mikr. Anat., Bd. 16, S. 618-625, 6 Fig. Maas, H. 1877 Uber das Wachsthum und die Regeneration der Rohren knochen mit besondere Beriicksightigung der Callusbildung. Arch. f.
klin. Chirurgie, Bd. 20, S. 708-770, 27 Fig. Mallory, F. B. 1911 Giant cell sarcoma. Jour. Med. Research, vol. 24, pp.
463-467, 8 figs. Maximow, A. 1902 Experimentelle Untersuchungen iiber die entziindliche neu bi Idling von Bindegewebe. S. Fischer, Jena, 7 + 255 S., 13 Taf.
1910 Untersuchungen iiber Blut und Bindegewebe. 3. Die embryo nale Histogenese des Knochenmarks der Saugetiere. Arch. f. mikr.
Anat, u. Entw., Bd. 76, Heft 1, S. 1-113, 48 Fig. Meyer, A. W. 1918 Some observations on megacytes in lymphatic tissues.
Amer. Jour. Anat., vol. 24, no. 1, pp. 91-107, 18 figs. -Morptjrgo, B. et Satta, G. 1908 a Sur quelques particularites de l'autolyse
de l'os. Arch. Ital. de Biol., vol. 49, pp. 380-382.
1908 b Presence d'un ferment actif sur les composes de calcium dans
les os malaciques. Arch. Ital. de Biol., vol. 49, pp. 383-384. Morrison, A. 1873 Bone absorption by means of giant cells. Edinburgh Med.
Jour., vol. 19, pp. 305-311. Murisier, J. 1875 Uber die Formveranderungen, welche der lebende Knochen
unter dem Einfluss mechanischer Krafte erleidet. Arch, f . exp. Pathol.
u. Pharmakol., Bd. 3, Heft 5 u. 6, S. 325-347, 3 Fig. Pommer, G. 1881 Uber die lacunare Resorption in erkrankten Knochen.
Sitzungsber. d. k. Akad. d. Wissensch., math.-naturw. Kl., Bd. 83,
Heft 1, Abt. 3, S. 17-120.
1883 Uber die Ostoklastentheorie. Arch. f. path. Anat. u. Physiol.,
Bd. 92, S. 296-363; 449-516. Prentiss, C. W. 1915 The origin and fate of the osteoclasts. Surg., Gynecol.
and Obstet,, vol. 20, pp. 678-679. Ranvier, L. 1889 Traite technique d'histologie. 2d ed., Masson, Paris. 871
pp., 414 figs. Renault, J. 1893 Traits d'histologie pratique. Lecrosnier et Babe, Paris,
T. 1,39 + 973 pp., 354 fig. RiNDFLEiscii, E. 1872 A text-book of pathological histology. Trans, by W. C.
Kloman and F. T. Miles. Lindsay and Blakiston, Phila., 695 pp., 208
figs. Robin, C. 1849 Sur l'existence de deux especes nouvelles d'elements anato miques qui se trouvent dans le canal medullaire des os. Compt. rend.
d. 1. Soc. Biol., Annee 1, pp. 149-150.
ORIGIN AND FATE OF OSTEOCLASTS 337
Rustizky, J. 1874 Untersuchung liber Knochenresorption und Riesenzellen.
Arch. f. path. Anat. u. Physiol., Bd. 59, S. 202-227, 8 Fig. Schaffer, J. 1888 Die Verknocherung desUnterkeifers und die Metaplasief rage.
Arch. f. mikr. Anat., Bd. 32, S. 266-377, 26 Fig. Shipley, P. G., and Macklin, C. C. 1916 Some features of osteogenesis in the
light of vital staining. Araer. Jour. Physiol., vol. 42, no. 1, pp. 117 123. Todd, T. W. 1913 A preliminary communication on the development and growth
of bone and the relations thereto of the several histological elements
concerned. Jour. Anat. and Physiol., vol. 47, pp. 177-188, 9 figs. Wegner, G. 1872 Myeloplaxen und Knochenresorption. Arch, f . path. Anat.
u. Physiol., Bd. 56, Heft 4, S. 523-533. Wells, H. G. 1910-'ll Calcification and ossification. The Harvey Lectures.
J. B. Lippincott Co., Phila., 146 pp. Ziegler, E. 1878 tjber Proliferation, Metaplasie und Resorption des Knoch engewebes. Arch. f. path. Anat. u. Physiol., Bd. 73, S. 355-379, 7 Fig.
