Talk:Book - Contributions to Embryology Carnegie Institution No.27

By Charles Clifford Macklin, Associate Professor of Anatomy, University of Pittsburgh.

With four plates.

INTRODUCTION.

The vital-slmning route as an ap})roach to the problem of bone-repair came as a natural consequence of the recent work of Shipley and Macklin (1916'-^) on osteo- genesis. By subjecting very }^oung, growing animals to trypan-blue, one of the azo-dyes belonging to the benzidine series of colors, these investigators were able to show that the regions of active bone-growth took a more intense stain than the remainder of the bone; and, furthermore, that the heightened coloration was largely referable to the presence in these areas of innumerable phagocytic cells, within whose cytoplasm the dyestuff was stored in multitudinous tinj^ segregations known as "dye-granules."

These phagocytes were identified as the reticulo-endothelial cells of the young bone-marrow\ Their reaction to the dyestuffs of the benzidine group is the same as that of th(^ host of cells found throughout the bod}-, which have been extensi\'ely studied by different authors, and to wliich various names have been given, such as "pyrrhol-cells" (Goldmann, 1909), "clasmatocytes" (Ranvier, 1899-1900), "resting-wandering cells" (IMaximow, 1906), etc. Recently Evans (1915) has employed Mctschnikoff's term "macrophage" to cover this entire group of phago- cytic cells which are united by a uniform functional response to these colloidal dyestuffs, and it is now well recognized that the term "macrophage" is a physiological designation, including within its compass very diverse morphological elements. This similar staining reaction, indeed, is but an expression of the phagocytic potentiahtj^ which these cells hold in common (Evans and Schulemann, 1914) and which mani- fests itself during their every-day existence in the ingestion and storage of certain elements of the surrounding tissue-fluids.

In consideration of the vigorous phagocytic properties attributed to these cells, and also of their being present in large numbers where temporary' bone and cartilage were being absorbed, it seemed evident to Shipley and ^lacklin (1916^) that they were a very important factor in active osseous development and that their peculiar role under these circumstances w'as played in connection with the resorp- tion of the provisional cartilage and bone.

Now bone-resorption is an active process in the later stages of bone-repair, for it is well known that the excess of provisional callus which is built up following a bone-injury, such as a fracture, is gradually removed. Since the resorption of this provisional callus is quite similar to that of provisional new bone it was decided to investigate the vitally stained cells in the callus of healing bone-wounds and to compare the findings with those in young, growing bone.

In tlio onrlicr stages of the roparative process following bone-wouiuls, too, there is a great deal of deliri? to be eliminated, such as injured bone, blood-clot, damaged muscle, and other devitaUzed tissue, and it is plain from the writings of other workers that the potentialities of the macrophages eminently fit them for the performance of tlxis duty. That they play a part in the healing of wounds of soft tissues, such as skin, kidney, and liver, may be inferred from the work of Gold- mann (1912), who demonstrated by the aid of vital-dyes that they were increased in tlie regions where repair was proceeding. Indeed, the evidence of numerous investigators jioints to the macrophages being concerned in all inflammatory con- ditions. Maximow (1902, 1909^), notably, has made a special study of these cells in inflammatory areas, where he finds them increased in number and size, and speaks of them as "i)olyblasts"; and Tschaschin (1913) has recorded similar findings.

The jiroblem of the heahng of wounds of bone, therefore, seemed to offer a particularly favorable field for the application of the vital-staining method, for it was expected that in the earlj^ stages of bone-repair, where damaged soft parts must lie cleared away, as well as in the later stages, where provisional bone has to be eroded, the trypanopliil cells — i. e., the macrophages — in the pursuance of their physiological vocation as phagocytes, would become locally very numerous and would show hypertrophy and intensified phagocj^tic power. These expecta- tions, as will be seen, were realized, and the following pages are devoted to the discussion of the gross and microscopic appearances jiresented in the progressive stages of healing of fractures and trephine wounds in rats in whose tissues the macroi^hages were made visible by the introduction of the dj'estuff (trypan-blue) into the circulating fluids shortly before death.

MATERIAL AND METHOD.

For the most part, the exjieriments w^ere carried out on the albino rat, though the black and cros.sed breeds were also used. The material was collected at the admiral»le rat colony of the Wistar Institute of Anatomy and Biology, Philadel- phia,* and tlus insures that the rats were all iierfectly healthy and were kept under the most favorable conditions during the time of experimentation. A complete series of stages was secured, covering the entire period of repair.

The operations were conducted as follows: The animal having been anesthet- ized with ether, the top of the head was carefully shaved and sterilized; a median incisif»n was then made and the skin reflected over the parietal areas. The jiarietal bone having been laid bare, a .small tre])lune of 5 mm. diameter was used to per- forate it. In some ca.ses the jjiece was removed altogether; in others it was replaced, sometimes upside down; and in still other cases a piece of Uving or dead bone from another rat, or even dead bone from an animal of a different species, was inserted. As a rule two areas were trephined, one in each jiarietal bone. The wounds were

, t, .*' '^'" '^"^'^ ^'^' '^•'^"•'"","'1 ""'! IJi'tiiildBon. of tho Wistnr Inslilulo, for lahonitory facilities and access to the rat r ' "'*■•'">■ of I'enniiylvnniB, for placing his lalxiratory at my disposal for the operations.

, ' .Miidue Uut;. 'riiurluw my most cordial tlianks for the invaluable assistance rendered by

de\t:lopment and function of macrophages in bone-repair. 5

careful!}" sutured ^^dth sterDe silk, and the most rigid asepsis was observed through- out. In the same animals, before recover}^ from the anesthetic, the tibia and femur of the left hind leg and a few of the ribs on the left side were fractured. No attempt was made to spUnt the bones, so that heaUng took place under the same conditions as in natural life.

There were sUght variations from this general type experiment; in some animals only trephining was done and in others only fracturing. In all, 20 animals were used, which furnished 16 trephined skulls, 15 fractured tibiiB, 15 fractured femora, and 12 sets of fractured ribs. In table 1 a full description of the material is given.

Table 1. — Material. ■

se = specimen examined.

The ariimals were killed at various periods during the repair process, the specimens ranging from the second to seventy-first day of healing, as shown in table 1. The vital-staining technique was the same as that used by Shipley and Macklin (1916'- ') in their work on developing bone. Shortly before the time selected for killing, trypan-blue was administered intraperitoneally in the form of a sterile 1 per cent aqueous solution. As a rule, the dyestuff was given 48 hours before kiUing and repeated after 24 hours, so that the macrophages were exposed to its action for 2 days. Occasionally the period of exposure was as short as 1 or as long as 3 davs. No ill effects followed the exliibition of the dye.

At tho time selected the animals were anesthetized and bled, the required tissues heiiip at once dissected out and fixed in 10 per cent formalin neutralized witli magnesium caH^onate. Care was taken to leave the soft i)arts surrounding the hones undisturbed. After fi>dng for 24 hours and washing in running tap-water for the wmie length of time the tissues were i:)assed through a graded series of alco- hols entling with two changes of absolute. They were then cleared in benzine and oil of wintergreen. after the method of Spalteholz (1914), and examined. Of the cleari'd six-cimens the ribs and skulls were most satisfactory, for it was impossible to make the larger bones transparent becau.se of their greater density and thickness. Certain of the skulls and long bones were then decalcified, embedded, and sectioned (table 1), .so that the various stages could l)e studied microscopically. A few were cleared after having been decalcified; some of these also were afterwards sectioned. The most valuable sections were those simply cleared and mounted with no stain other than the trj'pan-blue and those lightly counterstained with carmine, which afforded a satisfactory contrast with the blue. Hematoxylin and eosin for cellular detail were used, and also (in special cases) methjl-green and safranin.

OBSERVATIONS.

In the description of the gross and microscopic findings in the vitally stained healing wounds of bone, the tlifferent stages, as shown l)y a studj^ of cleared gross specimens of ribs and skulls and of cleared and stained sections, will be taken up in order. Later on, the various aspects of vital staining will be analyzed, con- solidated, and finally reduced to a summary. But before proceeding to a portrayal of the changes in the vital staining of wounded bone and the tissues immediately surrouncUng the .site of the wound it will be well to describe briefly the ajipearance of the vitally stained normal bone and its tissue environment.

A good example of this is seen in figure 1, drawn with the aid of the binocular microscope from a cleared rib taken from a rat vitally stained for 2 days with try|)Mn-l)hie. It is intended as a control of the stages afterward to be described.

The clear shaft of the bone (b) is easily seen surrounded by the periosteum (p) and inclosing a well-marked medullary canal (mc). To the left the intercostal vessels and nerve (n) ar(> faintly sketched in. The fasciculi of intercostal muscle (mu.s) are suggested by the intercrossing lines.

It will be nr)ted at once that black dots appear throughout the drawing. These are .seen as l)lue granules in the original specimen and represent individual cells which have i)hagocytize<l and stored the dyestuff. These cells are more concen- trated \u the medullary cavity, the periosteum, and in the connective tissue around th(! bone. In the intermuscular septa they are consi)icuous as granular streaks. These cells are macrophages and their presence in the regions describ(>d as is well known. IS normal. (;.,ldmann (in09), for instance, has recorded similar findings, a.s Ml hl-sTaf. XI, Xo. 2, where he shows the hi.sl„l„gioal ap|)earance of the vitally stained niterhbrillar cells in the iniiscle of (he tongue.

There are, then, both within the shaft of the bone and in the tissues of its immediate environment, under normal conditions, apprecia})le numbers of macro- phages. In the next section it will be found that, following bone injury, their numbers are locally very much increased.

Second-day Stage.

The earliest stage of bone repair examined was that at the end of 2 days (animals 18-1).

On inspection of the cleared ribs with the binocular microscope the most obvious change, as compared with the control, is the marked blue staining of the broken ends of bone and of the material surrounding them. The ends of the medullary canal are plugged with it. This staining is shown in black* in figure 2. It is very dense and irregular in character and quite different from the granular type of staining presented in the control rib (fig. 1). The imiDression conveyed is that the tissue injured by the trauma, and the associated exudate, have absorbed the dye. There is as yet very httle swelling at the site of the fracture, so that the contour of the bone, apart from the distortion of the break, is the same as that of the control.

Under the highest power of the binocular the blue granules, representing macrophages, are seen, as in the control, in the periosteum and marrow cavity of the bone, and in the surrounding connective tissue. At the site of the fracture, however, it is noted that they are more numerous than elsewhere and they have apparently been increased in this region. In the drawing (fig. 2) they appear as a small cloud of granules near the broken bone-ends and around the densely colored material. This granular appearance is a true intra-vital dyeing, and stands in sharp contrast to the diffuse and dense staining above described. The periosteum as yet shows no obvious changes, such as thickening.

In the cleared skidl, as in the ribs, a dense and diffuse blue staining is seen in the bone fragments and in other dead tissue resulting from the operation. Macro- phages are not definitely increased.

In the cleared long bones the tissues of the fracture-precinct are densely stained, but the specimen, on account of its thickness and opacity, is unsatisfactory for inspection of the macrophage tissue.

As would be expected from the cleared specimens, the uncounterstained sections of long bone (tibia) show areas near the broken ends where the misshapen and apparently injured tissue has taken a dense, irregular, and diffuse blue stain. Under higher powers such areas appear as shapeless blue masses of fragmented muscle-fibers, a little fibrous tissue, blood, and invading phagocytes, interspersed with clearer areas of exudate. Often individual muscle-fibers appear stained blue throughout. There is evidently much cell Uquefaction going on.

Diffuse staining of this type is an indication of cell death (MacCurdy and

• A11 blue staining is represented diagrainmaticallv in the illustrations by the denser black.

S DKVKUtPMK.NT AM) !• I. NCTION OF MACUOPHAGES IN BONE-REPATH.

Evans. 1912; Evans and Schulcmann, 1914), and hence it may be concluded that tlii^ difTusely stained material cnnsists of devitaUzed protoplasm.*

The sections also confirm tlie cleared preparations in revealing at the fracture- site an increase in the vitally stained phagocytes as comj)ared with corr(\sponding areas in control preparations. These blue cells are present among the degenerate tissue-remnants, extravasated blood, and exudate. Often large macrophages filled with blue granules api)ear in the blood-clot, and here the cori^uscles have almost disappeared, showing that there is a direct relationship between the pres- ence of the macrophages and the disappearance of the blood-cells. The same association is emphasized in the damaged muscle and other tissue.

In addition to the large, irregularly shaped cells, wliich we may identify as the clasmatocj-tes — the normal mononuclear tissue-resident phagocytes — which show the most distinct blue granulation, there are great numbers of smaller dye- containing cells. These are of various sizes, extending from a small, round, lym- phocyte-hke cell to the large polyblast. In the smaller cells the dj^estuff is very slight, even absent, and as a rule it is increased in amount progressively in the larger sizes. It would seem that the larger cells are developed from the smaller and that the cj'toplasm not only hypertropliies, but with this acquires the power (if imbibing and storing the colloidal d^'estuffs in granule form. The macrophages are always larger and more numerous where muscle and other tissue is evidently damaged. Later stages will show that there is always an excess of macroiihage tissue where protoplasm is degenerate, as evidenced by its diffuse staining with trypan-blue and by its histological characters.

Polymorphonuclear leucocytes are often seen in the tissues of the fracture- area. No dye-granules were observed in them. Quite a number of filM-oblasts were found; occasionally a few small grains of blue were noted in them, but they take the dyestuff very sparingly. There are, too, many tiny blood-vessels — in fact we have here to deal with granulation tissue.

On the second day the sections show the beginning of the callus. This is a fairly thick layer of basopliihc cells lying along the original bone in the region of the injury. These cells are evidently young osteoblasts, and it is jilain that there has been an extensive proliferation of them. Overlying tliis osteoblastic precallus is a layer of fibrous connective tissue continuous on either side with the periosteum.

In the i)recallus there are traces of new bone in the form of slender plates, lying along the original bone, and, in places, short delicate trabeculse arising from

■ '"'"K "■'•• o^ura ill (a) (Icvitdliacd tissue has several ouUtandins aud distinctive characteristica.

'"' ' "' "I" appeiira to l>e deKerierate tissue, in wliich the cells are often misshapen and broken up,

J* " "^ '" " disorderly manner and mixed with eicudatc. The .stiiiiiing is dense and irregular and

'°^ ' '"■ "ii'-l<"iiH. It is eimiily due to the absorption of the dyestuff by the necrotic protopl.osm.

}*' •"•'• "f the pre^R■ncc of di-funct tissue and exudate. Liviiiji protoplasm is able to protect

•t*" ilie dyi>. exeept as described Mow in "c."

