THE HISTOLOGICAL REMODELLING OF ADULT BONE.

AN AUTORADIOGRAPHIC STUDY

P. LACROIX Institute of Anatomy, University of Louvain

THE main purpose of this study has been to take advantage of isotope techniques to obtain a better knowledge of the histological remodelling of the adult bone. Furthermore, it was hoped that the research would provide some information on bone matrix.

Compact bone in the dog Microradiography has certainly been one of the most useful

tools in recent studies on compact bone (Engstrom, 1949; Amprino, 1952; Amprino and Engstrom, 1952). The pictures provided by this method are now familiar to all of us. They prove that calcification of compact bone occurs in two stages. At first, about three quarters of what will be the final load of calcium is stored by the osteon (Haversian system) while i t is built up. Then, after ostcogenesis has ceased, calcification of the ostcon is slowly completed.

The microradiograph docs not tell the whole story of the formation of an osteon. I n fact, calcification begins in a layer which already existed some time before. The information is obtained by comparing the microradiograph with the histo- logical picture (Fig. 1) . Two concentric zones may be ob- served in an osteon which is being laid down. The inner zone is seen only with the microscope (long arrow of Fig. 1 ~ ) . Since it does not appear on the microradiograph (Fig. l ~ ) , its calcium content is nil or very low. The peripheral zone (short arrow of Fig. 1 ~ ) corresponds to a rather heavy fixation

56

Bone Structure and Metabolism G. E. W. Wolstenholme.Cecil,ia M. O'Con,ner

Copyright 0 1956 Ciba Foundation Symposium

THE HISTOLOGICAL REMODELLING OF ADULT BONE 87

of calcium. It is obvious that the combined illustration expresses the sequence of events.

Histological observations have been made on ground sec- tions decalcified in a solution of sequestrene a t pH 7.

The inner zone, which we shall call the preosseous layer, is orthochromatic with toluidine blue, it is PAS and Bauer positive, it takes selectively Alcian blue and ruthenium red and it is made metachromatic by the action of sulphuric and chromic acids; methylene blue extinction occurs a t p H 4. The peripheral zone is metachromatic and methylene blue extinction occurs a t pH 5 . 6 . Most of these features are those of mucopolysaccharides.

In the fully deposited osteon, rnetachromasia is inversely proportional to the content of calcium.

This histological study (Vincent, 1954) is a development of previous remarks made from a comparison between the microradiographic and the histological pattern of undecalci- fied ground sections (Cohen and Lacroix, 1953).

In a short note on bone growth in rabbits and rats, Arnold and Jee (19544 have reported some observations which are in good agreement with the preceding description.

The main point seems to be that a new layer of bone changes its orthochromasia into metachromasia when i t begins to manifest a strong affinity for calcium. Related observations have been made by Levine and co-workers (1949).

Let us go one step further and compare microradiographs with autoradiographs. With the relevant histological ob- servations in mind, we are in a better position, and the micro- radiographic reference will, from now on, mean much more to us than a mere map of calcium deposits.

The first isotope that we used was 45Ca (Lacroix, 1952,1953, 1954). Seven days after administration to an adult dog, the areas of radioactivity (Fig. 2 ~ ) , when superimposed on the microradiograph (Fig. 2n), are found in the layers which begin to calcify. With adequate methods, a very faint radio- activity may be detected in the osteon5 which are completely built but not yet fully calcified. The distribution in vivo of

38 P. LACROIX

45Ca, therefore, entirely confirms what the microradiograph indicates about the dynamics of calcification : very fast a t first, very slow afterwards.

Analogous autoradiographs have been recorded with 32P (Engfeldt, Engstrom and Zetterstroni, 1952) and with 90Sr (Jowsey, Owen and Vaughan, 1953; Engfeldt et al., 1954).

Autoradiographs obtained seven days after administration of 3sS are similar a t first sight to those produced by 45Ca, but they are not absolutely identical. Here, the areas of radio- activity (Fig. 3 ~ ) are surrounded by the layers which begin to calcify (Fig. 3~), instead of corresponding exactly to them; in other words, they belong to the preosseous layers, the exist- ence of which is proved by the observations exemplified by Fig. 1.

