The effect of cortisol on bone cell differentiation.

The literature contains inconsistencies concerning the effect of corticosteroids on skeletal tissues. An in vitro system was developed to isolate the effects of cortisol on bone cell populations from those effects due to a secondary hyperparathyroidism. One hour prior to sacrifice, newborn mice were injected with tritiated-thymidine. The radii were placed into organ culture for four days. Effects of cortisol (0.0001, 0.01, 1.0 and 100.0 ug/ml media) and parathyroid extract (0.1, 1.0 and 10.0 units/ml media) on the bone cell populations were studied. Low doses of cortisol (0.0001 ug/ml media) stimulate progenitor cell mitosis and induce bone remodeling through increased differentiation of osteoblasts and osteoclasts. Higher doses of cortisol (above 1.0 ug/ml media) decreased progenitor cell mitosis, inhibit bone formation through decreased osteoblasts differentiation and preferentially protect and increase the resorbing cell population. Parathyroid extract, except at high levels (10 units PTE/ml media), stimulates progenitor cell mitosis and preferentially increases resorbing cell differentiation to the exclusion of osteoblast production. Cortisol and parathyroid extract can be seen to oppose each other in most effects on bone cell populations. While both increase resorbing cell populations, 45Ca metabolism of the whole bone in culture indicates that the activity of the individual cell is depressed by cortisol. Possible inter-relationships of the different bone cells through differentiation and modulation are discussed.

THE EFFECT OF CORTISOL ON BONE CELL DIFFERENTIATION by Susan Ann Schafer A dissertation submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Anatomy University of Utah August 1971 This Dissertation fo r the Doctor of Philosophy Degree by Susan Ann Schafer has been approved July 1971 Dean, �e Gradu School ACKNOWLEDGMENTS I would 1 ike to express honest appreciation to my friends, Drs. Robert Huseby and Barbara Jacobs, without whose encouragement and support I would not have entered graduate school, and Dr. Walter Stevens, without whose counsel I would not have finished. A simple thanks is extended to Dr. Thomas Dougherty, for himself; Dr. Ira Pilgrim (and Dr. David Steinmuller) for healthy, fertile and free mice; and Dr. Lowell Woodbury, for instantaneous statistical analysis. TABLE OF CONTENTS ... ... . ACKNOWLEDGMENTS ABSTRACT. Page iii vi CHAPTER 1• I NTRODUCT ION. 2. HISTORICAL REVIEW •• 2 EFFECTS OF ADRENAL CORTICAL STEROIDS ON NON-SKELETAL TISSUES • • • • • • • • • 2 EFFECTS OF ADRENAL CORTICAL STEROIDS ON BONE CELL POPULATIONS IN VIVO. • • • ••• 3 -- 3. SECONDARY HYPERPARATHYROIDISM. 11 BIOTRANSFORMATION OF ADRENAL CORTICAL STEROIDS. • • 16 EFFECTS OF ADRENAL CORTICAL STEROIDS ON BONE IN VITRO 18 MATERIALS AND METHODS 27 GENERAL INFORMATION 27 EXPERIMENTAL ANIMALS AND TREATMENTS. 29 The effect of cortisol on the release of previouslyincorporated 45Ca from mouse 1 imb-bones ~ vitro. 29 The time required for bone cell differentiation in infant mice in vivo. • • • • • • • • . • • • • • 30 The effect of cortisol on bone cell differentiation in vitro •• 31 The effect of parathyroid extract on bone cell differentiation in vitro. 32 -00. • • • • • • • • • • • ANALYTICAL METHODS • • • • • 32 Method of organ culture. 32 Plexiglas-methylmethacry1ate embedding •• 36 iv Chapter Page Autoradiographic procedure. 38 .... Cell counting. 4. 5. RESULTS • • • • • • 47 THE EFFECT OF CORTISOL ON THE RELEASE OF PREVIOUSLYINCORPORATED 45Ca FROM MOUSE LIMB-BONES IN VITRO. 47 THE TIME REQUIRED FOR BONE CELL DIFFERENTIATION IN INFANT MICE ~ VIVO. • • • • • • • • • . • • 47 THE EFFECT OF CULTURE ON BONE CELL POPULATIONS. • 60 THE EFFECT OF CORTISOL ON BONE CELL DIFFERENTIATION IN VITRO • • • • • • • • • • • • 63 THE EFFECT OF PARATHYROID EXTRACT ON BONE CELL DIFFERENTIATION IN VITRO • • • • • • 73 ...... .... .... DISCUSSION • • • • • • • • • • • 81 THE EFFECT OF CORTISOL ON THE RELEASE OF PREVIOUSLYINCORPORATED 45Ca FROM MOUSE LIMB-BONES IN VITRO. • 81 THE TIME REQUIRED FOR BONE CELL DIFFERENTIATION IN INFANT MICE IN VIVO. • • • • • • • • • • • • • • 82 THE EFFECT OF CULTURE ON BONE CELL POPULATIONS. 88 THE EFFECT OF CORTISOL ON BONE CELL DIFFERENTIATION IN VITRO • • • • 88 THE EFFECT OF PARATHYROID EXTRACT ON BONE CELL DIFFERENTIATION IN VITRO. • • • • • 90 -0 6. 