• ..( Ti-rotic or morilmnd ti».sue toward trypnn-bluc is to be sharplv distinKuishod from the (6)

'"" " '"'""' '" "'" macrophaues. Hero the dyestuff is housed within the cytoplasm in the form of

""" '"■ t'f-""!'!'"- Often the dye is found in the fluid of a vacuole. The nucleus does not talte the

• | '" *l^^ l0 l'>"o'ulnr microscope, the entire ceU is seen as a single blue Rranule, standing

"^^, ""^' ""rroundiiiKs. Such an appearance is represented in the drawings of tlic cleared ribs

I "■)• dimim-l from l>oth of the almvc, may be referred to here. It is found in certain

' liistic liimiiia' of blood- vessels), in fibrous connective tissue (as tendons and liga-

,," I""' '■*'"■" '•"'"■■ation and is easily distinguishable from the true vital slaininif in

""■ ' ■■■II. ■.■• Min ir..rM ir,. 'i.iiiiiiH' ..1 ilevilaliied tissue.

these into the precallus. This material is directly continuous with the old bone. Ossification is so slight that it is diflRcult to find manifestations of it. Occasionally spaces, containing one or two osteoblasts, occur between the spicules of bone. Vessels have hardly begun to invade this precallus, a small capillary being found but rarely in its peripheral laj-er.

The principal features of the second-day stage, then, may be summed up as follows: (1) the presence of increased numbers of vitally stained macrophages in the tissues surrounding the fracture which have been damaged by the trauma, and in the exudate associated with this; (2) the diffuse blue staining of dead and damaged tissue; (3) the first indication of the callus.

Third-day Stage. (S 11-1).

The next stage studied was that at the end of 3 daj's' repair. In the cleared ribs (fig. 3) it is at once seen that there has been a great change at the site of the fracture, which appears very much more intensely stained than at the earher stage. As at the second daj', the position of the approximated ends of the bone is marked by an opaque blue irregular mass, representing the cUffusely stained areas of injured bone and soft parts; beyond tliis the outline of the bone-shaft may be easily followed.

But the most characteristic thing is the dense, cloud-hke, blue sheath wliich surrounds the fracture-area and gives to it a swollen appearance. Upon e.xamining the specimen critically with the binocular, the blue staining may largely be resolved into granules, each of which represents a macrophage. The appearance is as though the normal resident macrophages had become immenseh' multiplied in the pre- ceding 24 hours, or as though great numbers of macrophages had invaded the area from other parts. IMany of the cells occur in rows between the muscle strands, giving rise to the appearance of blue granular streaks.

Between this investment of macrophagic tissue and the bone there is a clearer area, and as this is followed in either direction along the bone it is seen to underhe a membrane which is continuous with the periosteum. No definite periosteum is found on the bone which it covers. It represents the young callus. Its vigorous proliferation around the ends of the bone has caused the swelling which has resulted in an outpushing of the macrophagic zone.

In the long bones an intense blue staining at the site of the bone-wound is extremely conspicuous in the fresh condition. As in the ribs and skuU, it is due to the diffuse staining of injured tissue as well as to the macrophagic invasion. The sections of the long bones show a diffuse blue-staining of the broken ends (shown in black in fig. 7, b), and also a dense blue-staining of the exudate and loose debris, of which there is abundance. This dense staining is even more marked than on the second day.

These sections are very strildng. A good example of a specimen field is seen in figure 7. This is a low-power photomicrograph from a tangential section through the fracture-area; in it m3Tiads of blue cells (shown as black dots m) crowd about the broken bone and through the surrounding injured tissues and extravasated blood. There is a great deal of fragmented muscle and the blue macrophagic cells surround the remnants of this, and even .squeeze in between the fibers. Under thr hi^h i)<)\\('r th('\- swarm among the fil)robla.st.s and iwjlymorphonuclear hnico- cytes. arouiui (he l>roken bone-ends, in the open sijaees— everywhere. In the more intact part of the muscU', l)etween the fibers, the macrophages are often spindle- shajKHi or stellate. Many of tlie.se are doubtless the so-called resting-wandering cells or chusmatocytes. In all areas, but particularly where the tissue is fragmented, cells of more roimded or oval shape are found. These are similar to those described f<»r the .M'cond day, but are much more abundant.

It is at onci- seen that there are great variations in the dimensions of tlic macro- phages. The largest are enormous cells, studded with granules of irregular size; the average long diameter of 10 of these was 23.3 microns. They are distinctly more volumiiious than the greatest macrophages of the second day and contain more dyestuff, thus indicating a gain in phagocj^tic efficiency. Their shape varies; some are rounded or oval, while others are very irregular in outline — elongated, with long proce.s,ses sometimes constricted from the main part of the cell. They resemble the "polyl^lasts" which Tschaschin found in areas of inflammation in the rat vitally stained with isamin-blue and in the rabbit vitally stained with trj-- pan-blue. The cytoplasm, aside from the dye-granules, is for the most part clear.

It is significant that there are all degrees of size in the dye-containing cells, from tliis extremely large macrophage to quite small cells which harbor very little dye; this type again grades off into a small round cell with a relatively large nucleus and but little cyt()i)lasm, which resembles a Ij'mphocj'te. In the carmine-stained preparations they jircsent a large, deeply staining nucleus and little cytoplasm. They contain no dyestuff. We may .select transitional cells from almost any part of the field and arrange them in an order of size, as in figure 8. From the ajipear- ance presented by such a series it will be at once inferred that an hypertrophy of the cytoplasm of tliis small round cell has taken place and that coincident with this hypertroi)hy there has been an acquisition of the power of jihagocytosis; for dyi'-granules, at first small in number and size, begin to make their appearance in the enlarged cytoplasm. These granules gradually become larger and more numer- ous as the cytoplasm increases in size, and reach their maximum development in the largest polyblasts. There seems to be a direct correspondence between the size of the macrophage and the amount of dyestuff it contains, on the one hand, and the extent of its phagocytic activities on the other; for it may be assumed that the reaction of the phagocyte toward the dyestuff — in degree as well as in kind^ is an index of its behavior toward the material wliich it is s])ecialized to ingest.

During this metamori)hosis the nucleus undergoes little change in size, so that it is extremely small in coni|)arison with the enormous mass of the cytoplasm. In the early changes of the lymphocytoid cell the nucleus often becomes bilobed or even binucleate. Sujjerficially it may resemble a polymorphonuclear leucocyte. In the mature forms no multinucleate or giant-cells were observed.

The tran.sitional stages wliich liave been described suggest that at least some <.f the macrophages have been derived from the lymphocyte-like cells and this

DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 11

hypothetical origin will bo discussed later. IMitotic figures were extremely rare among these cells; thus it api)oars that the cells do not multiply in the fracture- area, but increase by immigration. It is of interest to ob.scrve in this connection that in the capillaries of the region of macrophage invasion there are large numbers of lymphocytes; these seem to be present mainly in the large sinusoids. Bay-like diverticula from the capillaries, representing the young vascular sprouts, were observed crowded with lymphocytes, and it may be that these represent important points for the exit of the embryo macrojihages from the blood-stream, and the circulation stasis here would lie favorable to such a proceeding. The finding of a small clump of lymphocytes in the tissues at the apex of one of these vascular pouches lends support to this supposition.

That there is an actual destruction of effete tissue is shown by the progressive disappearance of this in the consecutive stages; and this tissue-erosion is coincident with the presence of increased numbers of very large macrophages. Indeed, as will be seen, the numbers and size of the macrophages in a given area are a good index of the degree of i)rotoplasm demohtion occurring therein; in other words, there is a direct parallel between the exaltation of the potentialities of the macro- phagic tissue and the absorption of the products of proteolysis.

Though the macrophages are found at times lying ciuite close to — even in contact with — the tissue undergoing lysis, their characteristic position is rather one of complete separation from such tissue, for most of the cells are to be seen lying free in the colloidal fluid which bathes them. Tliis fluid, which stains faintly pink with eosin, is necessarily heavily charged with the products of proteolysis. The macrophages are thus favorably situated for functioning in the absorption of this waste material.

In the third-day stage a macrophage is occasionally found in which, though the cytoplasm is voluminous, the dye-granules are relatively few and scattered. The cytoplasm presents a vacuolate ai^pearance, and in some cases one gains the impression that the cell is filled with phagocytized tissue- waste. It is not unUkely that these cells have been active for a longer period than the well-stained macro- phages. They are most numerous in the areas of young scar tissue — where tissue destruction is largely over. Small and intermediate macrophages, as well as the larger sizes, show this meager type of staining. A few .seem to be falling to pieces.

Compared with the typical macrophages these exhausted or involution forms are as yet insignificant in number. They are more abundant in the later stages, as will be seen.

Polymorphonuclear leucocytes were present in appreciable numbers in the tissues of the fracture-region. They were somewhat irregularly distributed and were often quite closely associated \Aith heavily stained macrophages. Though favorably situated for imbibition of the dyestuflf. being at rest or wandering in tissue-spaces whose fluids were thoroughly impregnated with it (Downey, 1917). yet no dye-granules were found in them. Thus, if this cell functions in the absorption of colloids it must do so very sparingly. It also differs from the macrophages in that it (1(K« not undergo cytoj)l;\.smio hypertrophy; indeed, the only noticeable chnnRO in it is the usual increase in lobulation of the nucleus, this structure being quitt' complex at times.

The presence of these ueutrophiles in an area of inflammation, even of the aseptic ty|)e, which we have here to deal with, is of course to be looked for, and it may be that one of their functions here is the elaboration of proteolytic enzymes; more commonly they are found closely associated with the disappearing tissue than in the open spaces.

Fibrobhusts were fairly abundant throughout the wound-area. In some of these a few fine round dye-granules were observed; the most, however, appeared un.stained.

The skuU sections show substantially the same picture as the long bones. There is a good deal of fragmented and pulped debris which stains diffusely and densely blue, thus confirming the findings for this specimen in the gross cleared condition. Tliis material is the bone dust and other tissue injured in the operation. There is also quite a lot of extravasated blood and exudate. Though macrophages were not discerned in the cleared specimen, in the sections they are very numerous, but the i)icture is hardh^ as striking as in the long bones. Transitional tj'pes of dye-containing cells are seen, as in the long bones, but are less abundant. Distinct staining of the ends of the cut bone is present. Polymorphonuclear leucocytes are fairly lumierous, but contain no dye, even where they ajjpear among the densely staining debris.

On the tliird day the sections, hke the cleared ribs, show the callus somewhat tliicker and more vascular than on the second daj'. It is i)articu]arly well seen in the sections of the long bones. There is much more ossification evident than here- tofore, and distinct trabecular, staining like bone and containing definite bone-cells, project into the outer, more cellular layer. Somewhat larger spaces, with osteo- blasts Uning their walls here and there, occur between the trabeculae. Many of these contain capillaries of small caliber. The largest spaces are situated close to the original bone. Often it is evident that they communicate with the Haversian canals f»f the bone, which have apparently undergone an enlargement in the regions close to the callus. I'Veciuently such a space is found to contain a blood-sinus, and in some si)aces there may be situated a multinucleated basophilic cell resembling a ma«s of osteoblasts. These cells are probably the so-called "osteoclasts." They are not numerous, and no dye-granules were found in them.

When the clctired and uncounterstained section is looked at with the low- IK)wer the calhis has a pale-bluish appearance with a few spots of darker blue representing scattered cells. Under the oil immersion these cells are found to occupy the interstices betweeji the young bony plates; they are reticular cells, and in them it is iK)8sible, by close scrutiny, to see a very faint blue granulation. They are to be regarded !us the youjig recruits of the army of macrophage's which will uihabit the exj)andijig callus spaces in the later stages. They are largest and their granulation is most marked in arejus near tlie original bone— indeed her(\ especially in tlio dilated extensions of the Haversian canals, a cell well studded with dye- f!;ranulos is often encountered. In size and general appearance these are quite similar to the macrophages which inhabit the actively growing regions of develop- ing bone described l)y Shipley and Macklin (1916^), as shown by a comparison ■with the specimens of these authors, and also with sections from the end of a growing bone from the same animal (S 11-1) from which the fracture-sections were cut. In the Haversian canals of the adjacent normal bone, cells containing dye-granules are rare; when thej do occur they are of small size and slight degree of staining. In no case are the trypanoj^liil cells of the callus at this stage at all comparable in size and staining intensity with the extraosseous phagocytes of the degenerating tissues; indeed, when compared with these they are quite insignificant.

As to the function of these trypanopliil cells, it is noteworthy that some slight breaking down of tissue occurs in association with the hollowing-out of the inter- trabecular spaces, and it maj^ well be that they are being developed to absorb the waste products resulting from this process. In any event their advent in the spaces is coincident with the enlargement of the latter. Of even greater significance, however, is their importance in these early stages as examples of phagocj'tic pre- paredness, for, as will be later seen, it is these cells which light up suddenly into \agorous action coincidently wdth the onset of very active bone resorption, which begins about the tenth day.

The source of the trypanophihc cells in the spaces of the callus would seem to be the tissue of the Haversian canals of the old bone. They are doubtless of the same type as the reticulo-endothelial macrophages found in ordinary bone marrow. Under the conditions of bone erosion thej' have increased in size and phagocj'tic ability, for their weU-developed representatives are obviously much larger and more deeply stained than the cells of the non-gro^Ndng bone marrow.

In the tliird-day stage, then, it is evident, judging from the structure of the bone, that osseous resorption is under way to a limited extent in the Haversian spaces immediately underlying the new callus and in the spaces of the callus itself. It is significant that trypanoi:)luhc cells should make their appearance in the spaces which are being hoUowed out simultaneously with the onset of this process and that there should be a direct relationship between their size and staining intensity, on the one hand, and the extent of the bone-excavation on the other.

The callus of the skull, though not so far advanced as that of the long bones, is of the same general character.

Summarizing the features of the staining at the third day, we may first mention the immense congregation of macrophages which are evidently engaged in clearing away the debris consisting of blood-clot, damaged muscle, bone, and other tissues. The great majority of these are apparently developed from the ljTnphoc}i;e-Uke cells, brought bj' the blood-stream to the site of the fracture, where they rapidly gain in size and phagoc}i;ic power. Their function is the phagocytosis of waste products from tissue breakdown. Diffuse stauiing of the defunct tissue is present. In the expanding spaces of the growing callus trypanophihc reticulum cells have apix-arod. which arc a-s yet. for the most part, not strongly stained; to these an important phagocytic role is assigned.

Fifth-day Stage. (S 12-1).

On the fifth day tlie cleared jireparation of fractured ribs shows the macro- phagic sheath still very conspicuous about the area of the wound, its blue lines of trypanoplxil cells stretching out into the muscle and invading the spaces between the fibers. Witliin it the clear zone of young callus is somewhat increased. The staining of the ends of the bones is less marked, and it is probable that this is bccau.M- the tleail material is being cleared away; there is also less blue debris around tlu' l)one. Large macrophages are seen about the ends of the bone and in tiie entrances of the marrow cavity. The macroj^hages are at least as numerous as on the tliird day and are distinctly larger in size, being quite conspicuous even under the low power of the binocular.