The life of adult bone implies therefore a metabolism of sulphur which precedes that of calcium. Whether the former directs the latter is open to speculation (cf. on this particular point the suggestions of Neuman, Boyd and Feldman, 1952).

From the foregoing, some conclusions have been drawn pertaining to the value of radioactivity measurements.

If we compare the specific activities of samples of bones, free of all traces of marrow, seven days after 3sS administra- tion, the figures will give directly a relative estimation of the osteogenesis going on in the samples.

Similar measurements with 45Ca amount to comparing the calcification which occurs in the samples. Calcification follow- ing in the wake of osteogenesis, we may deem that the measure- ments will also mean indirectly a relative evaluation of osteogenesis.

The short-term localization of 3% in the preosseous layer, which is orthochromatic, is a t variance with the histological localization found in other tissues. All the observations recorded up to now (cf. Bostrom, 1953) indicate a close relation- ship between 35S incorporation and metachromasia. We shall see presently that, in a may, the relationship holds true for bone also, because the radioactive layer will acquire metachromatic properties some days later.

FIG. 1. An example of correlation between microradiography (A) and histo- logy (B) in the study of compact bone. During the process of deposition, an osteon is made up of an inner zone (long arrow), the preosseous layer, which does not appear on the microradiograph because i t is uncalcified, and of a peripheral zone (short arrow) which calcifies. The inner zone is orthochromatic and the peripheral zone is metachromatic ( x 167).

F ~ G . 2. Distribution of 45Ca in compact bone seven days after administration, as shown by an illustration combining the autoradiograph (A) and the corre- sponding microradiograph (B). The radioactivity belongs to the layers which

begin to calcify ( x 85) . [facing page 38

FIG. 3. Distribution of 35S in compact bone seven dayr after administration. The pattern of radioactivity ( A ) is not identical with that of Fig. 2 A. The radioactivity belongs now to a structure which is not shown by the micro- radiograph (B), most likely to the preosseous layer. the existence of which is

proved by Fig. 1 ( x 85) .

FIG. 4. Autoradiograph (A) of compact bone, six weeks after 35S adminis- tration. The radioactive layers, i.e. the layers labelled at the time of injection, are now a t the periphery of osteons which, according to their calcium content

(B), have just been deposited ( x 105).

FIG. 5. Autoradiograph (A) of compact bone, eight weeks after 35S adminis- tration, with corresponding microradiograph (R). Some osteons (e.g. those indicated by arrows) may have practically the same calcium content altliougli

they are not contemporary ( x 93).

FIG. 6. Autoradiograph (A) of compact hone, eighteen weeks after 35S ad- ministration. Even a t this later stage, some radioactive osteons are not yet

fully calcified ( x 113).

FIG. 7. Autoradiograph (A) of metaphyseal trabeculae, seven days after * T a administration to an adult dog. The corresponding microradiograph (B) shows that the radioactivity belongs to the layers which begin to calcify

( x 114).

FIG. 8. Autoradiograph of a longitudinal section of the upper tibia1 extremity, seven days after *jCa administration to an adult dog. The radioactive layers

are located almost exclusively in the metaphysis ( x 4.7).

THE HISTOLOGICAL REMODELLING OF ADULT BONE 39

Six weeks after 3 5 administration (Fig. 4), the correlation is easy to read, if we admit that the initial distribution of the isotope has not been substantially altered, that, in other words, the radioactive layers are still those which were labelled a t the time of injection, Fig. 4 shows that the radioactivity is now at the periphery of osteons, which, according to their low calcium content, have just been built. The case seems fairly typical and we may say that the deposition of an osteon takes about six weeks in the adult dog. This is only a rough esti- mation because the process of remodelling may sometimes stop completely for a while.

Working with 45Ca, Arnold and Jee (1953, 19543) and Jee and Arnold (1954) indicate that the deposition of an osteon is generally a matter of more than three weeks in the growing rabbit.

