Investigations on Congenital and Induced Osteopetrosis

DONALD G. WALKER Department of Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Osteopetrosis, or marble bone roid body and bone obtained from thermore, the fact that bone ero­ disease, is a disturbance of skeletal a normal littermate, little or no evi­ sion failed to occur, even when the development in which the rate of dence of bone erosion was demon­ parathyroid body was in direct con­ bone resorption fails to keep pace strable at the end of the culture tact with the calvarial bone, rules with the rate of bone formation. period. out the possibility that a parathor­ Bone matrix accumulates exces­ In another experiment (Barnicot, mone-destroying tissue intervenes sively throughout the skeletal sys­ 1941) , ribs from a grey-lethal between the gland and its target. tem, causing damage to neighbor­ mouse were transplanted into a In seeking additional information ing tissues-particularly the dental, normal littermate. When exam­ as a basis for an alternative to hematopoietic and nervous tissues. ined histologically two weeks later, Barnicot's explanation, I first de­ The line of investigation on the rib grafts appeared normal. voted my efforts to measuring the osteopetrosis which has led to the In the reciprocal transplantation, plasma calcium concentration in current point of view that the thy­ in which a rib from a normal untreated animals of both sexes at roid gland represents the primary mouse was cultured subcutaneously various ages. All of the grey-lethals site of the disturbance was initiated in a grey-lethal littermate, the graft examined, including the younger soon after an experimental animal underwent osteopetrotic changes. well-nourished group, were found became available. In 1935 Hans From these results it was evident to be hypocalcemic, with a total Grlineberg described a mutant of that the failure of bone remodelling plasma calcium averaging 20% be­ the house mouse, named the grey­ in the osteopetrotic mouse was not low the mean value of plasma cal­ lethal, whose skeletal system explicable on the basis of incompe­ cium obtained from the normal showed manifestations of osteope­ tence on the part of either osteo­ littermates. In response to a single trosis which closely resembled clasts or the chief cells of the para­ injection of PTE at a dosage of those in man originally described thyroid gland. In an effort to 5 units/ gm of body weight, the by Albers-Schonberg in 1904. promote the resorption of the ex­ plasma calcium level of normal Barnicot (1948), working at Uni­ cess bone, Barnicot ( 1945) dis­ mice one to four weeks of age versity College, was the first to covered that the grey-lethal mouse increased by 100% in 18 hours show that osteopetrosis is an endo­ suffered no ill effects from repeated and 200% in 36 hours. Grey-lethal crine disorder. His most interesting injections of parathyroid extract mice given the same dosage of work involved the reciprocal trans­ (PTE) at a dose level that was PTE showed a maximum elevation plantation of parathyroid tissue and toxic enough to kill the normal of plasma calcium concentration of bone. In one experiment he took siblings after only one exposure. only 25 % , reached within 18 from a grey-lethal mouse a piece of Thus, Barnicot terminated his study hours. By 36 hours the plasma cal­ calvarial bone and a parathyroid of the grey-lethal mouse by sug­ cium fell to a tetanic threshold body, attached the gland to the gesting that osteopetrosis is a form level. In response to injections of surface of the bone, and implanted of hypoparathyroidism which can­ PTE administered at intervals of the graft intracerebrally in a normal not be cured by parathormone 12 hours for as long as eight days, sibling. A few days later, when the therapy alone. the grey-lethal mouse never be­ graft was removed for examination, There are two reasons why the came hypercalcemic, whereas the the calvarial bone was found to "hypoparathyroid hypothesis" is normal littermates invariably died have been perforated in the region not tenable. It has long been known by the second or third day with underlying the parathyroid gland. that normal mice which have been extreme hypercalcemia and nephro­ When the grey-lethal mouse was thyroparathyroidectomized at birth calcinosis. used as host for grafting a parathy- do not develop osteopetrosis. Fur- In an effort to locate the basis