In the sections of the lo7}g bones hosts of macrophages throng the areas of effete muscle and other tissue damaged by the trauma. Diffusely staining damaged tissue is found. l)ut is somewhat less marked. There is a .slight amount of extra- vasated blood and fluid exudate. The macrojihages are very large and are loaded with dye-granules. There are not so many transitional types as in the third-day stage. They resemble in form those already described. The exhausted or involu- tion forms, seen now and then in the third-day stage, are here somewhat more often found; as before, they are much more frequently' encountered in scarring areas. In some the cj'toplasm presents nothing more than a mere network in which an occasional dye-granule is seen.

Some of the cells .show a very few large granular masses, suggesting that the smaller dye-granules have coalesced; occasionally a macrophage is encoun- tered in wliich the nucleus stains diffusely, thus pointing to the death of the cell (MacCurdy and Evans, 1912). These results may be due to an overloading of the cell with dyestufT. Sometimes, too, diffu.sc staining of cell inclusions leads to such o|)tical efTects. Similar cell-staining has been described by other authors, as Tscha.schin (1913).

Polymori)honuclear leucocytes are not so numerous as on the third day. They were not found to contain dye-granules, although this animal was subjected to the vital dye for 72 hours.

Scar formation is in progress, and the fibroblasts are of a more mature type. Rarely intleed are dye-granules, even of small size, to be found in them.

In the skull secdons of the fifth-day stage there is still some damaged tissue, taking a difTu.sc blue stain. The ends of the bone arc tinged, but rather less strongly tlian before. Dead filx-rs of muscle and connective tissue .stain bluish and show^ stained nuclei. There i.s some extrav:usated blood and fluid exudate.

In a.ssociation with this debris, macrophages are abundant, but are not so marked as in the long bones. As in the long bones of tliis stage, there are compara- tively few transitional types.

The callus of th(^ fifth day shows evidences of development m its greater thickness and in its l()n<!;(>r traheciilic of bone. The spaces between the bony plates arc larger, and a pronounced feature of this stage is the presence of large blood- sinuses within these spaces, especially in the region next to the original bone. They are distinctly more capacious than those of earher stages. Many of these sinuses have strands of endothelium projecting into the lumen, or even completely crossing it; again, the wall is often irregular and instances are easily found where a smaller capillary is being incorporated into a larger one. Some of the intertrabecular spaces show several small capillaries, and it is evident that the larger sinuses are formed from the fusion of two or more smaller vessels.

As in the third-day stage, cells faintly stained with small blue granules are found in the spaces of the callus. They are somewhat more numerous and a little more distinctly stained than in the earlier stages, and are largest and most strongly stained in the intervals between the walls of the blood-sinuses and the bone, and hence the area close to the original bone is most generously suppUed with them. Often the endothelial cells contain dye-granules. None of these cells are at all to be compared in size and strength of staining with the large phagocytes of the degenerating tissue.

The same parallel is thus present here as in the last stage, viz, the excavation of the spaces of the callus which goes hand in hand with the number and phago- cytic ability of the trypanopliilic cells.

Quite a lot of cartilage is found in the callus of this stage, but it shows nothing of interest from the standpoint of vital staining.

Summing up the fifth-day stage, the most striking feature, as at the third daj', is the great number of large macrophages vigorously at work in clearing out the waste material at the site of the injury. There is not such an active development of macrophages, for fewer transitional forms are seen. That defunct tissue is being cleared away is evident from the decreased amount of diffusely stained debris. Fibrous tissue is developing.

Trypanophilic reticulum cells, showing a little increase in numbers and stain- ing powers, are found in the expanding callus spaces and often envelop the large thin-walled blood-sinuses, which are now a prominent feature of the larger spaces.

Sixth-day Stage. (S 11-2).

The sixth-day stage as seen in the cleared ribs is characterized by the same dense cloud of large macrophages enveloping the site of the fracture. Plugs of macrophagic tissue fill the open ends of the bone. The broken surfaces appear to stain only faintly and also to be becoming round and thin; in this we have e\ddence that the dead bone is disappearing. Debris is sUght in amount. The callus is more sharply outlined and is optically denser.

The cleared skull shows the same local increase in the trypanophil cells, the space between the insert and the regions about the edge of the trephine opening being literally crowded with them. Some diffusely staining debris is seen.

The xcclums of the hmi bone show dense collections of macrophages of similar (vpc to those of earlier staRcs (fig. 9), except that there are not nearly so many voung transitional forms, and there are more degenerate cells in i)laces. \Vherever tliere is fragmented tissue to be absorbed, especially shreds of muscle, there are mvriads of large macropliages stuffed with blue granules (fig. 10). The crevices between nnisde fibers are often crammed with veritable nests of these phagocytes. As formerly, the damaged ti.ssue stains diffusely blue. There is very little exudate.

It is evident, however, that in some regions tissue destruction is waning. Large patches of young scar tissue appear (fig. 9, s), and in the sections simply cleared without coimterstaining one has a striking demonstration that in such areas the phagoc>'tes are much less prominent— indeed, they are often relatively incon- siiicuous. In figure 9 an area of scar tissue appears contrasted with an area of degenerating muscle fiber, and it is easily seen that in the former the blue staining is very weak, whereas it is exceedingly marked in the latter. The macrophages may be looked upon as assisting to prepare the way for the scar tissue, and they are thus an important factor in repair.

Upon searching with the liigh-power lens through the i)atches of young fibro- blasts (fig. 9,s), one is struck with the great number of phantom-like cells, which appear to be macrophages undergoing degeneration (fig. 11). They resemble .similar cells described for the third and fifth days, but are much more numerous. Their appearance is very characteristic ; they often resemble a mass of fishing-net, the knots being represented by the scattered dye-granules. The cytoplasm, at first pale and vacuolated, is in the more extreme types reduced to a mere lacework, and here and there in this are to be found occasional irregular grains of blue, showing that the specific function has not altogether departed. Not infrequently cellular inclusions are found in them, often stained diffusely blue. The cytoplasm may be reduced to tattered remnants. Fragments of cytoplasm, containing a few scattered dye-granules, are sometimes found, marking the remains of a cell which has suffered dissolution. Often the nucleus is stained blue — an evidence of cell death.

The forms of degeneration which have been described are the most extreme. The vast majority of the macrophages in the scarred areas on the sixth day are in this condition. There are, however, cells which are not so advanced in degenera- tion, especially in regions where fibroblasts are less abundant. These contain more dyestuff and usually are less vacuolate. All grades of these are found con- necting the normal macrojihage at the one extreme with the disintegrating remnant at the other.

The reason why these cells drop out of the ranks and break up maj^ be because they have become exhausted in the course of their strenuous activities, for often their cytoplasm is clogged with phagocytized material; they are thus incai)acitated for ingestion of the dyestuff. Possibly, too, the cells have become intoxicated by tlw! materials taken up. Again, since these cells are foiuul characteristically in the scarring area.s, where waste material has been removed, and conse(iuently where the op|)ortunity for activity — even the stimulus thereto — has departed, it may be

that they have atrophied from disuse or perhaps from the encroachment of the vigorous 3'oung fibroblasts. It must be borne in mind that these degenerate forms are occasionally found in areas of active proteolysis, associated with densely stained cells, and that they give evidence of some phagocytic activity. They do not appear all to be old cells, for some of them arc quite small ; young cells, however, as well as mature ones, could, theoretically, fall victims to overwork, poisoning, disuse atrophy, or fibroblastic overgrowth.

The subsequent history, then, of an area such as that shown in figure 7 of the third day is as follows : while the fibroblasts go on developing scar tissue the macro- phages complete their work of clearing awaj^ the products of protoplasmic solution. This being done, they gradually disintegrate in situ. Certain it is that these cells lose their power of motility and literally die in their tracks. Becoming reduced to mere cell skeletons, they collapse, break up, perhaps under the influence of the enzymes of the neutrophilic leucocytes, and doubtless pass off in the tissue fluids. It is quite possible that any solid fragments that remain fall a prey to the neigh- boring phagocytes. This fate seems to overtake most of the macrophages.

Polymorphonuclear leucocytes were not infrequently found in this specimen, in areas where damaged tissue and macrophages were present. Thej^ were not \itally stained.

The skull sections of S 11-2 present no additional points of importance. The picture, though similar, is much less strildng than in the long bones, the macro- phagic tissue being comparatively slight in amount, although increased over normal.

The callus of the long bone of 811-2 is still more extensive on the sixth day, and consists, as before, of a rather deUcate network of trabecular inclosing spaces. The spaces of the interior, especially near the old bone, show enlargement, so that there has been some tissue destruction here. The trabecuL-e are not much thickened.

Under the low-power there may be seen, in the cleared uncounterstained callus, many more trypanophil cells than in earlier stages, and they are a Uttle more dis- tinctly stained, especially in the interior of the callus. As before, the dyestuff is less obvious in the younger outlying tissue. The trabeculse are more dense and show a fibrous structure.

Under the high-power the peripheral spaces, smaller and of more recent origin, are filled with cells and contain small capillaries. Farther back the somewhat older spaces are larger and in them is noted a loose, plexiform aggregation of cells (fig. 12) . As development proceeds, it is evident that the cells of the spaces separate to form a large-meshed reticulum, while the cai)illaries become larger. Later still, the capillaries coalesce to form sinuses (fig. 12 bs). While this is going on, either growth or breakdown of the trabecuhie may take place. If the former, the walls appear lined with osteoblasts; if the latter, these cells are absent and the walls of the trabeculse are roughened. Both processes may be going on in the walls of the same space. As has been noted, the reticulum cells are often phagocjiiic, as shown by their trvpanophiUc reaction (tig. 12 m), and it is apparently in association with the process of disintegration of areas of callus that these cells develop their gor-

inaiulizing propertios. Although reticuhim cells containing a few small blue gran- ules nre seen in the younger spaces, the staining is stronger and the cells more evi- dent in the older spaces. Here the dye is fairly well marked. There has been no increase in size, and but httle in staining intensity as compared with the third and fifth day stages. The blue-stained cells frequently lie between the endothelium of the blood-sinuses (fig. 12 bs) and the trabeculae of young bone (fig. 12 c). The cndothcUum itself often contains granules of dyestuff. The vital-staining is nowhere at all comparable with the brilliant coloration of the extraosseous macro- phages. The reticulum cells are very small as compared with the larger macro- phages, and the dye-granules are also comparatively small.

A feature of this stage, as shown in the carmine-stained preparations under the oil-immersion lens, is the large number of mitotic figures which occur in the try- panophilic cells (fig. 13). These are especially numerous in the outer regions of the callus, where the cells are multiphdng rapidly. They are reticulum cells, and it is evident that their manner of multipUcation is by karyokinesis. The presence of dye-granules within the cytoplasm is not incompatible with mitosis, as was shown for the KupfTer cells by Evans, Bowman, and Winternitz (1914).

Giant-cells arc extremely rare. Only one small giant-cell was found after an extended search. No dye-granulos were to be seen in it.

In the skull scdions of S 11-2 callus is sUght in amount. Reticular phagocytes are beginning to appear in it.

The other member of the six-day stage, 8 13-1, shows essentially the same features. The callus occupying the marrow cavity is particularly well developed and innumerable trypanophilic reticulum cells are found in the intertrabecular sjwces. The original bone in the vicinity of the callus has a worm-eaten appear- ance; in these spaces the vitally stained reticulum cells are quite conspicuous and numerous. In addition there are here a fair number of giant-cells, some of large size. None contain dye-granules. The blood-sinuses of this region are very large.

In brief, then, it is noted that at the six-day stage there is the same relation- ship apparent between resorjjtion of the callus and the presence of trj'panophil cells. Of special interest is the presence of well-stained cells in the spaces which have been eroded in the old bone. The obvious relationship between the large blood-sinuses and bone-resorption is also noteworthy.

In reviewing the sb;th-day stage we have to note the persistence (in areas where tissue destruction is evidently proceeding rapidly, especially in moribund muscle) of enormous numbers of large and very phagocytic macr(»phages. They are of the same type as in jjreceding stages, except that transitional forms are much less frequently seen than on the third day, so that fewer young cells arc being caHed out. In areas where destruction of tissue has ceased scar-formation is well under way, and here the loose fibrous tissue contains immense numbers of relatively weak-staining but often voluminous macrophages, which appear to be degenerate or involution forms. A few of them are found among the active phago- cytc-8. Thus, in this stage, tissue resorption is gradually ceasing, as shown by diminishing evidence of debris and by the extensive scar-formation, but in some areas it is still proceeding actively, as attested by the remaining tissue-waste, engaged by the persisting hordes of active phagocytes. The enlarging callus shows an increase in number of the trypanophilic reticulum cells and some heightening in their staining activity, especially in the older spaces.

Ninth-day Stage. (S 17-2). In the cleared ribs of the ninth day (fig. 4) no diffuse staining of the ends of bone, or the surrounding injured tissues, can be made out. Hence it may be inferred that the clearing away of debris and exudate is at an end. As would be expected, this process is completed sooner in a small bone Uke the rib than in a larger bone, such as the femur, where the tissue damage is, of course, much more severe.

The investment of trypanophil cells about the fractiu-e-area in the cleared ribs is still quite obvious (fig. 4), though less conspicuous than in the earher stages. As will be seen from later specimens, these cells persist at the site of their former labors in the fracture-area for a few days after tinctorial and liistological evidence of damaged tissue and exudate has disappeared. They gradually dwindle in num- bers and vital-staining ability.

The edges of the new callus are much more sharply defined and are distinctly seen throughout. In addition, the callus is considerably denser and is reticular in structiue, an appearance which may be interpreted as evidence of extensive ossification. In the rib from which the figure was made this reticular structiu-e is complete throughout, but in the others it is incomplete betw^een the bone ends. Apparentlj'^, movement has delayed ossification in these cases.

In the cleared shdl of the ninth day the bonj^ insert of the left side slightly overlaps one of the edges (fig. 6). This insert consisted of sterile dead bone from another rat. On the right side the space is unfilled. This opening crosses the mid- hne and occupies some of the territory of the left parietal bone. A Httle bluish debris is seen lying about. The edges of the bone are slightly blue. Clouds of macrophages infiltrate the membrane filling the open spaces, as shown in the drawing. The clear spaces in this represent areas of extensive scarring; here the macrophages are much less conspicuous. Some unstained ossified callus radiates from the edges of the openings.

The sections of this stage are not good.

The principal features to be noted on the ninth day are the gradual subsidence of the extraosseous macrophages and the occupation of their fields by the scar tissue. In the callus increasing density is evident.

Tenth-day Stage. (S 5-1), (S 6-1), (S 6-2).