Eight weeks after injection of the isotope (Fig. 5 ) , we begin to realize that some osteons (e.g. those indicated by arrows) may have reached practically the same level of calcification although, according to the autoradiograph, they are not contemporary. This means that the completion of calcifica- tion is so slow that we cannot rely on the microradiograph to ascertain the relative age of two fully deposited osteons.

At the eighteen-meek stage, some of the radioactive osteons are almost fully calcified, but some others (Fig. 6) do not show much progress froin the preceding stages.

Since Figs. 4-6 prove (by comparison with Fig. 1) that the radioactivity belongs now to metachromatic layers, i t appears that a relationship between 35S-incorporation and metachromasia exists indeed in adult bone but is more dynamic than static. This is an additional indication in favour of a basic function of mucopolysaccharides in bone metabolism.

One may surmise that an adult dog, given 35S a t regular and short intervals during six weeks and killed three weeks afterwards, would have most of the bone radioactivity in metachromatic regions. The biochemist who could extract from the bones of such a dog a radioactive identifiable

40 P. LACROIX

compound would provide us with valuable new data on bone matrix.

It is encouraging to note that dong the same line of work i t has been found possible to extract from the young epiphysis (Dziewiatkowski, 1951), from the costal cartilage (Bostrom, 1952) and from the skin (Bostrom and Gardell, 1953), a radioactive mucopolysaccharide, in these cases chondroitin sulphuric acid.

Engfeldt and Hjertquist (1954) have already shown that 35S is incorporated in the organic portion of bone and also in the inorganic portion. BClanger (1954) has suggested a similar distribution. Furthermore the presence of mucopolysacchar- ides in bone has been recognized for many years (cf. Glegg and Eidinger, 1955).

Cancellous bone in the dog I n research on cancellous bone, the main problem was to

prepare sections which would be even and thin enough t o be microradiographed. The difficulty has been solved by em- bedding the specimens in methylmethacrylate and then by sawing slices which were ground like compact bone.

The microradiograph shows that the trabeculae of cancell- ous bone are made up of layers with varying calcium content. The general design may be likened to that of compact bone.

The distribution of 45Ca (Fig. 7) and of S5S seems to obey the same laws in both types of bone tissue.

It seems also that the deposition of new cancellous bone implies two stages : the formation of a preosseous, uncalcified, orthochromatic layer, and the sudden appearance in this layer of metachromatic properties linked with a strong affinity for calcium.

AS a matter of fact, all these features are much more easily observed in the metaphysis than in the epiphysis. The difference is such that sometimes the radioactivity is found almost exclusively in the metaphysis, as is shown by Fig. 8, a 45Ca autoradiograph of the upper extremity of the tibia. Comparable aspects have been observed with 35S.

THE HISTOLOGICAL REMODELLING OF ADULT BONE 41

Knowing now the significance of S5S and 45Ca distribution in bone, we may safeIy state that the histological remodelling is more active in the metaphysis than in the epiphysis.

The higher specific activity exhibited by whole extremities, as compared with the diaphysis, is therefore an average which does not take into account the difference which we have just pointed out.

But the ratio between epiphysis and metaphysis put to- gether, on the one hand, and diaphysis, on the other hand, rough as i t is, remains nevertheless useful. Since, on the whole, the remodelling is more active in the extremities than in the diaphysis, i t follows that, in the long run, the specific activity of the extremities should fall at a faster rate than that of the diaphysis.

Data are still meagre on this question. Lontie (1953) has observed that in the adult rabbit injected with 45Ca, the proportional distribution of specific activities in the long bones does not change significantly in a period of about two hundred days. It seems therefore that the histological remodelling, i.e. the complete destruction of old bone and its replacement by new bone, is very slow indeed.

The subject deserves further study in relation to current theories on the r81e of the skeleton in calcium homeostasis. It is now technically possible to figure out the amount of bone which is morphologically renewed in a given time. A compari- son with the turnover of 45Ca would be of much significance and might throw some light on the way in which calcium is removed from the skeleton.

