THE EFFECT OF CHRONIC FLUORINE INTOXICATION UPON THE HEALING OF EXPERIMENTAL FRACTURES IN RATS* RICHARD T. ODELL, M.D.

AND J. ALBERT KEY, M.D.

ST. Louis, Missoum
FROM THE DEPARTMENT OF SURGERY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI

DURING RECENT YEARS it has been shown that a concentration of fluorine of about one part per million in the drinking water tends to lessen dental decay and does not cause undesirable changes in the teeth or bones. On the other hand, prolonged consumption of water containing three to six parts per million of fluorine or prolonged exposure to dust containing this element may cause not only mottling and deformities of the teeth in children, but marked changes in the skeleton of adults and children. The skeletal changes consist of generalized increase in density of the bones, with a variable amount of thickening, especially of the mandible, ribs and long bones of the extremities. The sclerosis and thickening are due to the deposition of new bone, both inside the bone and beneath the periosteum, and the marrow may be encroached upon. The skeletal changes can be seen in roentgenograms, and as the condition advances, osteophytes are formed around the margins of the vertebrae and on the long bones at the attachments of ligaments, tendons and muscles. They may lead to ankylosis of the spine, with fusion of some vertebrae and of the sacroiliac joints, fixation of the thorax, and to stiffening of other joints (Moeller and Gudjonsson,6 Roholm,9 Shortt and McRobert,10 Bishop,2 and Pandit, et a18). The last observers noted that the severity of the changes was enhanced by malnutrition, and

especially by deficiency in Vitamin C, and as many of the subjects studied by him in South India were on a semistarvation diet, they not only developed poker backs and crippling, but some of them became bedridden at between 30 and 40 years of age and died of intercurrent disease. In the case reported by Bishop, the patient died of luetic heart disease, and samples of the skeleton when analyzed for fluorine, were found to oontain from 0.29 to 0.70 per cent, as compared with 0.076 per cent, fluorine in normal human bone. Similar changes in rabbits (Largent, Machle and Ferneau4 ), dogs (Bauer'), monkeys (Pandit and Narayana Rao7) and rats (Sutroll) have been reported, and in the young animals, growth is retarded and the teeth are affected. Microscopic examination of the osteophytes and subperiosteal bosses indicates that they are formed of true bone, which resembles that formed in the provisional callus of healing fractures. Bauer noted that the changes were not prevented by an antirachitic diet, and that the density of the trabeculae of bone laid down beneath the periosteum and perpendicular to the long axis of the bones varied direotly with the duration and intensity of the fluorine action, and that the reaction was more rapid in growing bones than in those of old animals. The matrix of the periosteal and endosteal bone was not altered, but the osteocytes were larger and * Presented before the American Surgical Asirregularly arranged. There was also some 1953. sociation, Los Angeles, California, April 2, 461

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diffuse osteoporosis, which was mainly evident in young dogs, but this usually lagged behind the new bone formation, and the result was an increase in the bone density which was especially evident in old dogs. In some of the young animals the costochondral junctions and epiphyseal lines exhibited changes which resembled those of

MATERIAL AND METHODS

One hundred thirty-four albino rats (69 were adults and 65 were six weeks old or about half-grown) were placed upon a normal diet and to the water of 85 of the animals, sodium fluoride was added in the proportion of 226 parts per million (.0226 per cent). It is estimated that each animal in-

FIG. 2. Humeri of 12 rats. Young animals above and adults below. The six on the left 21 dlays after fracture. Six on the right 28 days after fracture. Bony union in all. Sodium fluoride started one month before fracture.