In the cleared shdl of the tenth day (S5-1) a local excess of macrophages is still evident, as well as a little stained debris. In the sections of this specimen the diffuse staining is found to be due to the presence of a small abscess, the result of an infection. The exudate has taken a diffuse blue color. IMacrophages are found in numbers in the periphery of this abscess. Some appear degenerate. Dyestufif was not found in the poljTiiorphonuclear leucocj^tes of the abscess.

The sections of the long bones of the tenth day (S6-1, S6-2) present very little renjiiininji dehris. ( )ccnsionally a few slireds of muscle are found, showing evidences of defeneration. Here macrophages are fairly numerous, though much reduced in number as comjjaretl with earher stages. These jihagocytes are of the mature tjix*, with very few transitional stages. Comparatively few involution forms are seen. Scar-formation is advanced.

The ca//«s of the long bones (S6-1, S6-2) at tliis stage shows little increase in volume. The most interesting feature is seen in the hollowed-out spaces near the original bone; for here, especially in the cleared and uncounterstained sections, one is struck with the conspicuous ajjpearance of the many vitally stained cells. Even under the low-jiowcr they are very evident, as shown by figure 14, drawn from a section from S6-1, and are much more brightly stained than in the earlier stages. .\s before, they are often distributed around the thin-walled blood-sinuses. They are not in actual contact \fith. the callus. Thej^ are found in the callus of the marrow ca\'ity as weU as in that of the external surface of the bone. The degree of staining intensity gradually diminishes as the outer district of the callus is approached, and in the more compact areas of callus, where apparently no resorption is occurring, the blue cells are verj^ few and insignificant in staining.

A descrijition of the general morphology of the tjqMcal reticulum macroi)hages, as seen with the high-power lens, may here be given. It applies, with slight ^•aria- tions, to the cells of the succeeding stages up to and including the twentieth day.

like all reticulum cells they are characterized by a number of processes — flat and wide, or extended and threadlike — through which they are directlj' continuous with their neighbors. The cell-body is oval or stellate, and oftcni elongated and flattened. The nucleus is fairly large and usually rounded or of oval outline, though it may be notched.

The trypanophiiic reticulum cells at the tenth-day stage are the same as at the earlier stages, except that they are often much more brilUantly stained and hence may be regarded as much more actively phagocytic. They are quite evi- dentlj' true reticulum cells, and not invading elements from other regions of the body, for their protoplasmic connections with other reticulum cells, which may or may not contain dye-granules, can easily be made out. The size of the well-st;iined cells varies; the tendency seems to be to develop a cell of fairlj'^ uniform dimensions The average diameter of ten of the largest cells at the tenth-day stage, as taken between the widest extremities of the dye-granule content, was found to be 7.6 microns. They are thus considerably smaller than the largest extraosseous macro- |)hages, though larger than the macrophages of ordinary bone marrow. The arrangement of the cells in the spaces is the same as that in stages already described, or as in the later .stages.

The degree of staining varies considerably; some cells have a mere sprinkling of small dye-granules, while others are stuffed full of granules of larger size (fig. 15, a), and there are all degrees of variation between these extremes. Usually the nucleus is completely inclosed in a zone of blue granules, but not infnviueiitly cells are

found in which areas of cytoplasm are without visible dj^estuff. In the typical phagocytes of this stage the granules are much larger than in the earlier stages, and there are more of them. There is much more dyestuff in the cell.

Some of the cells contain a very few quite large granules of dyestuff instead of a multitude of smaller ones, suggesting that there has been a coalescence of the latter. Again, in an occasional cell, the appearance is as though the entire nucleus were stained, pointing to the death of the cell. It is possible that these cells have succumbed to the action of the material ingested; or such appearances may be due to stained protoplasmic inclusions. Similar findings have been noted in extraosseous macrophages.

As the cell fills with its pabulum or with dye it becomes more rounded and the processes are reduced to fine tlireads. As a rule, the dj^estuff does not find its way far into the processes, so that the dj^e-granule contour is usually' oval, as is seen in figure 15, drawn from representative uncounterstained reticulum macro- phages of different periods.

A few poh'morphonuclear leucocj'tes are seen associated with the macrophages. No dyestuff was found in them. Osteoblasts are sometimes met with in their vicinit}^ but never take the dj^estuff. Giant-cells were not encountered.

In the skull of this stage (S 5-1) distinctly stained reticulum cells were found in the spaces in the callus and also in the old bone.

As will be seen from the examination of the subsequent stages, the resorption of the callus is most active during th*^ period from the tenth to the twentieth day or shortly after; before that period the main trend in the callus is constructive rather than destructive, although, as has been pointed out, there is, even during this evolutionary phase, some hollowing-out of the spaces, as seen, for instance, in the specimens of the fifth and sixth days. Furthermore, the stages from the tenth to the twentieth day show that the greatest amount of resorption occurs at first in the ^•icinit}' of the old bone, for the spaces there undergo the greatest expansion. It is thus evident that the most brilliantly stained, and hence the most phagocji^ic, cells are found where callus destruction is most active. It is plain, too, that these cells have undergone an exaltation of their powers of phagocji^osis coincidently with the acceleration of callus resorption.

Reviewing the tenth day it is noted that extraosseous macrophages are grad- ually disappearing, as shown by the many degenerate forms and by the relative inconspicuousness of the survivors. Their place is taken by scar tissue. In the callus the most striking point is the marked hypertrophy and increase in phago- cytic ability of the macrophages of the reticulum of the callus spaces undergoing expansion at the expense of their osseous walls.

Twelfth-DAT Stage. (S 5-2).

Little or no stained debris was noted in the cleared skull of the twelfth day, nor was it found after this stage. In this specimen the opening in the bone was left unfilled. The central part of this opening is almost clear, being occupied by

scar tissue, in which but few ni:icrophases may be descried. Surrounding this scar, and bordering the bone edges, there remain macrophages in considcral)le numbers. Callus in this si)ecimen is well advanced, and in the spaces of this, wliich are now quite large, considerable numbers of large-sized macrophages may easily lie seen with the higher power of the binocular.

The si'ction.s of the .skull show marked scarring, but nothing of note as to vital staining. In the loncj-honc Hediuns no blue-staining debris was noted. In the vicinity of the fracture tlie muscle fibers are scattered, and Ijetween them there is a great deal of scar tissue. Here, too, there is some excess in the number of macrojihages, but these cells are numerically insignificant as compared with earlier stages. Tran- sitionals and degenerate forms are very rare. It is evident that httle or no tissue destruction is going on. The synchronism between proteolysis on the one hand and heiglitened efficiency of the macrophagic tissue on the other is thus consis- tently maintained to the end, both phenomena ceasing practically at the same time. As will be seen by the subsequent stages, there is no fiu'ther evidence, beyond the twelfth day, that extraosseous tissue destruction is going on, and the macro- phages which remain in places where tissue resorption has once taken place soon disappear.

The callus is of greater dimensions than before, and is evidently still growing in places, as the outer layer of deeply basophihc osteoblasts is often seen to be quite thick. It is very olnious that erosion of the callus is proceeding rapidly at tliis stage, for not only do the spaces in the vicinity of the original bone show increase in size, but the spaces throughout the callus almost as far as the periphery are enlarged. These spaces are occupied by large, very brilliantly stained reticulum macrophages. The cells are very numerous and crowded, some fields being quite blue with them, so that they present an exceedingly striking appearance in the cleared section (fig. 15, h). They are even more marked than in the last stage, and have evidently develoix^d an intense avidity for the blue dye, for the cytoplasm is literally ]xxcked with granules in most of the cells. There are degrees of staining, however, and some cells are found with but a sprinkling of dye-granules, so that it may be assumed that as time goes on more and more reticulum cells are developed a.s phagocytes. Again, some of the cells have a few very large, rounded granules, looking as though they were produced by a concentration of dye from the rest of th(! cyto])lasm, as was noted in the last stage.

The distribution of the macrojjhages is more widespread tlian before and goes hand in hand with erosion of the callus, for they inhabit the expanding spaces almost as far out as the periphery. In size they show a slight increase over those of the tenth day, the average long diameter of ten of the largest cells being 9.15 microns. These are fairly uniform in dimensions. As before, they are found in the locj.se reticulum of the spaces (fig. IG), or are crowded between the plates of bone and the walls of the sinuses. They are not in actual contact with the bone. The blood sinuses are very large at this stage, pointing to a sluggish blood flow. Their walls are formed by a single layer of endothelium.

Since the macrophages are largest, most phagocytic, and most numerous where resorption of the provisional callus is going on most rapidly, it is quite evident that they are concerned very intimately with the clearing away of the callus.

A thickening of the trabeculaj which escape erosion is evident, and thus there have been going on, side bj' side, the antagonistic processes of bone-ero.sion and bone-building.

Giant-cells are sometimes found, but contain no dyestuff. Large areas of cartilage are seen in tliis specimen. Marrow tissue is present in some of the spaces.

In the callus spaces of the skull macrophages are found, as in the callus of the long bones.

Passing in review the twelfth-day stage, there is to be noted an almost com- plete cessation of tissue destruction, and the extraosseous macrophages are but little increased over the normal. The callus, on the other hand, shows an exalta- tion in staining and increase in size on the part of the macro|)hagic inhabitants of the areas where bone destruction is activelj^ proceeding. Bone-erosion and bone- building are combining to give shape and strength to the permanent callus.

Thirteenth-DAT Stage. (S 11-3).

The thirteenth-day stage shows essentially the same conditions. The staining here is not good. In the cleared ribs no evidence of damaged tissue is present. Macrophages are much reduced in number and staining intensity. Callus is well advanced.

In the cleared skull, owing to the poorness of the staining, neither stained debris nor extraosseous macrophages can be made out. The callus is well marked and a few blue-stained cells are seen in its spaces.

Fifteenth-DAT Stage. (S5-3).

In the sections of the long bone of the fifteenth-day stage there is no diffusely staining material to be seen. In the vicinity of the fracture, where tissue destruc- tion has been proceeding, the muscle-tissue is loose, and the scattered fibers are interspersed ^dth scar tissue and macrophages. Transitional types are very rare. No degenerate forms were noted, nor were any found in later stages. Scar tissue is abundant. It seems to be evident that resorption of dead tissue has ceased. The extraosseous staining presents a much less striking picture than in the earlier stages, and from this period it becomes less and less noteworthy.

The sections of the skull show no remaining dead tissue. It is not evident that there is any excess of macrophages at the site of the healed wound.

The callus of the long bones of S5-3 is quite extensive, but, judging from the almost entire absence of typical osteoblastic formations on the periphery, its expan- sion has practically ceased. The regions near the original bone are characterized by thickened osseous trabecular, often inclosing large tracts of loose cellular marrow tiseue, traversed by voluminous thin-walled blood-sinuses. Some macrophages are found here, but in regions where the marrow tissue has become well established they are relatively few and inconspicuous. The apparent reduction in their num- ber here is due in part at least to tlu- increased proportion of other cells as well as to the fact that the ti.ssue becomes more oi)en in structure. The phafijocytes are to be regarded as having a hand in the preparation of the marrow-tissue hal)itat.

Farther out in the callus the spaces are large, and many are occupied by enor- mous blood-sinuses with walls composed only of endothelium. Here many of the trabecuhe are thin and are evidently being broken down; others are thickened and are being built up. This is the most active area of bone resorption. In this region horcU's oi reticulum macroi)hages, brightly stained and of increased size, form a striking i)icture and are the most outstanding feature of this stage. Their district of greatest concentration, as comjjared with the last stage, has shifted jK-riplierally, in company with the district of greatest callus-cro.sion. The preference of the macrophages for regions of bone resorption is all the more significant because it is consistent with the behavior of these cells in earlier stages.

The morphology of the macrophages and their relationship to the surrounding structures need no special description here, for they are similar to those of the last two stages described. Careful measurements of the longest diameters of ten of the largest cells gives an average length of 9.95 microns — thus showing that the cells have become somewhat hypertrophied. The dye-granule content is similar to that of earlier stages (fig. 15 c). Some associated polymorphonuclear leucocj'tes are found in the tissue-spaces, but no dye-granules apjjear in them. Giant-cells are quite frecjuently found, liut they also contain not a trace of dye. Often they give the imi)ression of being a mere scrap-heap of old osteoblasts and bone-cells, the residue of bone-erosion (Arey, 1917).

In the skull of this stage macrophages of large size and conspicuous staining inhabit the callus spaces. Large blood-sinuses in the spaces are also a feature here.

On the whole it may be concluded that at the fifteenth day little or no tissue is being destroyed outside of the bone, for the number and staining intensity of the macrophages have been reduced almost to normal and there is an absence of demon- strable moribund tissue. In the callus, however, the reticulum macropliages are esjx'cially abundant and i)hagocytic in the regions of bone destruction, and ai)pear to be actively engaged in phagocytizing the products of this process.

TWKNTIETH-DAY StAGE. (S 12-2), (S 13-2).

The cleared ribs of the twentieth daj'^ (fig. 5) are very different from those of earlier stages, as the third and fifth days, for the blue investment has become very thin and but few gramiles, representing macrophages, are to be seen in it with the binocular. What blue there is seems to be largely a diffuse staining of the j^erios- tcuiii and .scar-ti.ssue. The size of the fracture-site is much reduced and the callus \\:is undergone a good deal of ri^.sorjjtion, the part now being of a .slender spindle- like form. The interior of the bone is occui)ied by a large-meshed callus, containing some blue cells, and tin* medullary canal is evidently being restored. Other ribs at this stage show less })erfect ai)pro\imati()n, but essentially the .same features.

In the cleared skull of the twentieth day the extraosseous macrophages show- little if any increase above normal. The membrane of connective-tissue fiUing in the intervals between the bone pieces stains diffusely blue, especially where it is thickened near the edges, but this staining is not strongly marked and resembles the staining of any similar fibrous connective-tissue, as tendon (foot-note, page 8, c). A few persisting macro])hages appear in it. The new bony callus is abundant and in its spaces a few tr}'panoi)hilic cells are seen.

In the sections of the lony bones of the twentieth day (S 12-2, S 13-2) large areas of callus and of scar-tissue are conspicuous. Near the fracture the muscle-fibers, as noted for the two last-mentioned stages, are often scattered and surrounded by the new scar. They are frequently rounded and dwarfed. Numbers of macro- phages still persist in jjlaces such as this; jjrobably they are not actively' functioning and graduall}' disappear, for, barring a few found at the thirtieth day, thej' are not encountered in future stages. No evidence of extraosseous j^roteolysis is to be seen, there being no blue-staining debris or degenerate tissue. Traasitional cells were not found in the twentieth-day stage.