MCV QUARTERLY 5(1): 17-19, 1969 17 OSTEOPETROSIS for the great resistance to parathy­ activity than to decreased osteo­ three other mutants with osteope­ roid hormone observed in the grey­ clastic activity. In attempting to trosis have recently been studied. lethal, a thorough cytological and locate an abnormally enlarged The implication of the findings ob­ histochemical assessment of osteo­ source of an osteoblastic-stimulat­ tained in microphthalmic and os­ clasts was conducted. Bone samples ing factor, the pituitary and thyroid teosclerotic mice is essentially the obtained from grey-lethals between glands were surveyed thoroughly same as the implication of the find­ 12 and 24 hours after the admin­ by means of light and electron mi­ ings obtained from the grey-lethal istration of parathormone showed croscopy. No evidence of increased (S. C. Marks and D. G. Walker, widespread or intense signs of os­ somatotroph or follicle cell activi­ unpublished data) . teoclastic activity, including an ex­ ties was disclosed; however, a Rabbits with hereditary osteope­ ceptionally high level of collagen­ parafollicular cell hyperplasia was trosis as well as the mice with con­ ase (Walker, l 966a). However, in discovered in the parathormone­ genital osteopetrosis are hypocal­ the untreated mice and at all time treated grey-lethals. In electron mi­ cemic throughout life. They fail points later than 24 hours in the crographs the parafollicular cell to develop hypercalcemia even parathormone-treated animals, os­ differs from the follicle cell in when placed on an intensive para­ teolytic activity was subnormal, several respects. It is of a larger thormone regimen. However, the whereas the signs of increased os­ size, has a shape that is oval instead abnormal tolerance · to parathor­ teoblastic activity were most im­ of polygonal, and occupies a more mone in the rabbit does not seem pressive. The endosteum was hyper­ peripheral (parafollicular) posi­ to be related to parafollicular cell plastic, containing several layers tion, resting against the basement · activity, as it was in osteopetrotic of osteoprogenitor cells as well as membrane of the follicle but never mice. The parafollicular cell popu­ a prominent layer of large osteo­ bordering directly on the colloid. lation is not elevated in the rabbits, blasts; adjacent to the endosteum Unlike all other epithelial cells, the and, even after thyroidectomy or was a thick seam of osteoid. The parafollicular cells are devoid of thyroparathyroidectomy, the osteo­ hyperplastic endosteum was dis­ intercellular attachment devices, petrotic rabbits are refractory to tributed over most of the trabecular such as desmosomes, light junc­ the influence of parathormone. Fur­ surface in the grey-lethal bones. In tions, and interlocking processes. thermore, the rabbit mutants never order to place the osteogenic eval­ The most distinctive feature of the show increased osteogenic activity; uation on a quantitative basis, I parafollicular cells is the abundance in fact, according to tritiated pro­ employed tritiated proline incor­ of cytoplasmic secretory vesicles. line incorporating studies carried poration studies. Tritiated proline These vesicles are about 0.2µ, in out at various ages from birth to was administered intraperitoneally diameter and appear to be empty one month of age, their capacity to at a dosage of 4µ,c to 8µ,c per but may contain thyrocalcitonin, make bone matrix is only about gm of body weight, and the animal a substance of homogeneous ap­ half that of the normal littermates. was sacrificed six hours later. pearance and low electron opacity. Osteopetrotic rabbit bones show re­ Samples of calvarial and tibial bone Since the parafollicular cell has duced numbers of osteoblasts, os­ were pulverized, extracted in ether, been shown to be the source of teoclasts of abnormal appearance suspended in scintillation fluid and thyrocalcitonin, the presence of a and an abundance of mesenchyme counted by means of a well-type parafollicular cell hyperplasia may throughout the intertrabecular automated scintillation counter. The represent an excessive capacity to spaces (D. G. Walker and R. R. counts obtained from the untreated produce thyrocalcitonin. Chronic Fox, unpublished data) . animals indicated that the grey­ hypercalcitoninemia would be as­ On the basis of the fragmentary lethal incorporated 50% more of sociated with sustained hypocal­ data available at present, the pri­ the 3H-proline into bone matrix cemia, hypophosphatemia, and a mary defect in hereditary osteo­ than the normal control. Parathy­ retardation of bone resorption. All petrosis of the rabbit appears to be roid hormone was found to depress of these signs are characteristic of located in osseous tissue rather incorporation in the normal by osteopetrosis as manifested in the than in the endocrine system. about 50% but in the grey-lethal grey-lethal mouse. The increased Recently, osteopetrosis has been by only about 15% (Walker, rate of bone formation observed in induced in normal mice (S. C. 1966b). the grey-lethal might indicate that Marks, unpublished data), rabbits According to our interpretation thyrocalcitonin has a dual action on and rats (D. G . Walker and M.A. of the results of the evaluation of bone-inhibiting osteolytic activity, Shepp, unpublished data) by means osteolytic and osteogenic activities yet stimulating osteogenic activity of daily injections of PTE at the in the osteopetrotic mouse, the im­ -a combination of effects di­ moderately low dose level of 0.5 balance leading to the excessive ac­ ametrically opposed to the effects of units/ gm of l:\ody weight. Through­ cumulation of bone is more likely parathormone. out the two-week or longer PTE to be due to increased osteoblastic In addition to the grey-lethal, regimen initiated at birth, the med-

