STUDIES ON THE REPAIR OF FRACTURES USING 32P

PIERRE H. CARTIER, BENEDETTO DE BERKARD AND JEAN LAGRANGE

Laboratoire d'Etude de 1'0s et de la Croissance, Facultd de Me'dicine de Paris

IN this paper we report on results obtained by a study of the biochemical mechanisms involved in bone regeneration after fracture. Chemical analysis and determination of radio- phosphorus fixed by bones were used jointly to follow the different phases of fracture repair.

Realization of a definite type of fracture and its adequate fixation are essential conditions for following successfully the evolution of an experimental fracture. It is also essential t o operate on a rather large number of animals in order to elimin- ate individual variations. To satisfy these conditions, we per- formed a fracture in the middle of a femoral diaphysis; fragments were then fixed by an intramedullary nail, in complete asepsis. This way of holding the fracture prevents any displacement and a perfect consolidation is assured within 30 to 40 days. KO pseudarthrosis has been observed under our experimental conditions. One cannot, however, exclude a modification of the evolution of the fracture by this treat- ment and a study of the influence of the nail on fracture evolution is actually going on. Under our experimental condi- tions, this method seems to be the most reliable, giving constant evolutions which are described in this first paper.

Experimental methods and procedures Adult rats (Wistar strain) weighing 200 g. were anaesthet-

ized with chloral. After depilation, the skin was cut and the 148

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

Copyright 0 1956 Ciba Foundation Symposium

STUDIES O N THE REPAIR OF FRACTURES USING 32P 149

fracture was performed by cutting the left femoral diaphysis transversely with a dental burr. An intramedullary nail of vitallium (10-15 mm. long) was then introduced into both ends of the fractured bone. Muscles were sewn together with catgut and the skin was fixed with clips.

This mild operation was accomplished under surgical asepsis : an initial experiment with less rigorously controlled asepsis was frequently followed by attenuated infections in the zone of fracture.

Animals were killed at regular intervals from the third to the fortieth day after fracture. Each rat received a total of

HOUR5

( b )

FIG. 1. Uptake of 32P by bone in aivo.

150 P. H. CARTIER, B. DE BERNARD AND J. LAGRANGE

200 pc of 32P (solution of NaH,PO,) intraperitoneally 24 hours before death. Complete fixation of isotopic phosphorus in the femoral diaphysis was established by measuring bone activity in the living anaesthetized animal : maximal activity was reached 1-4 hours after injection (Fig. 1) ; to avoid individual variations, we have chosen the optimum time of 24 hours. Repair of the fracture was also controlled by radio- grams and histological preparations.

FIG. 2. Mineralization of callus.

The following bone segments were removed from the sacrificed animal :

From the left fractured femur: the callus; both terminal parts of the fractured bone; a fragment of femoral diaphysis distant from the zone of fracture and near to the inferior epiphysis.

STUDIES ON THE REPAIR OF FRACTURES USING 32P 151

From the right femur: a fragment taken from the medial part of the diaphysis.

The wet weight of these bone fragments was immediately recorded and the phosphorus content determined by the method of Fiske and Subbarow. Samples of bone digested with perchloric acid were dried in nickel dishes by an infra- red “epiradiator.” To obtain a regular distribution of the

lo(

z 0

N t i z

8

Y

50

w Y = 8

0

AS.

10 10 30 40

Callus

A S

10 m 10 (0

A S.

L-4-U-4 DAYS

Extremities and Fmctured Femur Fractured Ends Dlaphyris

FIG. 3. Rate of mineralization and affinity in different segments of bone.

ashes, the residue was dissolved in a small quantity of 5 per cent acetic acid and again dried. Activity of all the samples measured with a Geiger-Muller counter, was recorded as the average value of three determinations of a t least 2000 counts each. By this method we have calculated: (1) phosphorus content of the samples, and (2) rate of mineralization of the fractured bone, which was calculated by comparison with the corresponding diaphysis. Comparison with the degree of mineralization of normal rat skeletons was impossible because of the great variability of the phosphorus and calcium content

152 P. H. CARTIER, B. DE BERNARD A S D J. L A G R A N G E

in the rats’ bones: this was also observed by Roche and Mourgue (1939) and by Dallemagne (1943). Because of the low rate of demineralization which is observed even in the corresponding diaphysis (less than 10 per cent, according to Dallemagne), the comparison between the fractured and the homologous bone is expressed as a ratio which is slightly lower than the real value.

To follow phosphorus released during fracture evolution, a balance study of phosphorus and calcium was made on 6 rats during the whole period of fracture repair (40 days). The

20 10 DAYS

FIG. 4. Elimination of szP in the fractured and control rat.

