- Email: [email protected]
Effect of roentgen rays on bone growth and bone regeneration
EFFECT OF ROENTGEN RAYS ON BONE GROWTH AND BONE REGENERATION AN EXPERIMENTAL STUDY * BARNEY
BROOKS, M.D. AND HARRY
T.
HILLSTROM,
M.D.~
NASHVILLE, TENN.
D
URING the fn-st year after the discovery of the that this form of energy x-rays, it was observed produced profound changes in Iiving matter. In 1903 Perthes found that the growth of one wing of a chicken was greatIy diminished if this wing onIy were exposed to the inffuence of roentgen rays when the chicken was one day oId. Since the appearance of Perthes’ report, numerous investigators have recorded their observations concerning the influence of the roentgen ray on the growth of bones in anima1 experiments and aIso from the therapeutic use of the roentgen ray. From a carefu1 review of a11 the avaiIabIe Iiterature it seemed that there was as yet a sufhcient Iack of cIearness concerning the amount of irradiation necessary to inhibit the growth of bone, the quantity of the effect produced, the duration of the inff uence, and the coincident histoIogica1 changes, to justify the repetition of many of the experiments previousIy performed. The possibiIity of inhibiting or stimuIating regeneration of bone has afso been investigated by experiments upon animaIs and by observations upon patients with bone defects due to trauma or other disease. From previous studies it seems as if it were definiteIy determined that extraordinariIy Iarge doses of irradiation have IittIe or no effect on the capacity of a bone to [regenerate new bone. There is a conflict of opinions concerning the stimuIating effect of the roentgen ray on new bone formation, but it wouId seem as if the maximum stimuIating effect which had been observed is not sufficient to be of importance. * From the Department of Surgery, VanderbiIt University. t Deceased. 599
600
BARNEY BROOKS AND HARRY T. HILLSTROM
The number of experiments which are the basis of this paper is too Iarge for a detaiIed report. OnIy the experimenta method and the resuIts obtained in severa groups of experi-
FIG. I. Photograph of bones of forearms of dog five months old in which right radius and uIna and dista1 end of right humerus were exposed to standard dose of irradiation. Note difference in length of bones of right and left forearms and particulady difference in size of distal ends of right and Ieft humerus.
ments wiI1 be brieffy summarized, and particuIar attention given to the resuks obtained in a group of experiments performed for the purpose of studying some factors which have not been previousIy discussed. EXPERIMENTAL
METHOD
Young rabbits were used in most of the experiments. In a few experiments dogs, guinea pigs, rats and mice were used. No important difference was observed in the resuIts obtained in these different animaIs. In each group of experiments, one or more Iitters of young rabbits were used. As far as possibIe, each Iitter of rabbits was cared for in such a manner that each rabbit of a Iitter was subjected to the same environment except for the age at exposure and the amount of irradiation.
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
601
The referred
factors constituting the standard dose subsequentIy to as IOO per cent s.E.D., were as foIIows: 180,ooo to 200,000 voIts, 20 to 30 ma., 60 cm. target-skin distance, 0.3 mm. copper combined with 1.0 mm. aIuminum fiIters, and forty-five minute exposure. The exposed fieIds varied from 1 X 2 cm. to 1 X 3 cm. Either the right tibia or the right uIna and radius were exposed. The remainder of the animaI’s body was carefuIIy protected with sheet Iead. After exposure the animaIs were cared for on a suburban farm. At reguIar intervaIs varying from one to twenty-eight days, roentgenograms were made of both the exposed and the non-exposed extremities. In each instance the extremity was pIaced as near the fiIm as possibIe and the x-ray tube at a distance of 7 feet from the fiIm. Measurements in growth in Iength of the bones were made from the roentgenograms. In experiments designed for histoIogica1 study, the exposed and non-exposed bones were removed, fixed in formaIin, decaIcified, sectioned and stained with hematoxyIin and eosin. I.
EXPERIMENT OF
ILLUSTRATING GROWING
BONE
THE TO
GROSS
EFFECT
ROENTGEN
OF EXPOSURE
RAYS
The dista1 end of the right humerus and entire Iengths of the radius and uIna of a dog four weeks oId were exposed to a singIe I00 per cent S.E.D. with the technique previousIy described. Four months after exposure the anima1 was sacrificed. The effect on bone growth is iIIustrated in Figure I. From this iIIustration it is cIear that there was a marked inhibition in the growth of those bones exposed to the roentgen rays. The right uIna increased 41 mm. in Iength and the Ieft uIna increased 102 mm. in length. The fact that inhibition of bone growth occurs onIy in the bone which is within the fieId of exposure is apparent from comparison of the proxima1 and dista1 ends of the right humerus. Note aIso that the genera1 contour of the bones in which growth has been inhibited is remarkabIy unchanged.
602 II.
BARNEY BROOKS AND HARRY T. HILLSTROM EXPERIMENTS
GROWING
BONES
THAN
SHOWING
THE
TO THE
ROENTGEN
THE
STANDARD
EFFECT
OF
RAY
SKIN
EXPOSURE
IN DOSES
ERYTHEMA
OF
SMALLER
DOSE
The right tibiae onIy in a series of young rabbits were exposed to doses of roentgen rays estimated at 40 per cent, 60 per cent, 80 per cent and IOO per cent S.E.D. AI1 animaIs receiving 40 per cent or more of the skin erythema dose showed definite inhibition of bone growth. Increasing the amount of irradiation aIways increased the amount of retardation of bone growth. The proportionate decrease in rate of bone growth was greater than the proportionate increase in dosage. III.