39 • 0 • • • • • • • • • 92 SUMMARY. BIBLIOGRAPHY ••• 93 VITA • • • . • • • 104 v ABSTRACT The 1 iterature contains inconsistencies concerning the effect of corticosteroids on skeletal tissues. An ~ vitro system was developed to isolate the effects of cortisol on bone cell populations from those effects due to a secondary hyperparathyroidism. One hour prior to sacrifice, newborn mice were injected with trltiated-thymidine. radi i were placed into organ culture for four days. The Effects of cortisol (0.0001,0.01, 1.0, and 100.0 Wg/ml media) and parathyroid extract (0.1, 1.0 and 10.0 units/ml media) on the bone cell populations were studied. Low doses of cortisol (0.0001 ~g/ml media) stimulate progenitor cell mitosis and induce bone remodel ing through increased differentiation of osteoblasts and osteoclasts. (above 1.0 ~g/ml Higher doses of cortisol media) decrease progenitor cell mitosis, inhibit bone formation through decreased osteoblast differentiation and preferentially protect and increase the resorbing cell population. Parathyroid extract, except at high levels (10 units PTE/ml media), stimulates progenitor cell mitosis and preferentially increases resorbing cell differentiation to the exclusion of osteoblast production. Cortisol and parathyroid extract can be seen to oppose each other in most effects on bone cell populations. While both increase resorbing cell populations, 45Ca metabol ism of the whole bone in culture indicates that the activity of the individual cell is depressed by cortisol. Possible inter-relationships of the different bone cells through differentiation and modulation are discussed. vi Chapter 1 INTRODUCTION Since Cushing first called attention to the skeletal involvement of hyperadrenalcortical ism in 1932, unsuccessful attempts have been made to elucidate the mechanism by which corticosteroids produce osteoporosis. The major problem has been the fact that cortico- steroids produce a variety of responses in skeletal tissue. The inconsistencies in the 1 iterature have been attributed to species differences and to time and dose dependent secondary reactions within a given specie. Two explanations are currently given serious consideration to explain the varied responses of skeletal tissue to cortisol. The first hypothesis involves the concomitant responses of the skeleton to secondary hyperparathyroidism. The second hypothesis concerns the possible conversion of the corticosteroid to a substance which is either inactive or which has an effect opposite to that of the corticosteroid. It is the purpose of this dissertation to determine dose dependent effects of cortisol on bone cell populations. An ~ vitro system was developed to isolate these direct effects of the steroid on bone cell differentiation from those effects due to a secondary hyperparathyroidism. Chapter 2 HISTORICAL REVIEW EFFECTS OF CORTISOL ON NON-SKELETAL TISSUES In 1936 Hans Selye made the fundamental discovery that any biological stress is followed by a specific sequence of events. The "general adaptation syndrome" he described includes enlargement of the adrenal cortex due to hyperemia, intense atrophy of lymphatic structures including the thymus, spleenJ and circulating lymphocytes, and gastic bleeding (Selye, '36). The lympholytic effect of adrenal extracts has since been described in great detail (Dougherty and White, 143, 145). Cortisol causes small and medium-sized lymphocytes to bud and shed their cytoplasm, resulting in eventual death (Illymphocytokaryohexis"). Adrenal cortical steroids also decrease the number of mitotic figures in lymphatic tissue. The anti-inflammatory action of cortisol is another consequence of its protective effect. Hyperemia and exudation are decreased in a wound area due to decreases by cortisol in capillary dilatation and permeability and to inhibition of cellular migration and infiltration. Histamine has been impl icated as the trigger to inflammation (Eyring and Dougherty, 155). Its intracellular synthesis is inhibited by cort i so 1 (Schayer, 163). The prime metabol ic effect of cortisol is gluconeogenesis (Drury, 142). This process is manifested by hyperglycemia, glycosuria, and 2 3 protein depletion. In addition to increasing the production of glucose, cortisol decreases its peripheral util ization. A decrease in glucose uptake and in production of glucose-6-phosphate is produced