The callus of the long bones (S 12-2) of the twentieth day is especially inter- esting. At no stage are there greater evidences of erosion. Enormous spaces characterize the cross-sections and in some of these but a mere shell of bone remains at the periphery, while the central region is filled with marrow-tissue, tunneled by large blood-sinuses. Again, in other sections the spaces are jjartitioned by stout trabeculae of bone. A corner of a tj^pical section is presented in figure 17. Here the most spectacular feature, brought out with singular sharpness in the cleared section, is the multitude of brilUantly stained reticulum macrophages at work in the smaller spaces. As a rule they are now massed in the more peripheral recesses, though some crowd into corners here and there throughout the bony spongework, where temporary osseous scaffolding still awaits removal. A few haunt the interstices of the marrow tissue. In many areas they are very incon- spicuous or absent. Here bone erosion has evidently ceased. It is obvious that the area of most active bone destruction has shifted from the central mass of the callus to the more outh^ing regions and, as in the last stage, the macrophagic army has kept pace with this onward march.

Striking indeed are the pictures presented under the higher powers. In figure 18 is seen a small field, magnified 190 times, from the section from which figure 17 was taken. The formations of macrophages (m), as before, are drawn up in the perivascular spaces or deployed through the loose reticulum. In these areas of most active bone-erosion the phagocytes are even larger in size than at the fifteenth day, for the average measurement of the longest diameters of ten of the largest cells was 12.6 microns, in contrast with 9.95 microns, the average at the fifteenth- day stage. These compare in size with the large extraosseous macrophages. Typi- cal uncounterstained cells are shown in figure 15, d. Mitoses in djx^-containing cells are present, but are not frequently found.

Again, the presence of numerous hypertroplued and highly phagocytic macro- phages in regions where active bone resorption is proceeding is significant. The rehitionship is similar to that in growing bone.

The same general morphology as heretofore is noted. Even here there are many non-tryiwmophiUc reticulum cells. As before, too, the voluminous thin- walled blood-sinuses are a feature of the areas of disintegrating callus. Bone growtli at this stage is limited to the reinforcement of the permanent osseous trabecuhe. But few osteoclasts were found. No dye-granules were discovered in

them.

SixKiimen S 13-2 presents essentially the same features in the callus. Here, too. the macrophagic picture is very striking. This specimen shows an interesting condition in the cartilage, which sometimes appears in the callus, as noted at earlier stages, for it is now becoming ossified. In this process the cartilage is modified and hollowed into spaces in wliich appear blood-sinuses whose enviionment is charac- terized by reticulum macrophages, similar to those found in osseous spaces. Osseous tissue is built around the remnants of modified cartilage as in developing cartil- age bone. Trabecuhe so formed are resorbed Uke the usual trabecule of the callus, and the macrophagic tissue is concentrated similarly here. A formation essentially the same as the familiar epiphysial plate, wdth typical rows of enlarged and modi- fied cells, was observed. The reticular macrophages play the same part here as in callus or growing cartilage bone, for they congregate in regions where tissue is being broken down, as noted by Sliipley and Macklin (1916^).

This period represents the high-water mark of the macrophagic activity in the callas, and from this tune forward there is a gradual diminution in numbers of the cells and in their phagocj^tic power, as shown by the brightness of the stain- ing. The decUne of the macrophagic tissue is coincident with the gradual fall in the rate of bone-erosion, and with the cessation of this process the macrophages of the reticulum revert to the state of the macrophages of ordinary bone marrow. Though retaining in some measure the power of ingesting and storing colloidal dyestuffs, they are then nevertheless relatively small, few, and weakly staining.

Summarizing as to the twentieth-day stage, it may be stated that there is no destruction of tissue in any of the specimens outside of the callus. Extraosseous ti.ssue destruction has ceased coincidently with the falhng away of the macrophage concentration. In the callus, however, the same interesting participation of the reticulum macroijhages in deahng with the waste j^roducts of callus destruction is emphasized. Even in the cleared gross ribs the cells may be described in the callus spaces. It is especially evident from the sections that the greatest concentration of macrophagic tissue is always found in areas of greatest callus destruction and that these cells change their location to accompany the erosive mechanism, thus gaining the position of greatest efficiency for the i)erformance of their duties.

A brief d('scri])tion will suffice for the remaining stages, since the more active proces.ses, out.side the bone, ha\e terminated bj' the twentieth daj', and in the callus the stained cells gradually become less and less conspicuous.

Thirtieth-day Stage. (S 11-4).

In the cleared rib of the thirtieth day the fracture is well healed. The perios- teum shows none of the thickening of the earlier stages. Some swelling due to callus is evident at the site of the fracture. There are no staining features of interest. In the cleared tibia, too, the bone ends are held together by firm callus.

The cleared shdl of the thirtieth day has stained but poorly. Trabecule of callus rachate from the site of the injury. Macrophages are noted within the spaces of the callus, but are not mimerous anywhere else.

The sections of the lo7ig bone of this specimen show that there is Uttle trace of the original injury in the soft parts except the abundant scar tissue. As in the last- mentioned stages, macrophages are present in larger numbers than normal in some regions near the bone, often among scattered and small muscle-fibers. They are comparatively small and pale-staining, and are the survivors from earlier and more active periods.

The callus of the thirtieth day is very extensive. In texture it is considerably coarser than that of the twentieth day, the trabecula; being stouter and the spaces larger. The area close to the original bone is occupied principally by marrow-tissue. Farther out there is more bone, and the spaces are smaller. Large, thin-walled blood-sinuses are frequent here, and around their walls are great numbers of reti- culum macrophages. On the whole, however, the macrojjhages arc much less numerous and striking than at the twentieth day. The principal change in the callus seems to be a strengthening of the permanent trabeculae, with a paring away, here and there, of the temporary bone.

Fifty-first (S 17-1), Fifty-eighth (S 15-1), and Fifty-ninth (S 12-3) Day Stages.

The cleared ribs of the fiftj'-first day show an almost normal contour, there being hardly any swelling due to callus and the medullary cavity being quite patent. Some blue staining is present around the site of the fracture, but this is referable to the diffuse staining of the thickened periosteum and scar-tissue at this point. The same faint diffuse blue appears in the fibrous membrane joining the insert to the edge of the trephine opening in the cleared skull at this stage and is also present in the fifty-eighth and fift3--ninth day skulls (foot-note, page 8, c). There are no macrophages to be discerned in the cleared specimens of these periods. In the sections of the fiftj^-first and fiftj-ninth daj's the staining of the soft parts presents nothing of interest.

The callus of the fifty-first day is very compact and thick and can hardW be distinguished from the original bone. Much of the space has been taken up bj' the new, compact bone. The surviving trabeculjE are stout and inclose large spaces containing marrow-tissue. There are in this tissue comparatively few dj'e- containing cells, and these are relatively small and weakly stained, resembling the macrophages of ordinary marrow. It is evident from the appearance of the bone that little or no osseous resorption is going on. A few giant-ceUs, containing no dj^e-granules, are found in the marrow-tissue.

The cdlltis of the fifty-ninth day is very dense. Tlie description of it and of the vitally .staiiK'tl ceils it contains is .similar to that of the fifty-first day.

Sixtieth (Sll-5) and Seventy-first d.\y (S5-5) St.\ges.

The ckarcd rib at 60 days shows Uttle change as compared with tliat of the fifty-first day. The site of the fracture can hardly be distinguished, so perfect is the rei)air. In the cleared >>kuU of tliis stage, and that of the .-:eventy-first day, there is the same diffuse staining of the connective-tissue membrane joining the bony fragments and the same absence of macrophages. The sections of the sixtieth day show notliing of interest in the soft parts. The callus is greater in amount than in the last two specimens described, and hence more time is reciuired for the resorp- tion of the redundant bone. The osseous structure is somewhat less compact and the trabecuUe are thinner. There is evidently some osseous resorption still going on. In keeping with tlxis the macrophages are somewhat more numerous and con- spicuous than in the fifty-first and fifty-ninth day specimens. These cells, are, however, comparatively small and weakly stained. Bone-building is going on here ami there, as the rows of osteoblasts attest.

The skidl scclions show nothing of interest.

Summarizing the stages from the thirtieth to the seventy-first day, it is to be noted that there is no excess of macrophages outside of the bone. No tissue destruc- tion has taken place during this jjeriod except in the callus. At the thirtieth day there are some macrophages to be found in the callus-spaces, but sections of the fifty-first and fifty-ninth days show very few of them, so that bone destruction here may be regarded as almost at a standstill. The spaces are large and the remaining trabecular are much stouter, so that the principal effort has been directed toward reinforcement of the bony trabeculie rather than destruction of them. The occurrence of a few persisting macrophages in the 60-day stage is to be looked upon as a special ca.se where, on account of the excessive amount of callus, the work of destruction was of longer duration.

DISCUSSION.

From the fcjregoing account of the appearance of the tissues at the site of fractures and trei)hine wounds in the vitallj'^-stained rat, during successive stages of healing from the second to the seventy-first day, it is jilain that there are two distinct phenomena to be considered. In the earlier stages the most notable feature is the tremendous increase in the nimiber of tiypanophil cells which occurs in the injured tissue and exudate of the region and about the damaged surfaces of the bone; at a somewhat later period an equally remarkable develoimient of phago- cytic tissue within the si>aces of the callus is seen. The discus.sion falls naturally, then, under two main headings concerned with (1) the phagocytes of the soft parts surrounding the injured bone, or extraosseous macrophages, and (2) the phagocytes of the callus spaces, or intraosseous macrophages.

EXTRAOSSEOUS MACROPHAGES.

A survoj' of the specimens from the experimental animals has shown that the macrophage tissue of the soft parts at the wound-site, after a brisk initial rise fol- lowing the injur}', remains at maximum for a few days and then gradually falls away. During the first two or three days these phagocytes may be considered as undergoing mobilization and development. Noticeably increased at the end of 48 hours, they are on the third day very abundant, and so continue during the fourth, fifth, and sLxth days, after which their numbers gradually diminish. In the long bones they are not very marked after the fifteenth day, and in the ribs and skulls, since their work is less, they do not persist so long, being reduced almost to normal by the tenth day, or shortly after. This rise and fall is graphically shown in the series of cleared specimens, especially in the ribs, where the area surrounding the wounded bone speedily becomes blue from the accumulated vitally stained cells and (after remaining so for a few days) gradually pales. Sections from the skull and long bones serve to support this finding.

In studying this increased macrophagic tissue from day to day, one is impressed by the close association which it has \\ith the waste material, the result of the trauma. Again and again has it been noted that, where tissue- waste is present in large amount the macrophages are enormously increased, and that as the debris disappears the macrophagic tissue gradually becomes less marked. Indeed, so close is this relationship that the curve tracing out the chronological record of macrophagic intensity roughly parallels that described by the slowh- disappearing waste tissue. These facts will be apparent from the following review.

The evidence j^resented by the cleared ribs shows that damaged tissue and exudate, as manifested by diffuse staining of material in the region of the fracture, was, on the second and third days, strongly marked; on the fifth day somewhat less conspicuous; on the sixth day still more reduced; and after this it was not observed.

In comparison, it is to be noted that the macrophages at the fracture-site were somewhat increased over the normal on the second day, and on the third daj^ were very markedly increased, remaining so until the fifth and sixth days, and being reduced on the ninth day. They then gradually diminished. This relation of the macrophages to the demonstrable debris at the fracture-site is graphically set out in table 2, which shows a synchronism, as evidenced by the cleared specimens and sections, between the occurrence of debris (representing dead and dying tissue) and that of macrophages in increased numbers in the vicinity of the debris. Some variations in individual specimens are noted. Thus in the fifteenth and twentieth day stages of the long bones the macrophages are unusually numerous, probablj' due to the severity of the injury. Again, the skull sections show more debris and as many macrophages on the tenth da\' as on the sixth; this is due to an infection in this case.

In the cleared skidls, as shown in table 2, the findings are very similar. Debris was strongly marked on the second, third, and sixth days, becoming less on the ninth and not appearing after the tenth daj'. Its removal was thus accomplished in the first 10 (lays following the wound. In association with this gradual removal of the waste material there was a marked increase in macrophages of large size and pronounced staining al)ilities, extending from the sixth to the ninth day; after this the phagocytes became less consjiicuous and had reached almost their normal condition on the twelfth day. Though not jjlainly seen on the third day in the cleared skull they were abundantly present in the sections of this, so that the Ijcriod of macroi^hagic activity extends from about the third to the tenth day inclusive. Probably they begin to increase, as in the ribs and long bones, before the third day.

The cleared skulls jiresent one or two minor points of difference as compared with the ribs. The debris, as the table shows, disappears somewhat more slowly, and this is associated with a longer duration of the macrophagic tissue. To explain tliis slowness of absorption we have to consider, in one skull (S5-1), the element of infection. Again, macrophages are never quite so abundant in the skulls as in the ribs and are perhaps a little slower in their mobiUzation.

Table 2

The plus siens indicate roughly the relative amounts of d6bris or macrophagic tissue, as the case may be. Thus + would denoto a diw-cmiblc increase in macrophages as compared with the normal, or the pre-sence of an appreciable amount of d/-bri.s: + + + + would mean an exceptional amount of macrophagic tissue or d6bris, etc. The sign — indicates an absence i)f d/'bris nr niacruphagcs. as the case may be. The sign means that no evidence is presented liy the .specimen. The brackctcJ signi indicate macrophage! which have persisted in regions where formerly tissue was being absorlcd. They are compamtivcly small and weakly staining.

The sections of the long hones show even more strikingly the same remarkable coexistt'nce of debris and abundant macrophagic tissue over a jjeriod from the si'cond to the tenth day, with macrophages remaining in the field u\ reduced amount as far as the thirtieth day.

In the xcdions of the skull the same parallelism of debris and macrophages is found, extentUng (in the specimens examined) from the tliird to the tenth day.

From this summary, the results of which are graphically set out in table 2, it is evident that the occurrence of excess of macrophage-tissue is strikingly syn- chronous with the iire.senre of waste material. Following rapidly upon the injury, macrophages become excessive in numbers in and around the damaged tissue; these cells are not only larger in size than the usual resting macrophages, but are of heightened phagocytic power, as revealed by their increased dye-content. It may be inferred, therefore, that this waste material, the result of the trauma, is con- cerned in some way with the mobihzation of the phagocytes and with their accel- erated activities; that, in fact, the increase in macrophagic tissue, in volume and in functional efficiency, is a response to the presence of dead or dying tissue.

From table 2 it is also plain that as the dead tissue vanishes, as shown by the tinctorial and histological evidence, the macrophages become less and less con- spicuous, their numbers being reduced and their staining weaker. This points to some connection of the phagocytes with the absorption of the debris. More will be said upon this point later.

Another evidence that tissue resorption has been proceeding coincidently with the presence of macrophagic tissue in excess is the thinning and rounding of the ends of the bones surrounded by the phagocytes. This, for instance, is seen at the sixth day (S 11-2) in the cleared rib.