EXPERIMENTS STIMULATING
DESIGNED BONE
IRRADIATION
TO
GROWTH
IN SMALLER
PRODUCED
TEST
BY
THE
DOSES
INHIBITION
THE
OF
POSSIBILITY
USE
THAN BONE
OF
THAT
OF
REPEATED WHICH
GROWTH
The right tibiae onIy in a series of young rabbits were subjected to various combinations of repetitions of roentgenray exposures estimated at from 2 per cent to 5 per cent of the skin erythema dose. The animaIs were observed over various periods of time and in no instance was any evidence obtained of stimuIation of bone growth. IV.
EXPERIMENTS EXPOSURE
TO
TO
DETERMINE
DOSES
OF
INHIBIT
THE
ROENTGEN BONE
RESULTS RAYS
OF
REPEATED
SUFFICIENT
TO
GROWTH
The right tibiae onIy in a series of young rabbits were exposed to various combinations of repeated irradiation in doses sufficient to inhibit bone growth. The resuIts of various combinations of amounts of irradiation, number of exposures, and time intervaIs between exposures were observed. The tota quantity of irradiation received aIways produced Iess inhibition of growth the Iarger number of times the dose was divided or the Ionger the interva1 between the exposures. For exampIe, 150 per cent S.E.D. given in 2 equa1 doses on successive days
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
603
had about the same effect as a singIe IOO per cent s.E.D.; whiIe 160 per cent S.E.D. divided into 4 equa1 doses and given during a six day period was not as effective in inhibiting bone growth
FIG. 2. Photomicrograph
of IongitudinaI section of norma proxima1 epiphyseal cartiIage of tibia of a rabbit twenty-seven days old.
as a singIe I00 per cent s.E.D. V.
EXPERIMENTS AND
The exposed
FOR
THE
CARTILAGE
STUDY PRODUCED
OF
THE BY
CHANGES
IN
BONE
IRRADIATION
right tibiae onIy in a series of young rabbits were to the IOO per cent S.&D. of the roentgen ray. The
604
BARNEY
BROOKS
AND
HARRY
T. HILLSTROM
animaIs were sacrificed at periods ranging from twenty-four hours to 148 days. The histoIogica1 changes which occurred in the epiphysea1 cartiIage are iIIustrated in Figures 2, 3 and 4.
FIG. 3. Photomicrograph of IongitudinaI section of proxima1 epiphysea1 cartiIage exposed tibia of same rabbit as iIIustrated in Figure 2, four days after receipt of
of IOO
per cent S.E.D.
A detaiIed description of these changes wiII be reported in a separate paper. In genera1 it may be said that immediateIy after exposure of the epiphysea1 cartiIage to the standard IOO per cent S.E.D. there is marked evidence of serious damage to the cartiIage
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
605
of the ceI1.sbecomes irreguIar. Some ceIIs ceIIs. The arrangement are swoIIen and others are obviousIy disintegrated. Some ceIIs appear norma and are reguIarIy arranged. The irregu-
FIG. 4. Photomicrograph of Iongitudinal section of promima1 epiphyseal cartiIage of tibia of rabbit fifteen days after exposure to 40 per cent S.E.D.
Iarity of arrangement persists throughout the entire period of bone growth. During the Iatter part of the period of bone growth the epiphysea1 cartiIage becomes irreguIar in thickness. In some instances the cartiIage disappears compIeteIy in some areas. It is particuIarIy during this period that growth deformities deveIop.
606 VI.
BARNEY
BROOKS
EXPERIMENTS
AND
DESIGNED
IRRADIATION
ON
HARRY TO
BONE
T. HILLSTROM
STUDY
THE
EFFECT
OF
REGENERATION
It has been previousIy pointed out by the author that growth in Iength and thickness of bones is a process which operates onIy during a Iimited period of Iife, and that the capacity of a bone to regenerate a defect is not Iimited to any period of Iife. It wouId seem, therefore, as if the two processes must be independent. Previous investigators have demonstrated that repair of bone was not perceptibIy inffuenced by exposure to reIativeIy Iarge doses of the roentgen rays. For our own satisfaction, however, ten experiments were carried out on young rabbits. EquaI defects in the right and the Ieft uIna were produced by subperiostea1 resections of the bone shafts. In each of the rabbits the right foreIeg was exposed to the IOO per cent S.E.D. In some of the rabbits this exposure was made immediateIy after operatiori, and in others on the third to sixteenth day after production of the bone defect. The animaIs were frequentIy observed throughout the process of repair. In each instance growth of the right uIna was inhibited. In no instance, however, was there any perceptibIe difference in the amount of new bone formation or in the duration or compIeteness of the process of repair. No experiments were performed in which smaIIer doses were used with the idea of stimuIating bone repair. VII.
EXPERIMENTS
ROENTGEN
RAY
FOR
DETERMINING
ON THE IN
FORM
LENGTH
THE
OF THE OF
INFLUENCE
CURVES
OF THE
OF GROWTH
BONES
Six white rabbits from the same Iitter, born May 14, 1932, were IabeIed A-I, A-2, A-3, A-4, A-5 and A-6. The entire right tibiae of rabbits A-I, A-2 and’ A-3 were irradiated on June 4, 1932 with IOO per cent S.E.D. AI1 of the rabbits were kept as nearIy as possibIe under the same conditions. The entire right tibiae of rabbits A-4, A-5 and A-6 were exposed to IOO per cent S.E.D. on JuIy 30, 1932. Roentgenograms of both
-
--
.._-. 49
77
3;. :
105
ST
Exp. A-z. FIG. f;. Serial roentgenograms made at intervaIs of twenty-eight days in rabbit in which right tibia and libuIa were exposed to IOO per cent S.E.D. at age of twenty-one days. LEFT
RlCHT
:: _._-
-
AGE
.
._
133
161
Exp. A-4. FIG. 6. Serial roentgenograms made at intervals of twenty-eight days in rabbit of same litter as illustrated in Figure 5, in which right tibia and fibuIa were exposed to IOO per cent S.E.D. at age of seventy-seven days.