It may be fitting t o recall here that the bone marrow shows some histological individuality in the epiphysis and in the metaphysis and that both regions derive their main blood supply from distinct sources (Trueta and Harrison, 1953 ; Judet et al., 1954).

These considerations are admittedly hampered by the fact that all available information comes from animals of different species and sizes. They apply of course to the adult only and not to the growing skeleton where the situation is quite

42 P. LACROIX

different (cf., on this latter point, Lacroix, 1951; Bauer, Carls- son and Lindquist, 1955).

Human bone In adult human bone, the formation of a preosseous layer

is less easily observed than in the dog. On the other hand, the relationship between the metachromatic properties of an osteon a t the successive stages of its formation and its affinity for calcium is more obvious.

Incidentally, we may confirm the statement of Engstrom and Engfeldt (1953) that, in the osteon itself, the calcium content is alternatively low and high from one lamella to the other. The differences fade out when the osteon is fully calcified. The observation seems to meet those made with the electron microscope by Rouiller and co-workers (1952), but it must be admitted that the bridge between both sets of facts is still lacking.

Generally speaking, comparison between man and dog indicates that the dog is a good experimental animal for research aiming at results which may be valid also for man.

Remarks We hope that our survey will have shown that compact

bone has several advantages of its own in the study of prob- lems of bone physiology and pathology. The material lends itself easily to correlated microradiographic, autoradiographic and histological examinations. It shows in the same field the expression of stages where the metabolism of various elements may be visualized a t the microscopic level. The succession of events being slow, it presents opportunities to dissociate from each other processes which possibly overlap elsewhere.

The interest of cancellous bone is different and lies in the fact that the adult keeps something of the structural differ- ences prevailing during growth between epiphysis and metaphysis. The observation might lead to an understanding of the acute post-traumatic osteoporosis of the adult, the

THE HISTOLOGICAL REMODELLING OF ADULT BONE 43

first stage of which is a radiographic translucency of the metaphysis.

Acknowledgements This presentation is based chiefly on unpublished research carried out

in the author’s laboratory by J. Vincent who will give a full account of his work in a monograph due to appear later.*

The author and his co-workers wish to acknowledge with thanks the help they have received from the Institiit Interimiversitaire des Sciences Nucle‘aires of Belgium.

REFERENCES AMPRINO, R. (1952). Z. Zellforsch., 37, 144. AJIPRINO, R., and ENGSTROX, A. (1952). Acla anat., 15, 1. ARNOLD, J. S., and JEE, W. S. S. (1953). Anat. Rec., 115, 276. ARNOLD, J. S., and JEE, W. S. S. (1954~). Anat. Rec., 118, 373. ARNOLD, J. S., and JEE, W. S. S. (19543). Stain Techn.. 29, 49. BAUER, G. C. H., CARLSSON, A,, and LINDQUIST, B. (1955). Kungl.

Fysiograjiska Sallskapets I Lund Forhandlingar, 25, 1. BELANGER, I,. F. (1954). Canad. J . Biochem. Physiol., 32, 161. BOSTROM, H. (1952). J. biol. Chem., 196, 477. BOSTROM, H. (1953). Ark. Iiemi, 6, 43. BosTROhf, H., and GARDELL, s. (1953). Acta chem. scand., 7, 216. COHEN, J., and LACROIX, P. (1953). Lab. Incest., 2, 447. DZIEWIATKOWSKI, D. D. (1951). J. biol. Chem., 189, 187. ENGFELDT, B., BJORNERSTEDT, R., CLEMEDSON, C. J. , and ENGSTRON,

ENGFELDT, B., ENGSTROM, A., and BOSTROM, H. (1954). Ezp. Cell Res.,

ENGFELDT, B. ENGSTROM, A., and ZETTERSTRORZ, R. (1952). Biochim.

ENGFELDT, B., and HJERTQUIST, S. 0. (1954). Acta path. microbiol.