in thymocytes as early as 15 min after exposure to cortisol in vitro (Munck, '68). In marked contrast to its depression of the metabol ism of connective tissues, cortisol stimulates synthetic processes in the liver, specifically of tryptophan pyrrolase (Knox, '51), amino transaminases (Rosen et al., '58) and RNA (Feigelson et al., '62). EFFECTS OF ADRENAL CORTICAL STEROIDS ON BONE CELL POPULATIONS IN VIVO Skeletal resorption and formation occur simultaneously in a systematic remodel ing process throughout an animal's entire lifetime. This fact has made difficult the study of bone metabol ism and has encouraged the development of methods which focus on isolated phenomena. One approach to the study of bone metabol ism directs attention to cellular changes in the tissue after given treatment. While care must be taken in drawing conclusions from such morphologic data concerning actual bone formation and resorption rates, information can be obtained on cellular prol iferation~ differentiation, modulation, and death. Of the few histologic studies made on corticosteroid-induced osteoporosis in humans, all concern the end-results of chronic, massive steroid burdens. In general, most of these descriptions agree with Reifenstein's anti-anabol ic concept of steroid action (Albright, 143),namely that cortisol depresses osteoblast numbers and cellular 4 activity. In contrast, there is disagreement about the state of osteoclast populations in bone of Cushingoid patients. These resorbing cells are reported to be anywhere from completely absent to tremendously increased. The first anatomical description of Cushingoid bone On record is that of Mooser (121). This was one case used by Cushing to illustrate skeletal involvement in the syndrome bearing his name (Cushing, 132). The remarkable aspect of the bone lesion described was the complete absence of osteoclastic resorption. FoIl is (ISla) found a contradiction between the numbers of bone cells in the osteoporotic patients he studied and the decrease in skeletal mass seen. In each of five patients with Cushing's syndrome, he noted extreme loss of bone mass in both trabeculae and cortex; nevertheless, osteoblasts were present and resorption cavities absent. In one 15 month old infant t the few trabeculae at the costochondral junction of a rib contained no cores of calcified cartilage, such as normally found. cartilage. In addition, there was no vascular invasion of the Despite this, the trabeculae present were covered with thin borders of osteoid and normal appearing osteoblasts. No osteoclasts were evident. The first quantitative report in terms of cell numbers and types in human osteoporotic bone was that of Storey (163). He looked at bone from the il iac crest removed at operation from patients with Cushing's syndrome. The osteoblasts were small and scarce and new bone formation occupied less than 3% of the endosteal margin. than half the bone surface was irregular in out1 ine due to the More 5 presence of small Howship1s lacunae occupied by uninuclear cells. Multinucleated osteoclasts were small and rare. Cortisone administration in man can induce most of the abnormalities of the Cushing syndrome (Urist, 160). Bone samples from a rheumatoid arthritic administered 20-40 mg prednisone daily for two years had no osteoid tissue and large resorption cavities on the endosteal surface of the cortex. Matrix incorporation of intravenously- infused 32p indicated that bone deposition had not, however, come to a complete standstill. Because it is difficult to study more than the pathologic endpoint in humans, investigators have become more interested in experimental osteoporosis. Animal models have unfortunately increased the contradictions on the subject. Some of the disagreements in the 1 iterature can be attributed to species differences, some are time and dose dependent, and still others remain inexpl icable. The rabbit has proved to have steroid sensitivities similar to man (Sissons, '55; Storey, '57). In Storey's experiment, two groups of rabbits were given 20 mg cortisone/kg/day for either seven days or a longer time period. After seven days, bone growth and apposition were inhibited while the vascularity and the extent of calcified bone margin undergoing resorption were dramatically increased. numerous multinucleated cells present in Howship's lacunae. There were Later in the process, some osteoid matrix reappeared, and resorption was less obvious at endosteal bone margins than after seven days treatment. At the endosteum, uninuclear cells were present in small indentations in the calcified boneo Large multinucleated osteoclasts were not evident. 