DAYS
608
BARNEY BROOKS AND HARRY T. HILLSTROM
tibiae of a11 rabbits were made every twenty-eight days for six months. The resuIts obtained in rabbits A-2 and A-4 are iIIustrated in Figures 5 and 6. The measurements of the Iengths
FIG. 7. Graph showing growth in length of shafts of right and Ieft tibiae in three rabbits of same Iitter in which right tibiae were exposed to IOO per cent S.E.D. at age of twentyone days.
of the shafts of both tibiae of a11rabbits are shown graphicaIIy in Figures 7 and 8. Figure g is a graphic representation of the average growth in Iengths of experiments A-2, A-3, A-5 and A-6. A study of these curves reveaIs certain important facts which have not heretofore been discussed in the various reports deaIing with the effect of the roentgen ray on the growth of bone : Growth in Iength of the shaft of the norma unexposed tibia was remarkabIy constant in a11 of the six rabbits. The contour of this curve in genera1 corresponds with “growth curves.” The rate of growth is greatest during the earIier periods and decreases geometricaIIy to zero in the Iater periods.
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
609
An attempt was made to fit this curve to a simpIe equation. It became easiIy apparent that any equation which wouId even approximateIy reproduce the curve wouId have a compIexity
/I
FIG.8.
49
Graph showing growth in Iength of shafts of right and left tibiae in three rabbits of same litter in which right tibiae were exposed to loo per cent S.E.D. at age of seventy-seven days.
entireIy out of proportion to the accuracy of the experimenta method. The fact, however, that suitabIe simpIe equations for curves cannot be obtained for comparison does not precIude the possibiIity of obtaining vaIuabIe information by comparing the genera1 contours of the curves themseIves. In Figure 7 it wiI1 be noted that the growth in Iength of the unexposed tibiae in rabbits A-I, A-2 and A-3 was nearIy the same. AIso the growth in Iength of the exposed tibiae in rabbits A-2 and A-3 was nearIy identica1. The growth in Iength of the exposed shaft of the tibia in rabbit A-I is much more abnormal than that occurring in the exposed tibiae of rabbits A-2 and A-3, in spite of the fact that presumabIy the same amount of
610
BARNEY
BROOKS
AND
HARRY
T. HILLSTROM
irradiation. was used at the same age. On going back over the protocoIs of the experiments, it was interesting to find that rabbit A-I was first treated, and it is possibIe that a gross
FIG. 9. Graph showing average length of right and Ieft tibiae in rabbits A-2, A-3, A-5 and A-6.
inaccuracy occurred in the estimation of the dosage used. This supposition is supported by examination of Figure 8 which shows the growth in Iength of the norma and exposed tibiae of rabbits A-4, A-5 and A-6. Here, again, it is noted that rabbit A-4, which was first treated, shows a more marked effect than rabbits A-g and A-6, in which the effects were aImost the same. It wouId, therefore, seem probabIe that the differences observed might be attributabIe to the differences in dosage resuIting either from the initia1 operation of the apparatus, or to the oft-repeated experience of finding that in the repeated appIication of a pIan, error is most Iikely to occur with the first effort. Otherwise, it is necessary to assume that the same quantity of irradiation produces different effects in different individuaIs with the same hereditary and environmenta conditions.
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
611
A comparison of growth curves in both normaI and irradiated bones in a11 experiments shows concIusiveIy that the inhibitory effect of the roentgen ray on bone growth is a change in rate of growth and not a change in duration of growth. No instance of compIete cessation of growth in the irradiated bone before compIete cessation of growth in the corresponding norma bone has been observed. This observation might be expIained if it were assumed that the capacity for rate of growth in Iength of bones beIonged to the bone aIone and that the duration of growth was determined by inffuences arising eIsewhere in the body. There is, however, another explanation which, aIthough more fancifu1, perhaps best fits with the resuIts obtained in these experiments. By comparing Figures 7 and 8, or best by studying the composite curves of Figure 9, it wiI1 be noted that the effect produced during the Iater period of growth is very nearIy the same if the bone is exposed either at the age of twenty-one days or seventy-seven days. The curve “RO" is approximately paraIIe1 to the curve “sP." In other words, the effect of a certain amount of irradiation on the growth in Iength during any period is about the same regardIess of the time before this period at which the exposure occurred. This is not onIy important from its cIinica1 appIication, but it may have important significance in the consideration of the possibIe mechanism by which irradiation inhibits growth of bones. EIongation of the shaft of a bone may be considered as periodic finite increments in Iength, as periodic proportiona increments in Iength attained, and, finaIIy, as periodic proportiona1 attainment of its uItimate Iength. If a singIe inffuence of short duration produces a continuous effect throughout the entire period of growth, then it is best to study the effect of this inffuence from the viewpoint of tota future Iength to be attained, or growth capacity. If the capacity for growth of a bone Iies within the bone, it must be assumed to be determined by some component of the bone which, for purposes of discussion, we may further assume