FIG. 1. Humeri of 8 rats ten days after fracture. Young animals above and adults below. The medullary pins removed, one broken. Callus visible. No union. Sodium fluoride started 30 days before fracture.

ricketts. Largent, et al., produced fractures of one tibia in six rabbits, and four of these were fed fluorine for from 14 to 60 days. Whether or not these fractures healed is not recorded, but they state that the callus of the controls could not be distinguished structurally from that of the fluoride fed rabbits. In view of the fact that chronic fluoridosis tends to cause new bone to be formed in man and in other animals, it seemed possible that the ingestion of this element might exert a favorable influence upon the healing of fractures. Consequently, we have fed fluorine to a series of rats and have produced experimental fractures in these animals and compared the healing of these with that of a group of control rats with similar fractures.

gested about 4 mg. of sodium fluoride each day. This is a relatively small dose where the object is to produce fluoride intoxication, and the animals appeared to remain in good condition and the younger group seemed to grow nonnally and exhibited no changes in the hair or teeth. We lost several animals from an epidemic of diarrhea of unknown etiolo-gy, but these included both controls and experimental animals, and apparently this was not due to the fluorine and these were not included in the experiment. In 48 animals (23 adult and 25 young) the administration of fluorine was started one month before the fracture, and in 37 it was started at the time of the fracture. In 49 control animals, the fracture was produced in the usual manner and they remained on the same diet as the others, but received no fluorine. In all animals, the right humerus was fractured manually by bending it over the sharp edge of a container after the animal had been anesthetized with ether. In 106 of these, an intramedullary pin or wire was

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placed in the bone before it was broken, and the wire was then straightened. In previous work (Key, Odell and Taylor3), we have found that the fragments were usually maintained in satisfactory position and the rate of healing was quite uniform, and that of the experimental animals could be com-

Union, as determined by manipulation of the fragments, was slightly more advanced in the ten and 21 day fractures of the fluoridated groups than in the controls. In the earlier fractures, no difference could be detected between the two groups, and in the 28 day fractures, all were solidly united by bone. In the three day fractures the fragments were united by a delicate connective tissue callus and in the five day fractures, the callus was large and firm, and consisted of cartilage and bone. In the ten, 21 and 28 day fractures, there was a progressive diminution in the size and an increase in the firmness of the callus.

FIG. 3. Humeri of 9 young rats 32 days after Above: Controls. Below: Sodium fluoride started 30 days before fracture and in these union slightly more advanced.
fracture. No fixation.

pared with the controls. In 28 animals (18 fluoridated and 10 controls) the medullary pins were not used because we wished to determine whether or not the fluorine would hasten the healing of fractures with marked displacement of the fragments. The animals were sacrificed in groups of four or five on the 2nd, 3rd, 5th, 10th, 21st and 28th day after the fracture, and the fractured humeri were removed and examined in the gross and tested for union, then they were fixed in formalin, and after being roentgen rayed, the bones were decalcified, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, and studied microscopically.
OBSERVATIONS

FIG. 4. Humeri of 9 adult rats 35 days after fracture. Above: Controls. Below: Sodium fluoride started 30 days before fracture. Union about the same in all, delayed by displacement.

were

On gross examination of the specimens fixed by medullary pins, the callus was definitely larger in the fluoridated rats in five and ten day old fractures and slightly larger in the three and 21 day fractures, than they were in the controls. No difference between the two groups could be detected in the two and 28 day fractures.

The 28 animals in which the fragments not fixed with a medullary pin were sacrificed at 32 and 35 days after the fracture. Of these, the ten fluoridated animals in which the fluorine had been started 30 days before the fracture were all healed by bone, while only six of the controls were so healed and the other four were united by a firm callus, but slight springy motion could be detected at the site of the fracture. Four such rats in which the fluorine was started at the time of the fracture were sacrificed 45 days after the fracture, and of these, two were firmly healed, while slight springy motion was present in the other two. In many of this group the fragments were grossly displaced and were united by a large firm callus. No definite difference in