As table 2 shows, some of the phagocytes are found in numbers somewhat above normal after demonstrable debris has disappeared. These are to be looked upon as cells which have persisted in the field after their work was done. They undergo gradual diminution in number and in reaction to the vital dye and are probably to be looked upon as resting rather than as actively functioning.

To anyone famihar with the Uterature it will be quite obvious that the behavior of these phagocytes, in the reaction following bone injuries, is quite like that wliich obtains in the repair of any damaged tissue, and thus the problems involved are those common to inflammation. These problems have been investigated by \^arious writers, as JNIaximow (1902, 1906, 1909') and Goldmann (1912); the latter studied the tissues of the vitally stained animal following the application of turpentine and infection with the tubercle bacillus. Tschaschin (1913), too, investigated the reac- tion of the vitally stained cells in the neighborhood of foreign bodies in the loose connective tissue and in cauterized areas of the liver, spleen, and mesenteric lymph- nodes. Thorough discussions of the various aspects of macrophage behavior under these conditions are to be found in the literature, so that it is here sufficient to refer only to some of the more outstanding points and to emphasize the special application of macrophage function to the repair of bone-wounds.

\\e have seen that in the rise and fall of excess macrophagic tissue in the areas surrounding bone-wounds a curve is traced. From this three successive segments may be taken to block out periods of macrophage history in which the most out- standing features of the phagocytic tissue are consecutively development^ activity, and decline; but it must be recognized that these periods grade insensibly into one another, so that if arbitrary hmits be assigned to them there will be of necessity some overlapping. They serve, however, to separate the discussion into convenient subdivisions.

DEVELOPMENT.

Of special interest in connection with the development of the macrophage- tissue is the question of origin of these ]:)hagocytes of the soft parts. Among the possible sources there must be considered the macrophages found normally in the marrow-cavity and ixTiostcum of l)onc and in the tissue of the immediate vicinity. When an injury oorurs, such a,s a fracture, it is quite possil)le to think of these ■"rostinfi-wanderins" cells as undergoing extensive rapid multij^lication until the aggregation of phagocytes which is so marked a feature of th(! early stages of the repair process is produced. But to accomplish this result the i)roliferation would ha^•e to be enormous, and it is noteworthy that but few^ mitotic figures were to be found among the macrophages.

Again, it is known that endothelial cells can, under the stress of inflammation, take on phagocytic ])roperties, as shown bj'^ their reaction to colloidal dyestuffs (MacCurdy and ICvans, Tschaschin, and others), and hence it may be assumed that some of the macrophages are recruited from these elements. The reticular cells of bone-marrow, too, are known to be phagocytic, and these may contribute their quot^i to the sum total of the "polyblasts."

It would be difficult, however, to conclude that any or all of these sources could account for the tremendous local increase in trypanophilic cells, as seen, for instance, on the third day, even providing for the immigration of considerable numbers from adjacent tissues. Again, such an hypothesis w'ould have no place for transi- tional cells of the type seen in figure 8, and of these there are all grades, from the finished polyblast — large, filled with enormous blue granules, and with a relatively small nucleus — all the way down to what appears to be the parent cell. This is a small mononuclear element resembling a lymphocyte. The parent cells contain no dye-granules, but they soon gain j^hagocytic ability, as shown by their rapid increase in size and the larger and larger amount of dj'e which they take up (fig. 8). The transitional cells are particularly ai)un(lant in the early stages, and most of all on the third daj'.

The idea that some at least of the macrophages are really metamorphosed lymi3hoc}'tes has been steadily gaining in the literature. Even in normal connective tissue, transitional forms may be found hnking together the small round amoeboid lymphocytes with the clasmatocytes (Ranvier, Tschaschin) and suggesting the deri- vation of the latter from the former. The origin of the macrophages of the "taches laiteu.ses" in the rabbit is indicated by finding transitional cells connecting them with a small lym])hoid element (Tschaschin). Maximow (1907, 1909') has estab- lished the relationship of these wandering cells by embryological researches. The .siune author (1902, 1909-) has long maintained that the hypertrojihied mononuclear phagocytes or polyblasts of areas of inflammation are largely derived frt)m lyini)ho- cytes attracted thither from the tissue-s])a('es and from the blood-stream, and Tscha.'<chin brings forward evidence to confirm this view — the lym])hocytes, it is a.s'<umed, rapidly undergoing a mefamor])hosis, their powers of phagocytosis becoming intensified, as shown liy their i)rogressive increase in abiUty to ingest vital dyes. T.schaschin has illu.strated a series of transitional forms in his figure 11, Taf. vii, wliich strikingly resembles that shown in figure 8. Maximow (1916), according to Downey (1917), has even been able to bring about the develojjment of ty])ical vitally staining polyblasts from lymphocytes in ti.ssue-cultures of lymph-nodes of young

DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 33

atid adult rab})its by the addition of tissue-extracts. Similar views as to the lym- phogenic origin of the macrophages are expressed by other authors. In addition to the lymphoc\4oid cells, most workers recognize the participation, in the forma- tion of the wandering mononuclear phagocytes of inflammation, of the resting- wandering cells of the tissue-spaces, the endothelial cells, and, in the blood-forming organs, the cells of the reticulum, etc.

It is probable, therefore, that while some of the large macrophages of the inflammatory region at the fracture-site come from the reticulum of the marrow and from endotheHal cells, and that even more are mustered from the ranks of the resting-wandering cells of the surrounding tissues, yet the vast proportion develop from the small lymphocyte-like cells. Though the lymphocytes of the tissues (histiogenous l}'mphocji,es) doubtless supply some of these, by far the greater proportion probably arises from the lymphocytes of the blood-stream, which have wandered from the vessels into the inflammatory zone (hematogenous lymphocytes).

We may summarize the discussion of the source of the cells by saying that at the beginning of the inflammatory process cells of the types above mentioned are already present in the wound-area, and although these function activelj', and even increase in effectiveness, they soon become inadequate to the demands made upon them. Cells from the surrounding tissues, too, may be presumed to wander to the inflammator}' region; m these we may recognize rejjresentatives of the resting- wandering-cell type and also the closely related "histiogenous" h^mphocyte. But even these reinforcements are insufficient for the performance of the work, and the vast bulk of the phagocytes, as we have seen, come bj^ waj'^ of the blood-stream.

Thus the macrophages of the inflamed tissue in the vicinity of wounded bone, though derived from cells of diverse morphological type, are yet united by the possession of a common physiological potentiaht}' which manifests itseK in a uni- form response to a common call to arms. This response consists in the metamor- phosis of these cells into enormous and rapacious phagocj'tes and in the assump- tion bj'^ the latter of an important service in the treatment of the waste products occasioned by tissue-injury. This phagoc^'tic response is graphicalh' demonstrated by the trypanophil reaction. We must postulate a progressive adaptation on the part of the cytoplasm of the mobilized cells until there is produced a mechanism of the highest efficiency in the function of phagocytosis.

It is a striking fact that the working units of this phagoc}i;ic tissue, although derived from different sources, resemble one another so closely that the rijier forms are indistinguishable. Indeed, this fact has been commented upon by Tschas- chin (1913), who, speaking of the resemblance of the "polyblasts" derived from resting-wandering cells to those from the IjTBphocytes, states (p. 388) : "gegen Ende des zweiten Tages der Entziindung die Polj^blasten nach der Quelle ihrer Entstehung nicht mehr unterschieden werden konnen."

MOBILIZATION.

Whj^ it may be asked, do the macrophages congregate at the fracture-site? What is the influence which causes a cell situated near the area of inflammation to move toward it, or wliich brings about the dislodgment of a lymphocyte from its ordinary depot into the l)lood-stream (for we can hardly regard the blood-stream as normally containing sufficient mniibers of these cells to supply the requirements without replenishment)? The specific stimulant is undoubtedly the jiroduct of tissue breakdown. It may operate through its chemical properties, in which case its action would be described as a chemiotaxis, or its effect may be due to its pecu- liar physical condition and its influence be more accurately designated as a physi- cotaxis. Be that as it may, it is certain that the cells themselves are in some way specialized to react to this form of stimulation, not only by moving toward the source of the attraction — in the soft parts of the vicinity by ama-boid movement through the tissues, in the blood by movement of lymphocytes from their resting- places into the blood-stream and from this into the inflammatory area — but by developing prodigious phagocytic abilities.

In connection with the hematogenous lymphocytes the attraction must be thought of as acting to get the cells from their depots into the blood-stream, where the attracting influence may be assumed to be circulating. But the latter would not control their course in the blood-current, for in this the\^ float passively until they happen to reach the region of inflammation. Here the circulatorj^ conditions favor their arrest, the blood-current in the dilated capillaries being very slow. Diapedesis ensues, and the embryo phagoc^i:es are thus assembled on the field of their opera- tions. The inflammation-area, as it were, "screens out" the lymphocytes from the blood, just as, in certain types of septic inflammation, the leucocytes which are brought in the blood to the focus of inflammation are sifted out and retained there by the mechanical and other conditions wliich they encounter.

The interesting fact is noted by Tschascliin and others that the local lym- phoid elements of adenoid-tissue do not normally stain with vital dj^es and even in inflammation they are very slow in developing phagocytic power. It appears, however, that if these cells gain entrance into the blood-stream they soon become .sensitive and react promptly to the stimulus of inflammation by wandering from the vessels and becoming metamorphosed into typical "polyblasts."

ACTIVITY.

The method of action of the macrophages has long been the subject of much study on the part of cytologists. There is nothing to suggest that these phagocytes actually break down the tissue. It may be that they secrete some enzyme which a.s.sists in this proce.'JS, but no fact.s were discovered in support of tliis view. Their function is concerned with the clearing away of the waste products rather than with tis.sue solution. In the section describing the observations it has been noted that the cells are not typically in direct contact with the tissue being absorbed. !More- over, they do not seem to operate merely by engulfing fragments of the moribund tis,sue, although their ability to act in tliis way on r)ccasion is not questioned; rather, they arc concerned with the imbibition of a colloidal solution of the tissue. In this .solution they lie, their outer walls bathed with it. The way in which they absorb the material may be inferreil from the way in wliich they are known to take up colloidal dyc'stuffs (Evans and Schulcniann, 1914) . Tho ultranvicrons of such dyestuffs ill some way pormeate the cell-memhrane and are combined in agj^regations which soon become visible by the aid of the higher powers of magnification as multi- tudinous, isolated granules. It appears, too, that often an aggregation of dye- molecules is situated within a vacuole in the cytoplasm. This fact has led some workers to suppose that the cell was attempting to subject the material so housed to a form of digestion in which it would be made useful, or at least harmless, and that its treatment of this colloidal dyestuff was an example of its behavior toward any material in the same physical condition.

The same reasoning may be applied to the interpretation of the behavior of the macrophages in fractures, and we may postulate that the colloidal waste products resulting from the breakdown of the tissue are similarly acted upon within the cell economy. This may be a productive mechanism. It is a well-known fact that certain of the products of protein-splitting, if generally scattered throughout the circulation, will bring about great harm. Now it is quite jjossible that some at least of the products of proteolysis in fractures and other wounds are of this noxious character, and it may well be that the macrophages are called out to form a barrier against the escape of these materials into the general circulation. There is a stasis of fluids in these regions, and thus the conditions are most favorable for phago- cytosis (Downey, 1917). Indeed, in emergencies such as the heaUng of wounds and clearing awaj' of damaged tissue resulting from gross insults, the macrophages may be considered as expressing in an exaggerated form the same function they express every day under normal routine conditions of metaboUsm — as in the breaking-down of red blood-cells, or it may be of the protoplasm of muscle-fibers. Here it is note- worthy that Goldmann (1909) finds these cells especiallj'^ numerous in the heart — a hard-worked muscular organ. Not all tissues, however, are to be looked ujion as producing materials which are dealt with in this way; for instance, nerve-tissue contains few or none of these cells (Goldmann, 1909), and hence the functional waste- products of this tissue may be considered as being treated in some other manner.

DECLINE.

As to the fate of the macrophages of the inflammatory area, it has been noted that, shortly after the disappearance of the tissue-waste, the macrophages, in all the specimens, become less and less evident, finally dwindhng to their normal numbers (table 2). The examination of the sections throws some light on the final end of the individual cells. As early as the third day we have noted that certain of the phagocytes had a rlegenerate appearance, and these were more numerous on the fifth day. It seems evident that some of the cells start to degenerate quite early, even at the stage where new macrophages are developing and where the macrophagic tissue is, on the whole, increasing. On the sixth day the degenerate cells were present in enormous numbers, especially in the young scar- tissiie. and it is at this time that most of the phagocytes undergo dissolution. After the first week they disappear more slowly, for but few degenerate forms are noted; indeed none was seen after the twelfth day.

The morpholop;.v of tliese degenerate cells has been described in the text (sixth-day stage). It ai)i>ears that many (probably most) of the phagocytes fall to pieces, their liquefied content of ingested material (which is assumed to be changed in character) being returned to the lymph-spaces, from wliich it is passed on to the blood-stream, to be excreted or utilized; and any fragments which remain arc probably devoured by the tissue-phagocytes.

A few macrophages, however, as has been noted, persist on the field after absorbable material has disappeared. In the fractures of the long bones these were ol)ser\ed in numliers more or less above normal as far as the tliirtieth day. They haunt the surviving muscle-fibers and new scar-tissue. Their size and staining ability gradually diminish, and they are quite inferior in these respects to the active phagocytes; their numbers soon fall away to normal.

It is difficult to say, from the evidence, whether or not these cells ever become transformed into fibroblasts, as Goldmann (1912), Maximow (1902, 1906), and Tschaschin (1913) suggest. The typical fibroblasts plainly are quite different from the typical macrophages, and it seems probable that most, if not all, of the scar-tissue arises independently of the macrophages. In the fifth-daj'^ stage, for instance, when there is no jierceptible diminution in the number of macroi)hages, there is much new fibrous material. They, however, give place to scar-tissue, and, in a sense, may be said to prepare the way for the scar by assisting in the removal of waste material (Maximow, 1902).

INTRAOSSEOUS MACROPHAGES.

The second outstanding fact brought to light by the study of vitally stained, healing lione-wounds is that the reticulo-endotheUal cells of the callus-spaces develop marked phagocj'tic power coincidently with the apjoearance of the erosive processes concerned in the enlargement of these spaces; the intensity of this power, too, seems to be roughly proportional to the amount of the caUus breakdown. There is, indeed, a most obvious parallelism in the curves tracing the degree of activity of callus destruction, on the one hand, and the degree of phagocytic efficiency of the macrophagic reticulum-tissue on the other, wliich is consistently maintained throughout the entire history of the callus.