612
BARNEY
BROOKS
AND
HARRY
T. HILLSTROM
to be the ceIIs of the epiphysea1 cartiIage. With this assumption, it foIIows that diminution in the capacity for growth might resuIt from either of the foIIowing mechanisms : I. The capacity for growth may be equaIIy distributed between the ceIIs and be dependent on the number of ceIIs present. Destruction of any definite number of ceIIs wouId thus proportionateIy diminish growth capacity. 2. Growth capacity might be diminished by a diminution in the capacity of each individua1 ceI1. 3. The ceIIs may have varying degrees of growth capacity and different periods of existence. If such were the case, then the growth rate at any period wouId be a summation of the growth capacities of a11 existing ceIIs. Furthermore, ceIIs of different capacities for growth might have different degrees of vuInerabiIity to roentgen rays. Destruction of ce1I.s of Iesser growth capacities and Ionger Iife wouId diminish the rate of growth and shorten the period of growth. Destruction of ceIIs with greater growth capacities and shorter Iife wouId diminish the rate of growth but wouId not shorten the period of growth. The Iast of these methods, fancifu1 as it may seem, conforms best to the resuIts obtained in these experiments. Since connective tissue ceIIs so easiIy reproduce themseIves, and because the roentgen ray apparentIy has IittIe or no inAuence on bone regeneration, it is diffIcuIt to assume that the inhibitory influence of the roentgen ray on bone growth couId be brought about by destruction of ceIIs onIy pecuIiarIy vuInerabIe because of being in a particuIar phase of ceI1 Furthermore, if growth inhibition were produced division. in this way, it wouId seem as if there shouId be some evidences of recovery. Our knowIedge at the present time of the vita1 characteristics of a ceI1 is not sufficient for a discussion of the possibiIities of producing in the cell changes which wouId be constantIy reproduced in successive ceI1 generations. Furthermore, it wouId seem as if a difference in the vuInerabiIity of ceIIs were
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
613
evident from the histoIogica1 appearances of tissues which have been exposed to the roentgen ray. Some of the ceIIs at Ieast show no evidences of abnormaIity and others are obviousIy disintegrated. Instead, therefore, of assuming that ceIIs in the act of division are more susceptible to the destructive infIuence of the roentgen ray, we are assuming that ceIIs with a capacity for rapid division are more susceptibIe than those with a capacity for sIower division. It is obvious that this assumption is not contrary to the facts observed. AIso it is not a distinction without a difference. REFERENCES AI-BEE, F. H. Am. J. M. SC., 159: 40, Ig2o. ANSCHITZ,W., and BECK, A. Zentralbl. j. Cbir., 56: Part II, I180, Igzg. BAENSCH,W. Fortscbr. Geb. a. d. Rhtgenstrablen, 36: Part 2, No. 6, 1245, 1927. BAISCH,B. Ergebn. d. Cbir. u. Ortbop., 7: IIO, 1913. BECK, A. Strablentberapie 32: 517, 1929. CLUZET ~~~DUBREUIL, D. J. depbysiol. et depatb. gh., 15: 327-341; 367-372, 1913. EWING, J. Acta Radiol., 6: 399, 1926. FOSTERLING,K. Arch. j. klin. Cbir., 8 I : Part 2, 506, Igo6. FOSTERLING,K. Zentralbl. j. Cbir., 56: part II, I 178, 1929. FRAENKEL,M. Zentralbl. j. Cbir., 41: part I, No. 26, I 105, Igr4. GOTTHARDT,P. P. Deutscb. Riintgen-Gesellscb., 13: 85, 1923. GRASMANPZ, M. Muncben. med. Wcbnscbr., 74: part II, No. 46, 1960, Ig2T0 HOFFMANN,V. Strablentberapie, 13: 285, 1922. HORNICKE,C. B. Muncben. med. Wcbnscbr., 70: part II, No. 29, p. 943, 1923. HUECK, H., and SPIESS.Strablentberapie, 32: 322, Igzg. ISELIN, H. Ztscbr. j. Cbir., 103: 483, Igro. JUNGLING.Muncben. med. Wcbnscbr., 67: No. 40, part II, 1141-1144; No. 41, p. 1168; 1920. KARGER. Jabrb. j. Cbir., 93: 294, 1920. KRUKENBERG.Deutscb. Rijntgen-Gesellscb., 5: p. 70, Igog. LATTEN, W. Arch. j. klin. Cbir., 163: 427, 1921. LEIST, M. Strablentberapie,24: 268, 1927. LEPPIN. Deutscbe med. Wcbnscbr., No. 28, 1896. MARCUSE.Deutscbe med. Wcbnscbr., No. 30, 1896. Fortscbr. a. d. Geb. d. RGntgenstrabl., 1897. MULLER, W. Deutscb. Ztscbr. j. Cbir., 212: 350, 1928. NAGEOTTE,J. Compt. rend. Sot. de biol., 87: 913, 1922. PERTHES,G. Arch. j. klin. Cbir., 71: Igo3. PHEMISTER,D. B. Ztscbr. j. ortb. Cbir., 55: 161, 1931. PLAGEMANN,H. Deutscb. Riintgen-Gesellscb., 6: 63, 1910. RAHM, H. Strablentberapie, 25: 338, 1927. RECAMIER,D., and TRIBONDEAU, L. Comp. rend. Sot. de biol. Paris, 59: Part
2,621,
1905,
614
BARNEY
BROOKS AND HARRY T. HILLSTROM
REGAUD, C. Compt. rend. Sot. de biol., 87: 427-429; 629-632, 1922. SALVE~I, K. Ztscbr.f. Cbir., 128: 130, 1914. SCHAUDINN, F. Arch. j. Pbysiol., 77: 29, 1899. SCHEDE, F. Ztscbr. j. ortb. Cbir., 31: 497, 19x3. SCHMERZ, H. Beitr. f. klin. Cbir.. 81: 634, 1912. SCHMIDT, W. Bruns’ Beitr. z. klin. Cbir., 145: 441, 1928. STORDEUR, K. Muncben. med. Wcbnscbr., 71: part I, No. I g, 617, 1924. TAMMANN, H. Bruns’ Beitr. f. klin. Cbir., 128: 536, 1923. VE~LCHENFELD. Berl. klin. Wcbnscbr., No. 3, 1896. WILMS. Deutscbe med. Wcbnscbr., 36: part I, No. 6, 259, 1910. WYNEN, W. Muncben. med. Wcbnscbr., 76: Part I, No. 6, 244, 1929.