EXPLANATION OF PLATES
The figures of these plates were made with a Leitz ^s homogeneous immersion objective and a no. 1 Leitz ocular, at an original magnification of 785 diameters. With the exception of figures 8 and 11, which are from human embryos, the illustrations represent stages from the jaws of pig embryos.
b.c, bone cell obi., osteoblast
cap., capiHary ocl., osteoclast
c.t., connective tissue str., striate edge of osteoclast
mix., matrix of bone *, junction of confluent osteoblasts and
EXPLANATION OF FIGURES
The preparations, from which the illustrations of this plate were made, are from pig embryos, fixed in Zenker and stained with hematoxylin and eosin. The reduced magnification is now 590 diameters.
1 The basophilic syncytium, ocl., illustrates the first stage in osteoclast development by the fusion of osteoblasts.
2 The immature osteoclast, ocl., exhibits a transitional stage in stainability between the basophilic osteoblasts, obi., and a typical oxyphilic osteoclast like figure 4.
3 An asterisk, *, marks the continuity between an osteoclast at the left and the osteoblastic syncytium on the right.
4 At the right is a bold osteoblast-osteoclast junction. On the left a process from an osteoclast, in contact with the capsule of the bone cell, b.c, illustrates an early stage of phagocytosis.
5 Two later stages in the ingestion of bone cells by an osteoclast; the engulfment of, b.c.', is farther advanced than of, b.c.
6 Advanced osteoclastic degeneration. The nuclei are pyknotic, the cytoplasm highly vacuolated and palely staining.
7 An osteoclast within a blood capillary. Such usually show evidence of degeneration and suggest a method of filial removal.
ORIGIN AND FATE OF OSTEOCLASTS
LESLIE B. AKEY
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EXPLANATION OF FIGURES
Figures 8 and 11 are from human embryos, the others from pig embryos. The reduced magnification is now 630 diameters.
8 An osteoclast showing a finely striate border in apposition with the bone.
9 A striate border of coarse, block-like composition.
10 An osteoclast wrapped around an eroded spicule and conforming closely to its irregularities. The bone cell, b.c, is nearly incorporated.
11 Abrupt transitions. *, between an osteoclast and adjacent osteoblasts. The bone cell, b.c, is confluent with the phagocytosing osteoclast.
11' On the right, the bone cell, b.c, has practically lost its identity and become a part of the osteoclast. At the left, several encapsulated bone cells, b.c.', lie within the osteoclastic cytoplasm.
13 Two stellate, encapsulated bone cells, have been taken up during bone resorption.
ORIGIN AND FATE OF OSTEOCLASTS
LESLIE B. AREY
C. A. i<?
^*^ -iS fit ■—
EXPLANATION OF FIGURES
The reduced magnification of the figures of this plate is now 630 diameters.
14 An osteoclast with irregular processes, some of which are continuous with the marrow connective tissue.
15 An oxyphilic syncytium in the marrow, apart from the bone. Continuity with the connective tissue suggests a possible origin from the latter.
16 An early stage in the degeneration of osteoclasts. Nuclei are pyknotic; cytoplasm slightly vacuolated.
17 The isolated (?) spicule of bone is surrounded by a degenerating osteoclast which shows extreme vacuolization and cytoplasmic disintegration.
18 Vacuoles have nearly cut off that portion of the osteoclast indicated by a cross. Such an appearance has been interpreted by some as indicative of a resolution into reticulum cells.
19 A highly degenerated osteoclast apparently left behind after resorption was locally completed.
ORIGIN AND FATE OF OSTEOCLASTS
LESLIE B. AREY
YJ a •<>
\ oc/ *'J
EXPLANATION OF FIGURES
The reduced magnification of the figures of this plate is now 630 diameters.
20 A degenerating osteoclast within a blood capillary (compare fig. 7).
21 A large osteoclast measuring 40 ju xSOju- It is at the rear of the local region of bone dissolution and somewhat later might have become isolated like figs. 19 or 22.
22 Several huge giant-cells lie stranded in the marrow tissue in an area where bone destruction is locally finished; possibly serial sections would have shown these elements to interconnect. Similar nests occur in the near vicinity. The osteoclast at the left, ocl.', measures 65 /j. x 105 n and contains about 125 nuclei.
ORIGIN AND FATE OF OSTEOCLASTS
LESLIE B. AREY
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