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In order to compare the effect of fluorine the size of the callus or in the firmness of the union was noted between the young and on the diameter of the callus, this was measthe adult animals. This was true of both the ured on the roentgenograms of 30 fluorifractures fixed with medullary pins and of dated adult animals and of 28 controls in those without fixation. which the position of the fragments and the site of the fracture were such that comparROENTGENOGRAPHIC EXAMINATION able measurements were possible. It was The degree of union shown in the radiofound that the diameter of the callus of the graphs agreed quite well with that as deter- fluoridated animals averaged 118 per cent mined by gross examination of the humeri of that of the controls. The difference was when removed postmortem. The callus and greatest in the 28 day group where the cal-

c B A FIG. 5. (A) Thick subperiosteal new bone formation three days after fracture. (B) Large cartilagenous callus uniting ends of fmgments five days after fracture. (C) Extensive osteoclastic erosion of provisional callus five days after fracture. Cortex is below in all photomicrographs. All animals started on sodium fluoride 30 days before fracture.

subperiosteal new bone formation were visible in the five and ten day fractures, and most of the 21 day fractures appeared to be united by bone, and all of the 28 day fractures appeared to be united by bone, unless the fragments had been displaced. In fractures in which gross displacement had occurred either because the medullary pin had been broken or in which no fixation had been used, the bones united more slowly, and in two of the fluoridated group in which the fluorine was started on the day of the fracture, union by bone was not present 45 days after the fracture. The above was true of the controls, as well as of the fluoridated animals, and in both the young and the adult animals careful comparison of the roentgenograms of the fluoridated animals with those of the controls indicated a slightly more advanced state of union in the 21 day fluoridated group. In the others there was no constant difference.

lus in the fluoridated animals was approximately 1/3 larger (136 per cent) than it was in the controls.
MICROSCOPIC EXAMINATION

Study of microscopic sections revealed no

oonstant differences in the size and character of the callus and in the rate of healing

between the fluoridated animals and in the controls. In the two day fractures there was proliferation of cells beneath the periosteum, with formation of a thin layer of new bone which extended for a considerable distance (about twice the diameter of the bone) up and down the shaft on either side of the break, and near the ends of each fragment a few hyaline cartilage cells were present beneath the periosteum. The new bone was laid down in membrane and appeared to be calcified soon after it was formed. In the three day fractures, the collar of subperiosteal new bone was thicker than

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the cortex of the humerus, and the formation of the ring of hyaline cartilage around the end of the fragments was well advanced. In the sections studied, the subperiosteal new bone was most abundant in the five day fractures, and in some of these it was being eroded by osteoclasts and the ends of the fragments were united by a thick ring of

in size and replaced by cancellous bone from above and below until in some specimens, only a relatively thin disc of cartilage was left. In the 21 day fractures the cartilage had largely or entirely disappeared and the ends of the fragments were united by trabeculae, which sprang from both the subperiosteal and the en-

FIG. 6. (A) Bone and thin cartilagenous callus uniting ends of fragments ten days after fracture. (B) Bony union between ends of fragments 28 days after fracture. Sodium fluoride started 30 days before fracture.

hyaline cartilage, which was being replaced by bone on either side of the fracture. Also at this stage of healing, the endosteal new bone formation was well advanced and the trabeculae appeared to be laid down in membrane and calcified as formed. In some sections of the five day fractures there was marked osteoclastic activity whioh involved both the newly formed bone beneath the periosteum and in the medullary canal, and also there was some erosion of the original cortical bone. In the ten day fractures the thick disc or ring of hyaline cartilage which united the two fragments was markedly reduced

dosteal callus and bridged the space bethe ends of the bones to which it was applied as appositional bone. The reorganization of the provisional callus was well under way at 21 days, and at 28 days the union and reorganization of the callus were further advanced and the trabeculae were larger and the union was more firm. The details of the repair of these fractures were practically the same in the fluoridated and in the control animals, and in the young and in the adult groups. They have been described in a previous paper (Key, Odell and Taylor3). In none of the sections
tween