BROOKS, M.D. AND HARRY
T.
HILLSTROM,
M.D.~
NASHVILLE, TENN.
D
URING the fn-st year after the discovery of the that this form of energy x-rays, it was observed produced profound changes in Iiving matter. In 1903 Perthes found that the growth of one wing of a chicken was greatIy diminished if this wing onIy were exposed to the inffuence of roentgen rays when the chicken was one day oId. Since the appearance of Perthes’ report, numerous investigators have recorded their observations concerning the influence of the roentgen ray on the growth of bones in anima1 experiments and aIso from the therapeutic use of the roentgen ray. From a carefu1 review of a11 the avaiIabIe Iiterature it seemed that there was as yet a sufhcient Iack of cIearness concerning the amount of irradiation necessary to inhibit the growth of bone, the quantity of the effect produced, the duration of the inff uence, and the coincident histoIogica1 changes, to justify the repetition of many of the experiments previousIy performed. The possibiIity of inhibiting or stimuIating regeneration of bone has afso been investigated by experiments upon animaIs and by observations upon patients with bone defects due to trauma or other disease. From previous studies it seems as if it were definiteIy determined that extraordinariIy Iarge doses of irradiation have IittIe or no effect on the capacity of a bone to [regenerate new bone. There is a conflict of opinions concerning the stimuIating effect of the roentgen ray on new bone formation, but it wouId seem as if the maximum stimuIating effect which had been observed is not sufficient to be of importance. * From the Department of Surgery, VanderbiIt University. t Deceased. 599
600
BARNEY BROOKS AND HARRY T. HILLSTROM
The number of experiments which are the basis of this paper is too Iarge for a detaiIed report. OnIy the experimenta method and the resuIts obtained in severa groups of experi-
FIG. I. Photograph of bones of forearms of dog five months old in which right radius and uIna and dista1 end of right humerus were exposed to standard dose of irradiation. Note difference in length of bones of right and left forearms and particulady difference in size of distal ends of right and Ieft humerus.
ments wiI1 be brieffy summarized, and particuIar attention given to the resuks obtained in a group of experiments performed for the purpose of studying some factors which have not been previousIy discussed. EXPERIMENTAL
METHOD
Young rabbits were used in most of the experiments. In a few experiments dogs, guinea pigs, rats and mice were used. No important difference was observed in the resuIts obtained in these different animaIs. In each group of experiments, one or more Iitters of young rabbits were used. As far as possibIe, each Iitter of rabbits was cared for in such a manner that each rabbit of a Iitter was subjected to the same environment except for the age at exposure and the amount of irradiation.
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
601
The referred
factors constituting the standard dose subsequentIy to as IOO per cent s.E.D., were as foIIows: 180,ooo to 200,000 voIts, 20 to 30 ma., 60 cm. target-skin distance, 0.3 mm. copper combined with 1.0 mm. aIuminum fiIters, and forty-five minute exposure. The exposed fieIds varied from 1 X 2 cm. to 1 X 3 cm. Either the right tibia or the right uIna and radius were exposed. The remainder of the animaI’s body was carefuIIy protected with sheet Iead. After exposure the animaIs were cared for on a suburban farm. At reguIar intervaIs varying from one to twenty-eight days, roentgenograms were made of both the exposed and the non-exposed extremities. In each instance the extremity was pIaced as near the fiIm as possibIe and the x-ray tube at a distance of 7 feet from the fiIm. Measurements in growth in Iength of the bones were made from the roentgenograms. In experiments designed for histoIogica1 study, the exposed and non-exposed bones were removed, fixed in formaIin, decaIcified, sectioned and stained with hematoxyIin and eosin. I.
EXPERIMENT OF
ILLUSTRATING GROWING
BONE
THE TO
GROSS
EFFECT
ROENTGEN
OF EXPOSURE
RAYS
The dista1 end of the right humerus and entire Iengths of the radius and uIna of a dog four weeks oId were exposed to a singIe I00 per cent S.E.D. with the technique previousIy described. Four months after exposure the anima1 was sacrificed. The effect on bone growth is iIIustrated in Figure I. From this iIIustration it is cIear that there was a marked inhibition in the growth of those bones exposed to the roentgen rays. The right uIna increased 41 mm. in Iength and the Ieft uIna increased 102 mm. in length. The fact that inhibition of bone growth occurs onIy in the bone which is within the fieId of exposure is apparent from comparison of the proxima1 and dista1 ends of the right humerus. Note aIso that the genera1 contour of the bones in which growth has been inhibited is remarkabIy unchanged.
602 II.
BARNEY BROOKS AND HARRY T. HILLSTROM EXPERIMENTS
GROWING
BONES
THAN
SHOWING
THE
TO THE
ROENTGEN
THE
STANDARD
EFFECT
OF
RAY
SKIN
EXPOSURE
IN DOSES
ERYTHEMA
OF
SMALLER
DOSE
The right tibiae onIy in a series of young rabbits were exposed to doses of roentgen rays estimated at 40 per cent, 60 per cent, 80 per cent and IOO per cent S.E.D. AI1 animaIs receiving 40 per cent or more of the skin erythema dose showed definite inhibition of bone growth. Increasing the amount of irradiation aIways increased the amount of retardation of bone growth. The proportionate decrease in rate of bone growth was greater than the proportionate increase in dosage. III.
EXPERIMENTS STIMULATING
DESIGNED BONE
IRRADIATION
TO
GROWTH
IN SMALLER
PRODUCED
TEST
BY
THE
DOSES
INHIBITION
THE
OF
POSSIBILITY
USE
THAN BONE
OF
THAT
OF
REPEATED WHICH
GROWTH
The right tibiae onIy in a series of young rabbits were subjected to various combinations of repetitions of roentgenray exposures estimated at from 2 per cent to 5 per cent of the skin erythema dose. The animaIs were observed over various periods of time and in no instance was any evidence obtained of stimuIation of bone growth. IV.