From a review of the findings in the callus up to the sixtieth day it is possible to chvide the Ufe of its macrophagic tissue, like that in the degenerate soft parts, into the three phases: development, activity, and decline. Although these phases merge gradually into one another, yet arbitrary limits may be set for them, that of development covering the first nine days, that of activity, roughly, the period from the tenth to the twentieth day inclusive, and that of decline the remaining time. Ihiving made this division, it is a simple matter to recount the most important features of each j^liase.

DEVELOPMENT.

Upon referring to the records it will be noted that the callus rapidly develops, following its first indication on the second day, and by the sixth day is quite well marked. In tliis callus, spaces filled with cells (which are derived apparently from the tissue of the Haversian canals of the old bone) early make their appearance. First seen on the third daj^, they soon expand, the largest spaces naturally occupjdng the oldest part of the callus, situated, of course, in the vicinity of the original bone.

With the hollowing-out of these spaces numbers of the reticulum cells con- tained in them gradually acquire the power to ingest and store colloidal dyestuffs. Faintly stained cells were found in the spaces in the third-day stage. This mani- festation of phagocytic activity on the part of the cells of the reticulum is intimately associated with the breaking down of tissue conseciuent ui)on the excavation of the callus spaces. It is noteworthy that the brightest staining occurs in the cells of the largest spaces, and especially in those at the edge of the old bone — an indication that the greatest phagocytic activity is resident in areas of greatest tissue destruction.

As new spaces open out, trypanophiUc cells, at first veiy weakly stained, appear in them, so that the number of these cells gradually increases, keeping pace with the growing volume of the callus reces.ses. Cell multiplication is bj^ mito.sis, as is proved by finding karyokinetic figures among them, even in dye-containing cells. Also, with advance in age of the reticulum cells, there is usuall}' a shght concomitant progressive increase in their phagocytic potentiaUty wliich goes hand in hand with the gradually accelerated callus erosion; for, in the fifth and sLxth- day stages, the cells in the older and larger spaces are somewhat more brightly stained than corresponding cells of earUer periods. No noteworthj^ increase in size of these phagoc}i;es is so far apparent. Tliin-walled blood-sinuses of large size appear in the callus-spaces of this period, and are a marked feature. A favorite position of the macrophages is between the walls of these vessels and the osseous plates.

In no area of the callus during this period is the staining phenomenon of the reticulum cells at all comparable with that of the extraosseous macrophages; indeed, the reticulum cells are {juite small and inconspicuously stained as compared with the deeply dyed "poh^blasts" of degenerating soft tissue, so that it seems more rational to look upon them as cells whose powers are as yet potential rather than actively functioning — as elements, indeed, capable of developing very efficient phagocytic ability on short notice. Though their service in phagocytizing the products of tissue breakdown during this early stage is recognized, it is felt that the amount of colloidal waste resulting from callus erosion can not be very great, since the total callus destruction is as yet small. The main feature of the callus during tliis phase is construction (rather than destruction) of osseous tissue.

ACTIVITY.

The history of the callus during the period from the tenth to the twentieth day inclusive is of the greatest interest. The first part of this interval is charac- terized by both development and destruction of bone, for as the callus expands in its more outlying regions the older parts are worn away, as shown by the progressively widening spaces. Gradually peripheral growth decUnes, it being practically non- existent by the twentieth day, but very active destruction of the trabeculse is maintained thi'oughout. During the first few days the area of most active bone destruction is in the older portions of the callus around the original bone, but as the tissue here is eroded this active zone shifts outward and at the fifteenth day it occupies the interior oi the mass. From here the zone advances, in turn, to the periphery, and the redundant material is cleared away in that region. Certain areas of bone, necessary for the stabiUty of the callus, are conserved, and these are thoroughly reinforced through the application of layer ujjon layer of bone by t lie activities of the osteoblasts.

During this jierioil the intraosseous phagocytes, as demonstrated by vital- stjiining. are exceedingly striking. In regions where obviously bone erosion is l)roceetling most vigorously they now devour the dyestuff much more greedily and store it in the form of larger and more numerous granules. In their more' intense staining they form a striking contrast to the cells of the develojimental phase. Thus, in the earlier stages of this period, at the tenth and twelfth days, the largest macrophages are found crowded in the spaces near the original bone; on the fifteenth day they have shifted to the interior of the callus, and on the twentieth day (keeping pace with the outward movement of the zone of most active bone destruction) they have again shifteil their ranks to the peripheral regions of the callus, the more central areas containing relatively few of them.

This locaUzation of the demolition-zones is not absolute, for (especially in the later stages) detachments of hypertrophied macrophages may be found in nooks and corners throughout the callus wherever bone is being actively resorbed. Again, in some of the ribs (perhaps because there was less movement of the fragments) the callus was less and its removal was accomphshed apparently b.v a process of l)aring down from the i)eriphery, the bone becoming more and more slender, as the figures of cleared ribs indicate. It is quite obvious that concentration and hyper- trophy of macrophagic tissue are inseparably Unked with active bone erosion.

As time goes on, the macrophages of the reticulum undergo, in the areas of active bone destruction, a certain amount of progressive enlargement, the largest cells having an average long (hameter of 7.0 fx, 9.15 n, 9.95 n, and 12.6 /x on the tenth, twelfth, fifteenth, and twentieth day respectively. With tliis hypertrophy there is some increase in the amount of the dyestuff stored, indicating an exalta- tion of phagocytic power. Mitotic figures in reticulum cells, many of which contiiined dye, were found throughout tliis period of activity. A few smaller and less brilliantly stained reticulum cells are found in all parts of the callus tissue.

The manner of action of the reticulum macrophages presents some features f»f interest. There is nothing to sui)port the idea that they carry on, contribute to, or even initiate the actual process of callus destruction. Their jiosition in the reticulum never in actual contact with the bony structure — does not, to say the least, lend support to any such hyjiothesis; nor is there any evidence pointing to the elaboration, by these cells, of a secretion — such as an acid or a i^roteolytic enzyme— which would act in the liciuefaction of the callus. On the other hand, there is positive evid(;nc(! of the mwt convincing kind— the avidity with which these cells ingest colloidal dyestuffs-that they play the r61(> of i)liagocyt(>s; like the i)olybla.sts of degenerating .soft ti.s.sues, or the reticulo-endotlicli.il cells of develoi)ing lione, they ingest the ])roducts of tissue breakdown.

The particular tissue-destruction with which the reticulum macrophages have to do is that of callus. In this destructive process we recognize two a.spects: (1) the removal of the bone-salts; (2) the removal of the matrix with its contained cells.

It is becoming more and more clear that the process of removal of the bone- salts from the matrix is simply the reverse of that of their deposition in ossification ; that we have here to do with the reverse phase of a chemico-physical reaction whose direction is determined solely by the conditions of the immediate environment. Under certain circumstances, apparently centering around definite and well-ordered changes in the local blood-vascular sj'stem, there are precipitated from the circu- lating fluids into a special matrix (elaborated by the activities of definite specialized cells, the osteoblasts) certain insoluble building-materials, the bone-salts. These consist mainly of calcium phosphate and calcium carbonate, their quantitative relations being determined by their relative solubilities in the blood-plasma. There is here simply a cell-controlled calcification (Wells, 1911; ]\IackUn, 1917).

In deossification, or more accurateh' decalcification, the reverse process is encountered. Changes, particularly in the circulating fluids, cause the bone-salts to be released from the matrix and again taken up into the blood; the matrix and bone-cells remain; the former is Uquefied, by means at present obscure. As to the latter, there is ground for the view that they are sometimes left in heaps Uke drift- wood; that they even coalesce to form giant-cells (Arey, 1917). It is probable that many are disintegrated, to sweU the volume of the Uquefied waste-products.

That the liquefied bone-salts are phagoc^'tized by the macrophages seems doubtful. They are non-toxic and probably pass off into the circulating blood in a manner the reverse of their incoming. It does seem probable, however, that the phagocj-tes ingest some, at least, of the products resulting from hquefaction of the protein-content of the callus. Their function would thus be closely aUied with that assumed for the wandering macrophages of the degenerating extraosseous tissue. Here, too, it may be postulated that theu- service is protective; that they guard the organism from the harmful effects of toxic, nitrogen-containing compounds re.sulting from proteolysis. It is quite possible that only certain of the compounds arising from tissue breakdown are poisonous, and that only these are ingested. As in the case of the polyblasts, it may be assumed that the materials so phagocytized are digested and rendered innocuous — or even useful — within the cj^toplasmic laboratory of the macrophages.

It has been noted that the treatment by the macrophages of high-molecular dyestuffs, such as trypan-blue, is an expression of theu general behavior toward any material in the same physical condition; and it has been inferred, therefore, by Shipley and IMackhn (1916'-), that the material resulting from the erosion of pro- visional cartilage and bone, which is phagocj' tized by the macrophages, is in a finely dispersed state. Such an inference may also be made for the waste products resulting from the erosion of callus or from the breakdown of the soft tissues around the wounded bone.

The position of the macrophages of the demolition zones in the loose reticulum of the spaces is favorable to the exercise of then function, for they, hke the pol}'-

40 i)km;i.<)PMENT and function of macrophages in bone-repair.

blasts of moribund tissue, arc bathed by fluid containing colloidal waste products. EsiK-cially in the j)erisinusoidal spaces (where, as has been many times observed, the phagocytes are often thickly crowded) are they well situated to gather in waste products; for here not only do they have access to the materials coining directly from the dissolving tissues, but, owing to the thinness of the vessel-walls and the slowness of the circulation, diffusion of katabohtes from the sinuses into the spaces may easily occur, so that these macrophages are in a position to gain some of their pabulum from the blood-stream. In any event, toxic materials in the blood- sinu.ses are readily extracted by the phagocytic endothehal cells.

The enormous thin-walled blood-sinuses of the callus, which have been repeat- edly referred to, are a conspicuous feature of areas of active bone-demoUtion. They arise, as has been .shown, through coalescence of smaller vessels. So striking are they on account of their enormous size and great number, as well as because they are invariably present in areas of active callus destruction, that the conclusion is forced upon us that they must play an important part in the breaking-down and removal of the redundant osseous tissue. Certain it is that the rate of flow in these vessels is very slow and that, judging from the thinness and insecurity of their walls, the pressure is very low. It is i)robable, too, that the CO2 tension is high.

It is significant that a very voluminous blood-current of slow speed and low pressure, flowing through a thin-walled channel, enveloped thickly with phagocytes of high-grade efficiency, is so constant a feature of callus resorption, as it is also of the resorption of young developing bone. It suggests that this is part, at least, of the mechanism of l)one erosion.

DECLINE.

During the tliird phase, after the twentieth day, osseous resorption gradually ebbs and the destructive and constructive activities of the callus slowly subside. Reinforcement of the permanent trabecuhe is the principal industrj^ of the cells, but, esjx'cially in the earlier days, evidence of the work of the wrecking-gang is still seen in the clearing away of the few remaining provisional spicules of bone and the trimming of the rough corners. The result is a firm osseous structure formed of material almost indistinguishable from the bone of the original shaft, containing relatively few (but very large) spaces filled with vascular and marrow tissue. This bony formation occupies the fracture-site and thoroughly immobihzes the shaft of the bone.

In keeping with this falling away of destructive processes the macrophages gradually decline in size, concentration, and staining activity, and revert to the character of the ordinary bone-marrow reticulum cells. This does not take place uniformly in all stages, however, for in the specimens of the sixtieth day there was still evidence of bone erosion as well as of bone building, whereas in those of the fifty-first and fifty-ninth days these i)rocesses had ai)parently completely ceased. Involution and degenerate forms, hke those of the macrophages of the soft parts, were not found in the callus-si)aces.

Carlilagc was found almost constantly in the callus of the long bones and seems to be a.s.sociatcd with movement of the parts during repair, since they were not splinted. It wa.s not found in any of the skulls, where movement was absent. In the older stages the cartilage underwent changes similar to those of ossification of cartilage in normal skeletal developrntrnt. A case was found where the cartilage had even taken all the characters of a typical epiphysial plate. A-ssociated with the process of destruction of the cartilage, and of the trabeculie of bone built upon the calcified remnants, macrophages played the same part as in the normal endo- chondral ossification, as shown by Shipley and MackUn (1916-).

Giant-cells or "osteoclasts" were found not infrequently in the callus of some of the specimens, and rarely in others. Their numbers seemed to have no relation to the amount of bone destruction; thus none was observed on the tenth day, a few on the twelfth, they were fairly numerous on the fifteenth, but scarce on the twentieth day. In all of these stages there was undoubtedly a great deal of bone erosion progressing. Again, at the same stage but in different specimens, they were inconstant in number; thus in one of the specimens of the six-day stage there were very few, while in the other specimen a fair number was found. In no case was there a large enough number, nor were the cells sufficiently well distributed, to warrant regarding them as the agents of bone erosion. Careful search was made for dye-granules in them, but not a trace was to be found; hence they are not phago- cytes of the tj'pe of the macrophages. Indeed, no ingested material of any kind could be found in them. Thus the giant-cells of the callus and the underlying old bone in process of erosion are in these respects quite the same as the giant-cells of developing bone described by Shipley and MackUn (1916-).

COMPARISON OF EXTRAOSSEOUS AND INTRAOSSEOUS MACROPHAGES.

It is of interest to compare briefly the macrophagic cells of the callus with those of degenerating extraosseous tissue. They have maii}^ points of difference. The callus cells are fixed and belong to the reticulum tissue, whereas the cells of the soft parts are mostly wandering and are derived from the "resting- wandering cells" of the tissues and from certain lymphocjle-like cells brought in the blood-stream. It is probable, however, that cells originating from the reticulum and endotheUum of the bone-marrow of the broken bone-edges contribute to the forces of the extra- osseous macrophages in their vicinity. The callus cells develop in situ, whereas the cells of the soft parts begin their development probably for the most part out- side the zone of their operations; however, both no doubt undergo their hypertrophy mainly at the site of tissue-breakdown. The callus-cells multiply in situ bj^ mitosis (typical figures being found in dye-containing cells as early as the sLxth day and as late as the twentieth), whereas most of the cells of the soft parts undergo multipU- cation at their source. The largest of the extraosseous macrophages are of greater size than the phagocytes of the callus, and usually contain more dyestuff, their granules, as a rule, exceeding in magnitude those of the callus-cells.