STUDIES ON THE REPAIR OF FRACTURES USING 32P 153

skeleton was labelled with radiophosphorus 5 days before fracture, and faeces and urine were quantitatively collected and analysed daily. This balance was then compared with the normal phosphorus and calcium balance of control rats.”

During the chemical evolution of repair we checked, a t every stage, the capacity for phosphorus fixation of the different zones of bone. We defined this fixation by the term “affinity”, calculated from the proportion of 32P fixed after injection of NaH,32P0,, 24 hours before removal of the segments.

Days

4 7 10 15 ao 25 30 36 40

Results

Experimental results, average values obtained from a series of about 80 rats, are collected in Tables I, I1 and I11 and illustrated by Figs. 2 and 3, which indicate more clearly the evolution of fracture repair.

Phosphorus elimination in the fractured and control rats is shown in Fig. 4.

Rate of callus mineralization

4.4 5 . 0 6.15 10.75 11.2 18.6 28.1 45.2 68.9

Table I

represent yo values of P content of the homologous femoral diaphysis RATE OF MINERALIZATION OF CALLUS IN YOUSG AND ADULT RATS, figures

YOUNG ADULT

Days

8 10 13 17 25 30 35 40

Rate of callus mineralization

8.9 13.5 18.0 27.4 41 . O 55.3 76.1 100.5

* Only one curve for the elimination of phosphorus is given (Fig. 4). The rate of phosphorus elimination was the same for all the animals maintained under the conditions of balance study.

154 P. H. CARTIER, B. DE BERNARD AND J. LAGRANGE

Days

4 7

10 15 20 25 30 36 40

Rate of Mineralization

Fractured femoral

diaphysis Terminal parts

Callus of fracture

4 . 4 98.0 93.0 5 . 0 109.0 86.0 6.15 121.0 88.6 10.75 116.5 83.9 11.2 76.0 80.0 18.6 67.8 75.3 28.1 88.2 82.6 45.2 92.2 97.2 68.9 98.0 -

Table I11 S z P ACTIVITY IN DIFFERENT SEGMENTS OF BONE, figures represent yo x lo8

of szP injected

Days Rate of callus

mitieralization femoral diap h ysis

4 7

10 15 20 25 30 36 40

Temninal parts of fracture

4 . 4 5.03 6.15

10.78 11.20 18.6 28.1 45.2 68.9

Fractured femoral

diaphysis

23.8 25.1 21 *7 21.3 19.7 18.4 18.6 19.5 19.7

14.6 30.5 33.4 31.2 79.0 68.3 47.4 5 5 . 4

32P Activity 1

39.0 89.1 55.3 58.2 62.8 64.0 53.9 41.6

Callus

179.3 330.6 204.1 160.7 159.0 113-1 113.2 93.2 73.6

I I

STUDIES O N THE REPAIR O F FRACTURES USING 32P 155

Callus evolution Callus mineralization is accomplished in two phases : (1) during the first 20 days, fixation of phosphorus is small,

and it increases very slowly; (2) from the twentieth to the twenty-fifth day onward,

rapid mineralization is observed in the fibrocartil- aginous tissue formed during the preceding phase: the degree of mineralization is about 75 per cent within 10 days.

A striking increase of 32P affinity is demonstrated in the callus between the fourth and the eighth day after fracture; subsequently, in relation to the rate of mineralization, a decrease in this affinity has been observed.

Evolution of the terminal par ts of fractured bone The whole process is in three phases: (1) during the first 10 days, a significant increase in phos-

phorus content (20 per cent) is observed. The fact that more phosphorus is found than in normal bone indicates that bone condensation occurs in this zone;

(2) from the tenth day onward, the amount of bone salts quickly decreases : on the twenty-fifth day the degree of demineralization is over 30 per cent;

(3) finally, after the twenty-fifth day, the rate of phos- phorus fixation increases again in relation to the rate of callus mineralization.

Terminal parts of fractured bone show a moderate affinity for 32P, and are highest in the phase of strongest demineraliza- tion (twentieth to twenty-fifth day).

Evolution o f the diaphysis o f the fractured bone Fractured bone shows marked rearrangements during the

evolution of callus, as we have observed on analysis of a zone distant from the site of fracture (inferior part of diaphysis):

(1) demineralization of the diaphysis is an immediate conse- quence of fracture; the maximum effect is seen towards the twenty-fifth day (about 30 per cent);

156 P. H. CARTIER, B. DE BERNARD AND J. LAGRANGE

(2) subsequently, as has been observed in the other zones, mineral fixation begins again, and a normal content of phosphorus is observed a t the stage of consolidation of callus.

The specific activity of the diaphysis shows that, in regard t o affinity, its behaviour is similar to that of the terminal parts of a fracture: the lower the mineral salt concentration the higher the isotopic phosphorus concentration.