EXPERIMENTS EXPOSURE
TO
TO
DETERMINE
DOSES
OF
INHIBIT
THE
ROENTGEN BONE
RESULTS RAYS
OF
REPEATED
SUFFICIENT
TO
GROWTH
The right tibiae onIy in a series of young rabbits were exposed to various combinations of repeated irradiation in doses sufficient to inhibit bone growth. The resuIts of various combinations of amounts of irradiation, number of exposures, and time intervaIs between exposures were observed. The tota quantity of irradiation received aIways produced Iess inhibition of growth the Iarger number of times the dose was divided or the Ionger the interva1 between the exposures. For exampIe, 150 per cent S.E.D. given in 2 equa1 doses on successive days
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
603
had about the same effect as a singIe IOO per cent s.E.D.; whiIe 160 per cent S.E.D. divided into 4 equa1 doses and given during a six day period was not as effective in inhibiting bone growth
FIG. 2. Photomicrograph
of IongitudinaI section of norma proxima1 epiphyseal cartiIage of tibia of a rabbit twenty-seven days old.
as a singIe I00 per cent s.E.D. V.
EXPERIMENTS AND
The exposed
FOR
THE
CARTILAGE
STUDY PRODUCED
OF
THE BY
CHANGES
IN
BONE
IRRADIATION
right tibiae onIy in a series of young rabbits were to the IOO per cent S.&D. of the roentgen ray. The
604
BARNEY
BROOKS
AND
HARRY
T. HILLSTROM
animaIs were sacrificed at periods ranging from twenty-four hours to 148 days. The histoIogica1 changes which occurred in the epiphysea1 cartiIage are iIIustrated in Figures 2, 3 and 4.
FIG. 3. Photomicrograph of IongitudinaI section of proxima1 epiphysea1 cartiIage exposed tibia of same rabbit as iIIustrated in Figure 2, four days after receipt of
of IOO
per cent S.E.D.
A detaiIed description of these changes wiII be reported in a separate paper. In genera1 it may be said that immediateIy after exposure of the epiphysea1 cartiIage to the standard IOO per cent S.E.D. there is marked evidence of serious damage to the cartiIage
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
605
of the ceI1.sbecomes irreguIar. Some ceIIs ceIIs. The arrangement are swoIIen and others are obviousIy disintegrated. Some ceIIs appear norma and are reguIarIy arranged. The irregu-
FIG. 4. Photomicrograph of Iongitudinal section of promima1 epiphyseal cartiIage of tibia of rabbit fifteen days after exposure to 40 per cent S.E.D.
Iarity of arrangement persists throughout the entire period of bone growth. During the Iatter part of the period of bone growth the epiphysea1 cartiIage becomes irreguIar in thickness. In some instances the cartiIage disappears compIeteIy in some areas. It is particuIarIy during this period that growth deformities deveIop.
606 VI.
BARNEY
BROOKS
EXPERIMENTS
AND
DESIGNED
IRRADIATION
ON
HARRY TO
BONE
T. HILLSTROM
STUDY
THE
EFFECT
OF
REGENERATION
It has been previousIy pointed out by the author that growth in Iength and thickness of bones is a process which operates onIy during a Iimited period of Iife, and that the capacity of a bone to regenerate a defect is not Iimited to any period of Iife. It wouId seem, therefore, as if the two processes must be independent. Previous investigators have demonstrated that repair of bone was not perceptibIy inffuenced by exposure to reIativeIy Iarge doses of the roentgen rays. For our own satisfaction, however, ten experiments were carried out on young rabbits. EquaI defects in the right and the Ieft uIna were produced by subperiostea1 resections of the bone shafts. In each of the rabbits the right foreIeg was exposed to the IOO per cent S.E.D. In some of the rabbits this exposure was made immediateIy after operatiori, and in others on the third to sixteenth day after production of the bone defect. The animaIs were frequentIy observed throughout the process of repair. In each instance growth of the right uIna was inhibited. In no instance, however, was there any perceptibIe difference in the amount of new bone formation or in the duration or compIeteness of the process of repair. No experiments were performed in which smaIIer doses were used with the idea of stimuIating bone repair. VII.
EXPERIMENTS
ROENTGEN
RAY
FOR
DETERMINING
ON THE IN
FORM
LENGTH
THE
OF THE OF
INFLUENCE
CURVES
OF THE
OF GROWTH
BONES
Six white rabbits from the same Iitter, born May 14, 1932, were IabeIed A-I, A-2, A-3, A-4, A-5 and A-6. The entire right tibiae of rabbits A-I, A-2 and’ A-3 were irradiated on June 4, 1932 with IOO per cent S.E.D. AI1 of the rabbits were kept as nearIy as possibIe under the same conditions. The entire right tibiae of rabbits A-4, A-5 and A-6 were exposed to IOO per cent S.E.D. on JuIy 30, 1932. Roentgenograms of both
-
--
.._-. 49
77
3;. :
105
ST
Exp. A-z. FIG. f;. Serial roentgenograms made at intervaIs of twenty-eight days in rabbit in which right tibia and libuIa were exposed to IOO per cent S.E.D. at age of twenty-one days. LEFT
RlCHT
:: _._-
-
AGE
.
._
133
161
Exp. A-4. FIG. 6. Serial roentgenograms made at intervals of twenty-eight days in rabbit of same litter as illustrated in Figure 5, in which right tibia and fibuIa were exposed to IOO per cent S.E.D. at age of seventy-seven days.