6 This study emphasizes the importance of timing the sequence of treatment and tissue examination. Only the chronically-treated rabbits had histologic changes of bone resembl ing human osteoporotic bone. Bartley ('68) treated young rabbits for 28 days with doses of cortisol ranging from 0.2 to 5.0 mg/kg/day. There was a marked loss of osteoblasts in the primary spongiosa of the proximal metaphysis of the tibia at all dose levels. A maximum loss of 66% occurred at the 5.0 mg/kg/day dose. The response of the osteoclast population to treatment with cortisol was more severe than that of the osteoblasts, but only at the two highest dose levels and only within the periosteum. There was a reduction in the number of osteoclasts in the periosteum of the proximal tibia at 2.2 and 5.0 mg/kg/day dose levels. Five mg/kg/day produced an 88% reduction in the number of osteoclasts when compared with controls. Dose levels lower than 2.2 mg/kg/day produced no change from control values in the number of osteoclasts. In addition, the number of osteoclasts in the primary spongiosa was found to be constant at all doses of cortisol. This difference in response of metaphyseal and periosteal osteoclasts to cortisol calls attention to another source of contradiction in the 1 iterature, in addition to those of species, dose and time. Apparently particular cell types respond to a drug according to their anatomical location within a bone. More dramatic morphologic evidence for the osteopenic effect of cortisol in rabbits has been reported for doses lower than those used by Bartley (Blackwood, '69). In animals of the same age treated for 30 days, an increase in osteoclast numbers occurred at the extremely 7 low doses of 0.05 and 0.1 mg cortisol/kg/day. A reduction in osteoblasts occurred at all dose levels up to 10 mg/kg/day. Blackwood attributed the reduction in bone in the metaphysis of the tibia to enhanced osteoc1asia and depressed osteoblast formation. he In addition, 1t that cortisol induced a transformation of the osteob1asts into inactive or resting 1 ining cells. He did not consider the possibi1 ity of osteoblast modulation into the uninuclear resorbing cell mentioned i nth eli t era t u r e ( Dod d s, I 32 ; S tor e y , I 57, f 63; Uri s tan d De u t s c h , I 60) . While Fol1 is (15lc) and Storey (163) considered the mouse skeleton to be insensitive to corticosteroids, osteoporosis has been observed in the jaws of young-adult mice (G1 ickman and Shkl~r, maturation is inhibited in infant mice (Howard, 162). 155) and bone However, no systematic histologic study of the effect of corticosteroids on bone has been conducted in mice. Most of the work on the effects of corticosteroids on the skeleton has been conducted in rats. Un1 ike man, the rabbit, and the bird (Urist, 159), the rat does not develop osteoporosis when given cort iso1. I t would seem, therefore, a poor model for the study of the mechanism of action of corticosteroids in bone. Foll is (151b) demon- strated that, although cortisone inhibited growth in young rats, instead of causing rarefaction of the bone, it caused the metaphyseal bone to become dense. In bones of rats given 40 and 50 mg cortisone acetate/kg/day, there were no excess numbers of osteoblasts nor osteoc1asts, no increased activity of periosteal cells nor increased or decreased cortical bone. It is, therefore, difficult to understand how Foll is 8 arrived at the final conclusion that rat bone treated with certain doses of corticosteroids exhibits normal osteoblastic activity, but inhibition or retardation in normal osteolytic sequences. Some investigators disagree that corticosteroids increase meta physea 1 bone in the ra t (S i ssons, 155). On 1y one of two groups of animals given 20 mg cortisone/kg/day for 31 days developed the histologic picture described by Foll is. A possible clue to the source of variabil ity in response of rat metaphyseal bone to corticosteroids is the effect of calcium concentration in the diet during treatment (Storey, 160, '61). Rats fed diets with a low or unbalanced calcium- phosphorus ratio developed the histologic