18 D. G. WALKER ullary cavities of the long bones inhibiting bone resorption. There­ I. The effect of subcutaneous trans­ failed to enlarge, and the cancel­ fore, at the present time we are in­ plantation of bones of the grey­ lous bone of the epiphyses and me­ vestigating other possible sources lethal house mouse into normal taphyses became increasingly dense, for the osteoblast-stimulating influ­ hosts and of normal bones into grey-lethal hosts. Am. /. Anat. 68: gradually approaching compact ence. In order to determine whether 497-531, 1941. bone in appearance. The experi­ or not the growth-promoting effect mentally osteopetrotic animals, like was mediated by the pituitary gland, ---. Some data on the effect of parathormone on the grey-lethal the congenitally osteopetrotic mice, rats hypophysectomized at two mouse. /. Anal. 79:83-91, 1945. showed increased extent of osteo­ weeks of age were injected with ---. The local action of the para­ blastic activity in autoradiographic either growth hormone or PTE or a thyroid and other tissues on bone studies and elevated levels of triti­ combination of the two hormones in intracerebral grafts. /. Anat. 82: ated proline incorporation into bone for a period of three weeks. As 233-248, 1948. matrix as determined by counting compared with the untreated hypo­ MARKS, S. C. Experimental osteope­ of radioactivity. In general, the physectomized controls, hormone­ trosis in the mouse. / . Bone Joint counts indicated that the experi­ injected hypophysectomized rats Surg., in press. mental animals incorporated two showed approximately a tenfold in­ MARKS, S. C. AND D. G. WALKER. to three times as much of the ad­ crease in the number of parafollic­ The role of the parafollicular cell ministered label as did their normal ular cells of the thyroid gland of the thyroid gland in the path­ littermate controls. In addition, and a threefold to fivefold increase ogenesis of congenital osteopetrosis parafollicular cell hyperplasia was in the bone label-incorporating ca­ in mice. Am. I. Anal., in press. shown to exist in all of the animals pacity. The excessive accumulation WALKER, D. G. Elevated bone col­ with induced osteopetrosis. The of bone matrix was observed only lagenolytic activity and hyperplasia parafollicular cell population in the in those animals receiving PTE, of parafollicular light cells of the experimental animals was increased but the .osteopetrotic trend devel­ thyroid gland in parathormone­ about eightfold over that of the oped more rapidly in animals re­ treated grey-lethal mice. Z. Zell­ controls. ceiving growth hormone in addi­ forsch. Mikroskop. Anal. 72: 100- 124, 1966a. The explanation for the mecha­ tion to PTE. The disturbance of nism which induced osteopetrosis in calcium homeostasis that leads to ---. Counteraction to parathyroid therapy in osteopetrotic mice as re­ normal mice was as follows. The the excessive accumulation of bone vealed in the plasma calcium level daily injection of PTE caused a matrix occurs not only when the and ability to incorporate 3H-pro­ transient hypercalcemia. This, in endogenous source of parathor­ line into bone. Endocrinology 79 : turn, stimulated the secretory and mone is inhibited, but also when 836-842, 1966b. proliferative activities of the para­ thyrocalcitonin production is in­ follicul ar cells, leading to an over­ creased. This dual effect is created production of thyrocalcitonin. The by the administration of PTE at a explanation received additional sup­ low dose level. However, adminis­ port from the finding that osteo­ tration of growth hormone indi­ petrosis could not be induced in rectly stimulates the activities of mice that had been completely the chief cell of the parathyroid thyroidectomized. In those instances gland as well as the parafollicular where the thyroidectomy was not cell of the thyroid and, thus, cal­ complete, the degree of osteope­ cium homeostasis is not disturbed. trosis induced by the parathormone The fact that growth hormone acts regimen correlated directly with synergistically with PTE in pro­ the number of parafollicular cells ducing osteopetrosis can be ex­ counted in the remnant of thyroid plained simply by the fact that gland (S. C. Marks and D . G. growth hormone stimulates chon­ Walker, unpublished data). drogenesis, and the more rapid the From these investigations on rate of cartilage production by the hereditary and induced osteope­ epiphyseal plate, the more rapid trosis has emerged the theory that the bone accumulation under con­ the parafollicular cell is the source ditions where bone resorption has of a potent osteoblast-stimulating been inhibited. factor which may be an entity dis­ tinct from thyrocalcitonin. Accord­ References ing to general consensus, thyrocal­ BARNICOT, N. A. Studies on the citonin has but one action, namely, factors involved in bone absorption.

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