DAYS
608
BARNEY BROOKS AND HARRY T. HILLSTROM
tibiae of a11 rabbits were made every twenty-eight days for six months. The resuIts obtained in rabbits A-2 and A-4 are iIIustrated in Figures 5 and 6. The measurements of the Iengths
FIG. 7. Graph showing growth in length of shafts of right and Ieft tibiae in three rabbits of same Iitter in which right tibiae were exposed to IOO per cent S.E.D. at age of twentyone days.
of the shafts of both tibiae of a11rabbits are shown graphicaIIy in Figures 7 and 8. Figure g is a graphic representation of the average growth in Iengths of experiments A-2, A-3, A-5 and A-6. A study of these curves reveaIs certain important facts which have not heretofore been discussed in the various reports deaIing with the effect of the roentgen ray on the growth of bone : Growth in Iength of the shaft of the norma unexposed tibia was remarkabIy constant in a11 of the six rabbits. The contour of this curve in genera1 corresponds with “growth curves.” The rate of growth is greatest during the earIier periods and decreases geometricaIIy to zero in the Iater periods.
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
609
An attempt was made to fit this curve to a simpIe equation. It became easiIy apparent that any equation which wouId even approximateIy reproduce the curve wouId have a compIexity
/I
FIG.8.
49
Graph showing growth in Iength of shafts of right and left tibiae in three rabbits of same litter in which right tibiae were exposed to loo per cent S.E.D. at age of seventy-seven days.
entireIy out of proportion to the accuracy of the experimenta method. The fact, however, that suitabIe simpIe equations for curves cannot be obtained for comparison does not precIude the possibiIity of obtaining vaIuabIe information by comparing the genera1 contours of the curves themseIves. In Figure 7 it wiI1 be noted that the growth in Iength of the unexposed tibiae in rabbits A-I, A-2 and A-3 was nearIy the same. AIso the growth in Iength of the exposed tibiae in rabbits A-2 and A-3 was nearIy identica1. The growth in Iength of the exposed shaft of the tibia in rabbit A-I is much more abnormal than that occurring in the exposed tibiae of rabbits A-2 and A-3, in spite of the fact that presumabIy the same amount of
610
BARNEY
BROOKS
AND
HARRY
T. HILLSTROM
irradiation. was used at the same age. On going back over the protocoIs of the experiments, it was interesting to find that rabbit A-I was first treated, and it is possibIe that a gross
FIG. 9. Graph showing average length of right and Ieft tibiae in rabbits A-2, A-3, A-5 and A-6.
inaccuracy occurred in the estimation of the dosage used. This supposition is supported by examination of Figure 8 which shows the growth in Iength of the norma and exposed tibiae of rabbits A-4, A-5 and A-6. Here, again, it is noted that rabbit A-4, which was first treated, shows a more marked effect than rabbits A-g and A-6, in which the effects were aImost the same. It wouId, therefore, seem probabIe that the differences observed might be attributabIe to the differences in dosage resuIting either from the initia1 operation of the apparatus, or to the oft-repeated experience of finding that in the repeated appIication of a pIan, error is most Iikely to occur with the first effort. Otherwise, it is necessary to assume that the same quantity of irradiation produces different effects in different individuaIs with the same hereditary and environmenta conditions.
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
611
A comparison of growth curves in both normaI and irradiated bones in a11 experiments shows concIusiveIy that the inhibitory effect of the roentgen ray on bone growth is a change in rate of growth and not a change in duration of growth. No instance of compIete cessation of growth in the irradiated bone before compIete cessation of growth in the corresponding norma bone has been observed. This observation might be expIained if it were assumed that the capacity for rate of growth in Iength of bones beIonged to the bone aIone and that the duration of growth was determined by inffuences arising eIsewhere in the body. There is, however, another explanation which, aIthough more fancifu1, perhaps best fits with the resuIts obtained in these experiments. By comparing Figures 7 and 8, or best by studying the composite curves of Figure 9, it wiI1 be noted that the effect produced during the Iater period of growth is very nearIy the same if the bone is exposed either at the age of twenty-one days or seventy-seven days. The curve “RO" is approximately paraIIe1 to the curve “sP." In other words, the effect of a certain amount of irradiation on the growth in Iength during any period is about the same regardIess of the time before this period at which the exposure occurred. This is not onIy important from its cIinica1 appIication, but it may have important significance in the consideration of the possibIe mechanism by which irradiation inhibits growth of bones. EIongation of the shaft of a bone may be considered as periodic finite increments in Iength, as periodic proportiona increments in Iength attained, and, finaIIy, as periodic proportiona1 attainment of its uItimate Iength. If a singIe inffuence of short duration produces a continuous effect throughout the entire period of growth, then it is best to study the effect of this inffuence from the viewpoint of tota future Iength to be attained, or growth capacity. If the capacity for growth of a bone Iies within the bone, it must be assumed to be determined by some component of the bone which, for purposes of discussion, we may further assume