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did we find microscopic changes in the bone, either at the site of the fracture or in the bone above or below the fracture, which we could ascribe to fluorine intoxication.
DISCUSSION

In these experiments, we used a relaatively small dose of fluorine and the degree of intoxication was not sufficient to impair the health or inhibit the growth of the animals. It is possible that careful postmortem studies of the skeletons, and especially of the jaws, might have revealed some subperiosteal new bone formation, but our observations were limited to the fractured humeri, and in these we were not able to discover any evidence of fluorine intoxication. In regard to changes in the teeth and the inhibition of growth, these occur only when the administration of fluorine is started in very young animals, and our rats were halfgrown before this was begun. Consequently we would expect no such changes. Our observations prove that 226 parts per million of sodium fluoride in the drinking water of rats does not inhibit the repair of fractures, and they suggest that this may exert a slightly favorable influence on the process. We hope to investigate this subject further and determine the influence of more severe fluorine intoxication on the healing of bone.
BIBLIOGRAPHY Bauer, J. T., P. A. Bishop and Wm. A. Wolf: Pathologic Changes of Bone in Chronic

Fluorine Poisoning. Bull. Ayer Clin. Lab. Pennsylvania Hosp., 3: 68, 1937. 2 Bishop, P. A.: Bone Changes in Chronic Fluorine Intoxication. A. Roentgenographic Study. Am. J. of Roentgenology, 35: 577, 1936. 3 Key, J. Albert, R. T. Odell and L. W. Taylor: Failure of Cortisone to Delay or to Prevent the Healing of Fractures in Rats. J. Bone & Joint Surgery, 34A: 665, 1952. 4 Largent, E. J., W. Machle and I. F. Ferneau: Fluoride Ingestion and Bone Changes in Experimental Animals. J. Industrial Hygiene Toxicology, 25: 396, 1943. 5 McCollum, E. V., N. Simmons and J. E. Becker: The Effect of Additions of Fluorine to the Diet of the Rat on the Quality of the Teeth. J. Biol. Chem., 63: 553, 1925. 6 Moeller, P. F., and S. V. Gudjonsson: Massive Fluorosis of Bones and Ligaments: Acta Radiology, 13: 269, 1932. 7 Pandit, C. G., and D. Narayana Rao: Edemic Fluorosis in South India. Experimental Production of Chronic Fluorine Intoxication in Monkeys. Indian J. of Med. Res., 28: 559, 1940. 8 Pandit, C. G., T. N. S. Raghavachari, D. Subba Rao and V. Krishnamurti: Endemic Fluorosis in South India. A Study of the Factors Involved in the Production of Mottled Enamel in Children and Severe Bone Manifestations in Adults. Indian J. of Med. Res., 28: 533, 1940. 9 Roholm, Kay: Fluorine Intoxication. H. K. Lewis and Co., Ltd., London, 1937. 10 Shortt, H. E., G. R. McRobert, T. W. Barnard and A. S. M. Nayar: Endemic Fluorosis in the Madras Presidency. Indiana J. of Med. Res., 25: 553, 1937. Sutro, C. J.: Changes in the Teeth and Bones in Chronic Fluorine Poisoning. Archives of Pathology, 19: 159, 1935.

DIscuSSION.-DR. PHILIP D. WILSON (321 E. 42nd Street, New York 17, N. Y.): Mr. Chairman, Dr. Key has made a good study of the effects of the administration of fluorides on the healing of fractures in animals. He used a small, daily dose in these animals which is comparable to that used in the water supply of certain cities for the prevention of dental caries. His results are somewhat equivocal. I would like to see him repeat these experiments using larger doses, because this is the best

way to show whether the effects are injurious or not or whether they influence the metabolism of the bones. CHAIRMAN GIBBON: Is there any further discussion? Dr. Key, have you anything to add? DR. J. ALBERT KEY (Washington University, School of Medicine, St. Louis 10, Mo.): Only that I ought to thank Dr. Wilson.

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