ENGSTROY, A. (1949). Acta radiol., 31, 503. ENGSTROM, A., and ENGFELDT, B. (1953). Experientia, 9, 19. GLEGG, R. E., and EIDINGER, D. (1955). Arch. Biochem. Biophys., 55,

JEE, W. S. S., and ARNOLD, J. S. (1954). Anat. Rec., 118, 315. JOWSEY, J., OWEN, M., and VAUGHAN, J. (1953). Brit. J . exp. Path., 34,

JUDET, R., JUDET, J., LAGRANGE, J., and DUNOYER, J. (1954). Mkm.

LSCROIX, P. (1951). L’Organisation des 0s. Likge : DESOER ; English

LACROIX, P. (1952). Expehenlia, 8, 426.

A. (1954). Acta orthopaed. scand., 24, 101.

6, 251.

biophys. acta, 8, 375.

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19.

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Translation ; London: J. & A. Churcliill Ltd.

* NOTE ADDED IN PROOF : this work has since been published (Vincent, 1955).

44 P. LACROIX TACROIX, P. (1953). Bull. Acad. Toy. Mkd. Belg., 6 e sCrie, 18, 489. LACROIX, P. (1954). I1 Radioisotope Conference, vol. 1, 134. London:

Butterworths Scientif. Publ. LEVINE, M. D., RUBIN, P. S., FOLLIS, JR., R. H., and HOWARD, J. E.

(1949). Josiah Macy, Jr., Foundation, Trans. 1st Conf, Metabolic Interrelations, p. 41.

Jr., Foundation, Trans. 4th Conf., Metabolic Interrelations, p. 100.

(1952). Acfa anal., 14, 9.

442.

LONTIE, P. (1953). Rev. belge path., 23, 118. NEUMAN, W. F., BOYD, E. S., and FELDMAN, I. (1952). Josiah &lacy.

ROUILLER, C., HUBER, L., KELLENBERGER, E., and RCTISITAIJSER, E.

TRUETA, J., and HARRISON, M. H. &I. (1953). J . Bone Jt. Surg., 35~,

VINCENT, J. (1954). Arch. Biol., 65, 531. VINCENT, J. (1955). Recherches sur la Constitution de 1’0s Adulte

(Thkse de I’UniversitC de Louvain). Bruxelles: Arscia.

DISCUSSION

Fanconi: I have X-rays of bones from children with lead poisoning. These X-rays present a lot of lead just in the metaphysis; as you said, that is the most active part of the bone and not the epiphysis or the diaphysis.

Lacroix: Your remark proves that lead deposition is linked with osteogenesis. This suggests the technical possibility of using lead, instead of radioactive isotopes, as a tool in the study of the histological remodelling of adult bone.

Follis: We have studied the histological appearance of lead in the bones of children. At the Johns Hopkins Hospital we had a series of about GO cases. The dense shadow, as Dr. Park showed many years ago in lead poisoning in children, is due in large part to the persistence of excessive amounts of calcified cartilaginous matrix encased in large amounts of bone. The defect there apparently is a “poisoning” of the destructive mechanisms, so that the calcified, and probably plumbified, matrix remains; the bone encasing it remains, and both give rise to the bright line one sees in the X-ray.

Engstrom: We did some experiments with radiosulphate in dogs and radioautographs from longitudinal sections of the long bones showed, as was known before, a heavy deposition in cartilage but also in the bone tissue itself. If you take spongious bone and powder it and compare its radiosulphate activity with powder from compact bone, the total radioactivity is about twice as high in the spongious bone. Then we tried to separate two fractions containing radiosulphate and we found that about one half of the radioactivity goes to the inorganic portion and about half of it to the organic. You can take a bone section, non- radioactive, and incubate it in z‘itro, and you obtain uptake in the inorganic fraction of bone and nothing in the organic. This means that a fraction of the 35S0, goes to the inorganic portion and is probably surface-bound by exchange.

DISCUSSION 45

Meyer: How do you distinguish between inorganic and organic sul- phate?