Discussion Mineralization of callus of the growing and adult animal is

accomplished in two phases which are readily identifiable. These two phases, which parallel biochemical processes con- cerned with normal ossification, seem to be related to different mechanisms.

During the first phase, an equivalent of the initial period of bone formation is evident, i.e., the conversion of the undif- ferentiated proteid structures to the calcifiable matrix. The arrangement of matrix during this phase is almost without phosphorus fixation. Once mineral-binding capacity has appeared, calcium and phosphorus are readily and intensely incorporated, as we have observed in the second phase of fracture repair (twentieth day). During this second period (3 weeks), the phosphorus content increases from an initial value of 5 per cent t o the stage of complete mineralization.

This biphasic evolution seems t o be present in all the different zones of the fractured bone. A demineralization reaction, in agreement with the first proteid phase, is demon- strated in the diaphysis and in the terminal parts of the fractured bone. The latter segment is, moreover, characterized during the first 10 days by a rather marked condensation, which was also described by Dallemagne (1943) in a study of bone repair in rabbits.

We have demonstrated that a strict relationship exists between the rate of demineralization and the capacity for incorporation of isotopic phosphorus. Analogous conclusions

STVDIES ON THE REPAIR OF FRACTURES USING 32P 157

were drawn by Engfeldt, Engstrom, and Zetterstrom (19.521, and Lacroix (1953, 1954), who noticed that structures with a low content of mineral salts fix isotopic Ca or P to a high extent.

The determination of this isotopic affinity represents a very sensitive test for demonstrating demineralization processes." In fact, we have observed that from the first few days follow- ing fracture the affinity of the homologous diaphysis increases to a value which is twice the normal (average value : 2 * 3 *to * 2). This interesting phenomenon, which was also described by Wilkinsori and Leblond (1953) seems to be the expression of a general reaction of the whole skeleton to the lesion of a single bone.

Therefore, fracture evolution is not to be considered as a process only concerned with the local mechanisms of repair, but with a more general participation of bone apparatus. This idea is also supported by the result of the study on excretion of radiophosphorus previously fixed by the skeleton. During the first phase characterized by these demineralization processes, evidence is obtained of a remarkable increase in phosphorus excretion in the faeces and urine of rats.

The results obtained by this research have led us to under- take the study of a series of important problems:

(1) Different parts of the fractured bone have shown a marked variation in tricalcium phosphate content; the main problem is to discover the origin of the bone salts we have found in the terminal parts of fracture and the destiny of the salts released during the demineralization phase.

(2) Proteid and mineral phases of the repair of fractures seem to be related to different local mechanisms, the nature of which we do not yet know.

(3) The general participation of the whole skeleton in fracture repair seems to be a stress-like reaction: it is

* A striking increase in this afflnity (3 tinies the normal value) follows a degree of demineralization of about 5 per cent (Cartier, de Bernard and Lagrange, t o be published).

158 P. H. CARTIER, B. DE BERXARD S N D J. LAGRANGE

important to study the mechanisms (nervous, vascular, hormonal or others) with which it is correlated.

REFERENCES DALLFMAGKE, M. J. (1943). Thkse d’agrkgation, Universitk de Likge. ENGFSLDT, B., ENGSTROJI, A., and ZETTERSTROM, R. (1952). Biochim.

LACROIX, P. (1953). Bull . Acad. roy. MLd. Belg., 18, 489. LACROIX, P. (1954). 11 Radioisotope Conf., 1, 134. London: Butter-

ROCHE, J., andMovRcvE, M. (1939). Bull . SOC. Chim. biol , Paris,21,243. WILKINSON, G. W., and LEBLOND, C. P. (1953). Surg. Gynec. Obstet., 97,

DISCUSSION

biophys. acta, 8, 375.

worth.

143.

Lacroiz: The larger the experimental animal, the more localized are the reactions of its skeleton after a fracture. In man, between 20 and 50, a fracture of a diaphysis is followed by a definite radiolucency in the metaphysis and in a narrow zone just under the articular cartilage. The epiphyseal bone in between is involved afterwards. Everything returns to normal if the fracture heals well.

At present we are trying to study your subject, but in the dog and with microradiographic techniques. An aspect which puzzles us is that of some osteons with two concentric zones, as shown in an X-ray which I have here: an outer one poorly calcified, and an inner one highly calcified, just the reverse of the normal sequence of events. What it means, we do not know. Prof. Engstrom tells me that such aspects are sometimes observed here and there, but it looks as if they are more numerous in the vicinity of a fracture.

Harris: I should like to support what Prof. Lacroix said, from the clinical viewpoint. The X-ray he shows is a very common finding in fractures; occasionally we have had the courage to biopsy the rarefied area and in addition to meaning loss of Ca it means loss of bone sub- stance, true osteoporosis, Something is happening which inhibits the formation of new bone.