It seems probable that the demands upon the macrophages of the soft parts are much heavier than those which the callus-phagocjiies are called upon to meet, the waste material being much greater in the damaged soft parts than in the resolv- ing callus. Hence the cells of the callus never reach the phj^sical proportions nor the great numbers of the cells of the soft parts. The effort of the extraosseous cells is siulden, vigorous, and of short duration, their jjeriod of maximum efficiency being from the tliird lo the sixtli days inekisive; wliereas the effort of the intraosseous elements begins later, is more gradual in onset and less violent, and is of longer duration, the jx'riod of maximum efficiency of these cells ranging from the tenth to the twentieth days. Both types, however, are apparently stimulated to hyper- troi)hy and functional efficiency by the same type of material— waste resulting from iiroteolysis; but the call is ajiparently much more sudden, forceful, and per- emjitory in the soft parts than in the callus, so that here an expeditionary force of potential macrophages from distant regions must be rushed in. The needs of the callus, on the other hand, in the matter of its resorption, are apparently served adequately by the exaltation of the powers of the resident phagocytes. Both macrophagic types are concerned in hke manner in the treatment of dissolved tissue-waste, and they are thus physiologically similar. In both types phagocytic activity has been developed coincidently with the occurrence of tissue breakdown. Both have a reconstructive as well as a scavenger function, for they prepare the ground for scar-tissue or permanent callus, as the case may be. Most of the cells of the soft i)arts perish in situ, while the fate of the callus-cells is obscure; no involution forms, however, were found among them.

Macrophages were found in healing wounds of membrane-bone, not only in the surrounding soft parts but in the spaces of the callus itself. Here the cells were of the reticulum type and were related to blood-sinuses as in the long bones. The callus of membrane-bone was rather slower in develojjment and was relatively small in volume, so that the pictures presented by callus in the trephine-wounds of the skull were not so striking and instructive as those in the long bones. Thej^ were useful, however, in confirming the findings in the long bones.

It is worthy of emphasis that the method of vital-staining, as appUed to developing callus on the one hand and to developing bone on the other, demonstrates very f(jrcibly that the same cellular elements are at work in the performance of a like ta.sk. ^Macrophages of identical morphological type are found in mass forma- tion in each case, intimately related to areas of bone or cartilage resorption, and for them a common physiological significance is claimed — that of phagocytizing the products of disintegration of provisional cartilage and bone. ISIore than this, there was found absolutel}^ no trace (in the osteoclasts of either callus or developing bone) of any phagocytic activity. Thus the conclusions of Shipley and ]Macklin (1910-) in regard to develojjing bone are upheld by the findings in callus.

From an examination of all the cleared and sectioned skulls it may be said that no difference can be discerned in the staining reactions consequent upon putting the in.sert back right side up, or upside down, or inserting living or dead bone from another rat, or even foreign dead bone. Thus the macrophages, as far as could be made out, behave ahkc toward all these types of insert. In no case was the insert removed, although the edges were trimmed and rounded off; and, alth(nigh here and there in some of the older skulls there was some slight evidence of erosion in the inserts and in the surrounding bon}^ edges, it is remarkable how little of the in.sert disa])pcars. The absence of an increased macrophagic tissue, after the debris has been cleared up, is what would be expected, since so Uttle of the insert is eroded.

A note may here be made as to the condition of the healing areas of skin over the trephined bones. Several of these were cleared and some were sectioned. It was found that macrophages are increased in certain places, especially around the suture holes, in the earUer stages. This was noticed in the skulls of the following days: s?cond (S 18-1), ninth (S 17-2), tenth (S 5-1), and twentieth (S 12-2). Thus the macrophages appear to have a function to perform in the solution of the sutures and probably in the repulsion of infection as well. In the actual scar-tissue the trypanophil phagocytes are few in number. It is of interest to observe that Gold- mann (1912, p. 80) noted a similar increase of macrophages in healing skin wounds of the rat.

CONCLUSIONS.

The following conclusions are based solely upon investigations with the rat. It seems probable, however, that they would hold good in general principle for the other mammals, and no doubt for manj' of the lower forms.

In the heaUng of bone-wounds, macrophages, which stain brilUanth' with trypan-blue, soon congregate at the site of the injury and become very numerous, hypertrophied, and of increased phagocytic power. They assist in deahng with the tissue-waste resulting from the trauma. These phagocytes are developed prin- cipally from the lymphocj^e-Uke cells from the blood-stream, but also from local mononuclear cells with phagocji^ic potentiaUties. INIost of them ultimatelj' dis- integrate in situ.

During the structural changes attending the transformation of provisional into permanent callus, trypanopliilic macrophages develop in the callus-spaces from the reticulum cells and become numerous, large, and phagocj'tic. They function in the removal of redundant bony spicules, their particular role being con- cerned with the absorption of the waste products from the breaking down of the matrix. When cartilage is present in the callus they also play a part in its removal. Their action here is thus the same as that of the macrophages of developing bone.

The macrophages of soft parts and bone, though morphologically different, are physiologically similar. They phagocytize the products of proteolysis and segregate the material within their cytoplasm, where they probably subject it to a form of digestion.

Limited numbers of polymorphonuclear leucoc>i;es were encountered among trypanophiUc macrophages in areas undergoing repair. No dye-granules were found in them. Physiologically they are distinct from the macrophages.

The osteoclasts of the callus did not show dye-granules. Their numbers did not bear any relation to the apparent amount of bone-destruction which was going on. Osteoblasts, too, contained no dye-granules.

BIBLIOGRAPHY.

Aret, L. B., 1917. On the origin and fat* of the ostoo- cla^sts. Anat. R'.Tor(l, vol. 11, p. 319.

D»>WNBr, H., 1917. Reactions of blood and tissue cells to acid rollolihil ilycs under pxperimentiil condi- tion-s. An:it. R.'oonl, vol. 12, p. 429.

EvAN.s, H. M., and W. Soiiulf.m.wn, 1914. The action of vital stains ln-lon:.;ing to the benzidine group. Scienc3, n. s., vol. 39, p. 443.

Evans, H. M., F. B. Bowm.vn, and M. C. Winternitz, 1914. An exi)?riin;'ntiil studj- of the histogenesis of the iniliarv tubercle in vitallv stained rabbits. Jour. Kx|)er.".\Ie<l., vol. 19, p. 283.

EvAN'!«, U. M., 1915. The macrophages of mammals. .\mer. Jour. Physiol., vol. 37, p. 243.

GoLOMA.NN, E. E., 1909. Die ilussere und innere Sekre- tion dea gesunden Organismus im Lichte der vitalen Farbung. Beitr. z. klin. Chir., Bd. 64, p. 192.

GoLDUAN.N, E. E., 1912. Neue Untcrsuchun!?en iibcrdie aussere und innere Sekretion des gesunden und kranken Organismus im Lichti' der vitalen Fiir- bung. Beitr. z. klin. Chir., Bd. 78, t. 2, p. 1.

MacCordy, J. T.,and II. M. Ev.\n.s, 1912. Experi- mentelle Lasionen des Centralnervensystems, untersucht mit Hilfe der vitalen Fiirbung. Berl. klin. \Vi-hn.schr., vol. 49, p. 1695.

M.4CKLIN, C. C, 1917. Studies in calcification by the use of vital dyes. Jour. Med. Research, vol. 36, p. 493.

Maximow, .\., 1902. Experimentelle Untersuchungen iilxjr die entzundliche Neubildung von Binde- gewebe. Beitr. z. pathol. Anat. u. z. allg. Pathol., Bd. 5, Suppl. p. 1.

1908. Ober die Zellformen des lockoren Binde-

gewebcs. Arch. f. mikr. Anat., Bd. 67, |). 680.

Maximow, A., 1907. Uber die Entwicklung der Blut. und Bindegewebszellen beim Saugetierembrj'o- Folia Haematol., Bd. 4, p. 611.

1909'. Untersuchungen iiber Blut und Binde-

geuebe. I. Die friilicstcn F.iitwickhing.sstadien der Blut- uml Biinlcgewebszcllcii licim Siiuge- tierembryo bi.s zuiii .Viifani; der Mhitbildung in der Ixibcr. Arch. f. mikr. Anat., Bd. 73, p. 444.

1909*. Die Ilistio^enes,? der Entziindung. Ver-

handl. d. 16. Intern. Med. Kongr. Budaix-st 1909, Sekt. 4I>. (cited by Tschiuschin, 1913).

1916. The cultivation of connective tissue of

adult mammals in vitro. Arch. Rus-ses d'Anat., d'llistol. et d'Embryol., t. 1, f:isc. 1 (cited by Downey, 1917).

Ranvier, L. a., 1899-1900. Des chismatocytes. Arch. d'.-Vnat. micr., t. 3, p. 122.

Spalteholz, W., 1914. Uber das Durchsichtigmachen von menschlichen und tieri.schen I'riiparaten. Leipzig.

SHrpLKY, P. G.; and C. C. Macklin, 1916'. The demon- stration of centers of osteoblastic activity by use of vital dyes of the benzidine series. Anat. Record, vol. 10, p. 597.

Shipley, P. G., and C. C. Macklin, 1916'. Some features of osteogenesis in the light of vital staining- Amer. Jour. Physiol., vol. 42, p. 117.

T.scHA.scHiN, S., 1913. tlber die "ruhenden Wander, zellen" und ihro Bezeihungen zu den anderen Zellformen d&s Bindegewebes und zu den Lj'm- phozyten. Folia Haematol., Bd. 17, p. 317.

Weli.s, H. G., 1911. Calcification and ossification Arch. Int. Med., vol. 7, p. 721; also Harvey Lecture.?, 1910-1911.

DESCRIPTION OF FIGURES.

6, original bone. M, macrophage. N, nerve.

US, blood-sinus. MC, marrow-cavity. P, periosteum.

C, callus. Mxa., muscle. S, scar-tissue.

Plate 1.

Fig. 1. Portion of normal rib from rat vitally stained with trj-pan-bluo. The black dots represent macrophages

normally present in the marrow-cavity, periosteum, and tissue around the bone. X 27. This and the

next five figures are free-hand drawings from gross cleared preparations under the binocular microscope.

The macrophages arc represented somewhat larger than they actually are, for the sake of plainness. Fig. 2. Fractured rib of rat on second day of repair. Note the d6bris around the ends of bone and the slight increase

in macrophages as compared with figure 1. X 27. Fio. 3. Fractured rib of rat on third day of repair. Great increase in macrophages is seen, with swelling due to

young callus. Diffusely stained debris present. X 27. Fio. 4. Fractured rib of rat on ninth day of repair. Macrophages somewhat less numerous. Callus much more

dense. X 27. Fig. .5. Fractured rib of rat on twentieth daj' of rei)air. The contour of bone is almost normal, the medullary cavity

is being restored, and macrophages are but little in excess over the normal. X 27.

PL.4.TE 2.

Fig. 6. Trephined skull of rat on ninth day of repair. .\ bone-dLsk has been inserted on the left side, while the right has been left open. Note the crowds of macrophages in the open spaces and around the bone edges. Here and there are areas almost free from phagocytes; these are patches of scar tissue. Early callus spicules line the bone edg&s. X 19.

Fig. 7. Photomicrograph of area near broken end of long bone from rat on third day of repair. The edge of hone (B) and the fragments of dead tissue are stained blue. MjTiads of macrophages (M) are found through- out the figure. X 40.

Fig. 8. A few cells selected from specimen from which figure 7 was made, showing the development of the large macrophages from the small lymphocji.e-like cells. As a rule, more and more dyestuff is taken up with increase in size of the cell. Besides the dye the cells also contain the tissue-waste which they have phagocytized. Free-hand drawing. X 1,000.

Plate .3.

Fig. 9. Photomicrographs from a cleared and uncounterstained section of the sixth-day stage in fracture-repair. Exactl}' the same field is seen in the two pictures, but that on the left received a much longer exposure, so that practically the only objects seen in it are the macrophages. These are larger and more thickly distributed in the central and lower right regions. Here the greatest amount of diffusely stained tissue- waste is present, as is seen by the denser staining in this region in the right-hand picture. The field is from an area of degenerating muscle. Above and to the right is scar-tissue (S), while below and to the left the muscle is beginning to deteriorate. In the central and lower right areas the muscle is degen- erate, and here the macrophages (M) are largest and most numerous. X .50.

Fig. 10. High-power drawing (camera lucida) of area of degenerating muscle from a region similar to that shown in figure 9 {M). Fragmented muscle-fibers are .seen, together w-ith young fibroblasts, polymorphonu- clear leucocytes, and macrophages. Three small lymphoc\-toid cells appear. Some of the macro- phages contain more blue dye than others; the latter are usually stuffed full of phagocjtized material. One small macrophage has engulfed a poIjTnorph. X 1,000.

Fig. 11. .\ few degenerate macrophages from an area of scar-tissue, similar to that shown in figure 9 (5). They are of different sizes. Some appear vacuolate and ragged; others are mere fragments. They contain comparatively Uttle dye. Some young fibroblasts are shown. X 1,000.

Fig. 12. A portion of a callu.s-space from the sLxth-day stage. A thin-walled blood-sinus {BS) is conspicuous, and between its walls and the bone (C) are situated reticulum cells, some of which contain dyestuff {M) and are thus phagocj-tic. X 1,000.

Fig. 13. Three reticulum phagocytes from the callus of the sixth-day stage, showing mitosis in dye-containing cells. There are two metaphases and an anaphase. X 1,000.

Pl.-vte 4.

Fig. 14. Old bone (S) and callus from the tibia of the tenth day. Camera-lucida drawing, from cleared section. The spaces near the original bone are filled with the large.st and brightest macrophages (A/). Under high magnification these large phagocrtes are shown in figure 1.5 a. Here, too, there are blood-sinuses. Farther out the macrophages are less conspicuous and are absent at the peripherj-. X 80.

Plate 4 — Continued.

Fig. 15. The Bnni|>s a, b, c, and (/ (see plates) were drawn with the aid of the camera hicida from typical large pha- ROO.vtic n-ticulum cells selecte<l from the callus spaces of the tenth, twelfth, fifteenth and twentieth days resiMTtivcly. The sections were cleared without counterstaiiiiiig and the dyi-granule contour only is represented. In the interior of the cell the nucleus is indicated by the clear space. Only mature phagocyti'S were selected in each stage. They resemble one another closely and show a certain amount of increase in size with age of callus. In each case the drawings were made from cells situated in areas where active osseous resorption was going on. X 1,000.

Fig. 16. A space in callus, showing developing macrophages of the reticulum (M). Camera-lucida drawing from peripheral region of callus at the twelfth day of repair in long bone. Some of the cells are clear, while others contain varying amounts of dyestuff. The granules are at first small, but gradually increase in size and number. I^arge intercellular spaces will be noticed. Blood vessels are not shown in this figure, but in the older spaces at this stage they were large and abundant. X 1,000.

Fig. 17. Drawing made from a tracing from a photomicrograph of the twentieth-day stage of repair. Section cleared without counterstaining. In the lower left corner the old bone and marrow cavity is seen. Around it the callus is arranged, and in many of its spaces there are crowds of black granules, representing macrophages of the reticulum. X J>0.

Fi<;. is. .\ small field from the calliLS of figiu-e 17, much enlarged. Drawing traced from a photomicrosraph. Throngs of macrophages (A/), heavily loaded with dye-granules, present themselves between the osseous tra- becube (C). These cells arc seen more highly magnified in figure 15d. X 190.