Engstroin: The first fraction came out with simple water extraction after which there was still some radioactivity left in the bone.

Meyer: So one can only talk about insoluble sulphate, whether this is organic or inorganic you cannot say.

Engstrom: No , of course. We tried to extract this “organic” fraction but the amounts were too small to permit estimation of the specific activities.

Amprino: I would like to point out to Prof. Engstrom that it is not certain whether the radioactive broad band underlying the epiphyseal plate is only due to the sulphate fixed to the bone tissue, because there is a t that very level a lot of calcified cartilage which is the remnant of the lower part of the epiphyseal plate which has been partially resorbed. So we have there both bone and cartilage and we know that cartilage fixes a large amount of radioactive S. We should, therefore, be quite cautious when interpreting the autoradiographic aspect of the epiphyseal plate region.

Kodicek: Prof. Lacroix, your Fig. 1 showed the sequence which leads up to calcification; a very similar picture is obtained in healing wounds, skin or tendon wounds, where you also have this sequence, of course without Ca deposition. I would suggest that the true causes of calcifica- tion, or of eventual deposition of Ca, are not the structures which are revealed by sulphate uptake or metachromasia or PAS staining, because otherwise why should the skin or tendon not calcify as well?

Lacroix: Some data in the literature strongly suggest that muco- polysaccharides play a rBle in calcification.

Armstrong: You have shown us what I think is very good evidence for the time required for the formation of calcification of an osteon. Is it possible to indicate how long an osteon endures?

Lacroix: The question is now being studied. It is a problem of great importance, because what we need most a t the present time is a compari- son between the rate of the histological renewing and the biological half- life of 45Ca. Up to now, the biological half-life of 45Ca has been stitdied in the so-called “adult” rat, which does not stop growing, or in the “almost fully grown” animal; as far as I know i t has not been satis- factorily recorded in really adult bone.

Armstrong: One would assume that the number of osteons in an adult ought to be constant or nearly constant. This would then require that the number formed should equal the number removed.

Lacroix: Yes, roughly, It would perhaps be more accurate to state that, considering a long period of time in a young adult in perfect health, the total amount of newly formed lamellae equals that of re- moved lamellae.

Armstrong: Can you see anywhere except in the endosteum or perios- teum any evidence of the resorption of a radioactive osteon?

Lacroix: No. It would take, I think, several months before we would have a chance of seeing a radioactive osteon being eaten up: We may perhaps infer from what happens in radium poisoning in man. There are

46 DISCUSSION two papers on the question, one (Hoeclier, F. E., and Roofe, P. G . (1961), Radiology, 56, 89) showing that 7 years after radium poisoning, the pictures of radioactive osteons are still complete rings, and the other (Looney, W. R., and Woodruff, L. A. (1953), Arch. Path. (Lab. Mcd.), 56, l), in which there are instances of radioactive osteons partially destroyed 22 years after radium incorporation. However, the meaning of these facts remains uncertain because the poisoned bone tissue is certainly abnormal.

Lisco: I noticed from your pictures that the amount of radioactivity that you have used in order to demonstrate the activity by radioauto- graphs was fairly considerable, and the question arises as to whether or not such an amount of radioactivity could have interfered with the formation of such an osteon?

Lacroix: The fact that radioactivity may by itself impair the deposi- tion of the osteon remains open to question, and it is one of the numerous objections which may be raised against that type of work. That is why I wish that something other than radioactivity could be used, for instance lead, or vital staining.

Lisco: In other words, we may have to revise this figure of six weeks for the formation of the osteon a few years hence. Furthermore, I wonder whether you have given consideration to the possibility that marked species differences might exist for the time that is required for the growth of an osteon to be complete.

Lacroix: I completely agree. I was careful to point out that it was a rough estimation.

Lisco: I noticed that. Follis: If you were to study the difference which you showed between

the metaphyseal and epiphyseal trabeculae a t various stages while the cartilage was still there, would you again find this difference?

Lacroix: Yes, we may sum up by saying that the adult, a t least the young adult, keeps something of the differences which prevail during growth.