Lacroiz: It may be that the process of osteogenesis is stopped, ox that there is a leakage of Ca, or that there is an exaggerated bone absorption. I think it is bone absorption.

Harris: An easy solution is to regard the body’s energy as being con- cerned with the healing of one fracture rather than with maintaining the normal balance between bone formation and absorption in the rest of the bone.

Nassim: This is a problem we have been most interested in for a long time. There is no doubt that as soon as a fractured limb is immobilized, the patient goes into a very negative Ca balance. It does not matter whether the whole body is immobilized or not. A patient can have a fracture of the tibia and put on a walking plaster and still that patient

DISCVSSIOS 159

will be in a negative Ca balance and putting out about 300 mg. of Ca per day. I think that is well recognized, once you have normal stresses and strains you will not have osteoporosis occurring.

Nicolaysen: I am rather surprised to hear Dr. Nassim’s remark in view of the American results where they have people immobilized in casts. When Shorr put them in an oscillating bed the retention was back to normal. But you observe losses also when they are walking?

Nassim: Yes, as long as the limb is immobilized, even if they are in a walking plaster they are still putting out 300-400 mg. of Ca. Also, we have found that when a joint is inflamed, for instance a tuberculous knee, and it is not immobilized but the person is walking about on a crutch, we still find loss of mineral in that limb, not only adjacent to the joint but in the joint above it, in the hip.

Dent: You are suggesting, Rlassim, that i t is chiefly from the part in plaster rather than from the rest of the body?

Nassim: Not entirely. I am suggesting that for instance in a tuber- culous knee, which has got, say, synovial tubercle and which is not put in plaster where the patient is having only chemotherapy, you will not only have demineralization adjacent to the inflamed joint, but you will also get it around the proximal area, around the hip. He is not weight- bearing there.

Follis: Would you not have more osteoporosis if he were immobilized in plaster?

Nassim: Yes. Nordin: Surely this is too rapid to be explained by cessation of new

growth, although not very much is known about collagen turnover. I believe this thing appears very rapidly after the fractures and it seems to me to tie up very nicely with the work of Slack, who denervated and immobilized rat’s hind-limb and found that there was an increased uptake of labelled glycine into the collagen in the limb. This suggests that there is not cessation of new formation but an increased turnover and that the loss is greater than the new formation.

Armstrong: A fracture is a rather severe trauma. Are the effects which you observed and which have been previously noted specifically related to trauma to the skeleton? Would you obtain somewhat similar results with the same degree of trauma to soft tissues? Is this phenome- non a general physiological reaction which is not necessarily specific for the skeleton?

Howard: That can be answered very quickly, on overall balance at least, because the N loss ceases after about 10 days in an ordinary tibia1 fracture and the Ca loss continues for about 30-40 days. Furthermore, it does not happen if you take out a gallbladder or perform other major abdominal surgery; Ca does not follow the N balance; you get a little loss in Ca but nothing at all like the other.

Follis: That has to do with immobilization, because both with mobili- zation and with early mobilization everything goes back to normal.

Howard: It is less marked. Rutishauser: We have limited experience with this subject, on rhesus

monkeys. If mobilization is carried out too prematurely decalcification

160 DISCUSSION appears to be more pronounced than is the case when mobilization is carried out cautiously. We also injected the stellate ganglion with talc and the results have not been convincing, to date.

Howard: It is interesting that with the tuberculous knee the bone grows faster in a growing child than on the other side. That is an old observation which was made in Toronto by Dr. Harris’s father. You can demonstrate it quite readily.

Nicolaysen: You get that growth in length with aneurysm also. Is it all due to circulation?

Harris: It must be related to circulation, because every now and then we come across a massive arterio-venous communication that has a longer limb on that side.

Nicolaysen: It happens when you put patients to bed that they sud- denly have a very heavy urinary sedimentation with acute kidney attacks and they even become temporarily insane.

Blaxter: What happens if you produce a ikacture in a bone which normally does not bear much weight, for example, a skull fracture?

Howard: You will not get any more negative Ca balance than you would if you operate on a man’s stomach.

Dallemagne: Some years ago we performed a series of experiments of the same kind as those of Drs. Cartier and de Bernard. We resected sub- periosteally a fragment of bone (1 cm.) at the middle of the cubitus (or the radius) of the rabbit. Some days later we observed a reduction of the Ca and P content in the upper and lower fragment of the resected bone. Moreover, we regularly observed a decalcification in the diaphysis of the same bone on the opposite side; we thought a t the time that this phenomenon was depending on a sympathetic circulatory reflex. But probably this was not the only mechanism involved: indeed, this was true when new periosteal bone was formed in the resection focus. On the other hand, if the periosteum had been destroyed when the resection was performed, no new bone appeared and decalcification in the same bone on the opposite side was absent. So, probably, local and general factors were involved in this process.