612
BARNEY
BROOKS
AND
HARRY
T. HILLSTROM
to be the ceIIs of the epiphysea1 cartiIage. With this assumption, it foIIows that diminution in the capacity for growth might resuIt from either of the foIIowing mechanisms : I. The capacity for growth may be equaIIy distributed between the ceIIs and be dependent on the number of ceIIs present. Destruction of any definite number of ceIIs wouId thus proportionateIy diminish growth capacity. 2. Growth capacity might be diminished by a diminution in the capacity of each individua1 ceI1. 3. The ceIIs may have varying degrees of growth capacity and different periods of existence. If such were the case, then the growth rate at any period wouId be a summation of the growth capacities of a11 existing ceIIs. Furthermore, ceIIs of different capacities for growth might have different degrees of vuInerabiIity to roentgen rays. Destruction of ce1I.s of Iesser growth capacities and Ionger Iife wouId diminish the rate of growth and shorten the period of growth. Destruction of ceIIs with greater growth capacities and shorter Iife wouId diminish the rate of growth but wouId not shorten the period of growth. The Iast of these methods, fancifu1 as it may seem, conforms best to the resuIts obtained in these experiments. Since connective tissue ceIIs so easiIy reproduce themseIves, and because the roentgen ray apparentIy has IittIe or no inAuence on bone regeneration, it is diffIcuIt to assume that the inhibitory influence of the roentgen ray on bone growth couId be brought about by destruction of ceIIs onIy pecuIiarIy vuInerabIe because of being in a particuIar phase of ceI1 Furthermore, if growth inhibition were produced division. in this way, it wouId seem as if there shouId be some evidences of recovery. Our knowIedge at the present time of the vita1 characteristics of a ceI1 is not sufficient for a discussion of the possibiIities of producing in the cell changes which wouId be constantIy reproduced in successive ceI1 generations. Furthermore, it wouId seem as if a difference in the vuInerabiIity of ceIIs were
EFFECT OF ROENTGEN RAYS ON BONE GROWTH
613
evident from the histoIogica1 appearances of tissues which have been exposed to the roentgen ray. Some of the ceIIs at Ieast show no evidences of abnormaIity and others are obviousIy disintegrated. Instead, therefore, of assuming that ceIIs in the act of division are more susceptible to the destructive infIuence of the roentgen ray, we are assuming that ceIIs with a capacity for rapid division are more susceptibIe than those with a capacity for sIower division. It is obvious that this assumption is not contrary to the facts observed. AIso it is not a distinction without a difference. REFERENCES AI-BEE, F. H. Am. J. M. SC., 159: 40, Ig2o. ANSCHITZ,W., and BECK, A. Zentralbl. j. Cbir., 56: Part II, I180, Igzg. BAENSCH,W. Fortscbr. Geb. a. d. Rhtgenstrablen, 36: Part 2, No. 6, 1245, 1927. BAISCH,B. Ergebn. d. Cbir. u. Ortbop., 7: IIO, 1913. BECK, A. Strablentberapie 32: 517, 1929. CLUZET ~~~DUBREUIL, D. J. depbysiol. et depatb. gh., 15: 327-341; 367-372, 1913. EWING, J. Acta Radiol., 6: 399, 1926. FOSTERLING,K. Arch. j. klin. Cbir., 8 I : Part 2, 506, Igo6. FOSTERLING,K. Zentralbl. j. Cbir., 56: part II, I 178, 1929. FRAENKEL,M. Zentralbl. j. Cbir., 41: part I, No. 26, I 105, Igr4. GOTTHARDT,P. P. Deutscb. Riintgen-Gesellscb., 13: 85, 1923. GRASMANPZ, M. Muncben. med. Wcbnscbr., 74: part II, No. 46, 1960, Ig2T0 HOFFMANN,V. Strablentberapie, 13: 285, 1922. HORNICKE,C. B. Muncben. med. Wcbnscbr., 70: part II, No. 29, p. 943, 1923. HUECK, H., and SPIESS.Strablentberapie, 32: 322, Igzg. ISELIN, H. Ztscbr. j. Cbir., 103: 483, Igro. JUNGLING.Muncben. med. Wcbnscbr., 67: No. 40, part II, 1141-1144; No. 41, p. 1168; 1920. KARGER. Jabrb. j. Cbir., 93: 294, 1920. KRUKENBERG.Deutscb. Rijntgen-Gesellscb., 5: p. 70, Igog. LATTEN, W. Arch. j. klin. Cbir., 163: 427, 1921. LEIST, M. Strablentberapie,24: 268, 1927. LEPPIN. Deutscbe med. Wcbnscbr., No. 28, 1896. MARCUSE.Deutscbe med. Wcbnscbr., No. 30, 1896. Fortscbr. a. d. Geb. d. RGntgenstrabl., 1897. MULLER, W. Deutscb. Ztscbr. j. Cbir., 212: 350, 1928. NAGEOTTE,J. Compt. rend. Sot. de biol., 87: 913, 1922. PERTHES,G. Arch. j. klin. Cbir., 71: Igo3. PHEMISTER,D. B. Ztscbr. j. ortb. Cbir., 55: 161, 1931. PLAGEMANN,H. Deutscb. Riintgen-Gesellscb., 6: 63, 1910. RAHM, H. Strablentberapie, 25: 338, 1927. RECAMIER,D., and TRIBONDEAU, L. Comp. rend. Sot. de biol. Paris, 59: Part
2,621,
1905,
614
BARNEY
BROOKS AND HARRY T. HILLSTROM
REGAUD, C. Compt. rend. Sot. de biol., 87: 427-429; 629-632, 1922. SALVE~I, K. Ztscbr.f. Cbir., 128: 130, 1914. SCHAUDINN, F. Arch. j. Pbysiol., 77: 29, 1899. SCHEDE, F. Ztscbr. j. ortb. Cbir., 31: 497, 19x3. SCHMERZ, H. Beitr. f. klin. Cbir.. 81: 634, 1912. SCHMIDT, W. Bruns’ Beitr. z. klin. Cbir., 145: 441, 1928. STORDEUR, K. Muncben. med. Wcbnscbr., 71: part I, No. I g, 617, 1924. TAMMANN, H. Bruns’ Beitr. f. klin. Cbir., 128: 536, 1923. VE~LCHENFELD. Berl. klin. Wcbnscbr., No. 3, 1896. WILMS. Deutscbe med. Wcbnscbr., 36: part I, No. 6, 259, 1910. WYNEN, W. Muncben. med. Wcbnscbr., 76: Part I, No. 6, 244, 1929.