picture of cortisone-induced osteoporosis. Rates of bone resorption were increased to such an extent that pathologic fractures occurred after twelve days· treatment with 20 mg cortisone acetate/kg/day. Corticosteroid effects on bone cell kinetics have been reported only in the rat. Young and Crane (164) treated male rats with 25 mg cortisol acetate/kg/day for 28 days and tritiated-thymidine. th~n gave a flash label of The steroid reduced the number of prol iferating cells in the cartilage and the number of cells in the metaphysis, but did not change the percent of metaphyseal cells which could be flash1a be 1ed • Simmons and Kunin (167) gave male rats 50 mg cortisone acetate/ kg/day for various time periods up to 19 days. tritiated-thymidine one hour prior to sacrifice. All animals were given Cortisone reduced the numbers of both osteoblasts and precursor cells in the metaphysis and lowered the ratio of osteoblasts to precursor cells. The ratios 9 of the labeled nuclei to the numbers of osteoblasts (i.e., the label ing index) were similar in both groups. Cortisone lowered the label ing index of the precursor cell population more than 50%. Simmons and Kunin suggested that cortisone modifies the abil ity of the precursor cell to modulate to the osteoblast, but does not alter the capacity of fully-differentiated osteoblasts to become flashlabeled with tritiated-thymidine. tibial The number of osteoclasts in the metaphysis was depressed after two to three days of cortisone treatment. This change was transient and no significant difference was observed on the nineteenth day. Investigations on the effects of cortisol on rat bone at the University of Utah have recently been reviewed (Jee et al., 170). These investigators found that low doses of cortisol (0.5-2.5 mg/kg/ day for five days) reduced bone volume more rapidly than doses higher than 5.0 mg/kg/day (Roberts, 169). Us ing a rat periodontal 1 igament (POL) model, low doses of cortisol: 1) increased the number of progenitor cells labeled 1 hr post 3 HTdR 2) decreased the number of osteoblasts and 3) increased the number of osteoclasts. The usual bone forming surfaces were replaced by bone resorbing sites and the POL widened due to the resorption of alveolar bone. Doses higher than 5 mg/kg/day, decreased bone volume at a rate slower than that occurring with low doses. High doses: 1) decreased the label ing indices in POL fibroblasts 2) decreased the number of osteoblasts and 10 3) decreased the number of osteoclasts. Roberts felt there was still a preferential differentiation of precursor cells to osteoclasts, even though the number of osteoclasts produced were small. He postulated that the osteoclast number was low due to the depressed cellular prol iferation of the fibroblasts which, in turn, decreased the supply of stem cells available for differentiation. In summary, although the effect may occur only at given time periods after treatment, there is a good deal of support for the concept that all doses of cortisol ~~ decrease the differentiation and activity of osteoblasts, with a resultant depression in new bone formation. Low doses (less than 5 mg/kg/day) stimulate precursor cell division and preferentially increase the number of osteoclasts. The source of these osteoclasts has not been studied and it cannot be said whether they differentiate from an increased precursor pool, whether they modulate from old osteoblasts or osteolytic osteocytes, or whether they migrate from other sources, e.g., the blood stream or capillary endothel ium. Not always are the resorbing cells typical multinucleated giant cells, but sometimes uninuclear, possibly macrophagic in nature. In contrast, high doses (greater than 5 mg/kg/day) depress precursor cell division and decrease the number of osteoclasts. The resultant loss of total bone mass occurs at a rate slower than seen at low doses. Presumably the loss is due entirely to an anti-anabol Ie process, with no increased catabol ism. 11 SECONDARY HYPERPARATHYROIDISM The first and most popular explanation for the dichotomy of the effect of cortisol on bone is that of secondary hyperparathyroidism, The theory proposes that low doses of cortisol stimulate parathyroid gland secretion to such an extent that the effect of the steroid is masked by the effect of the parathyroid hormone. Acute treatments of both parathyroid hormone and parathyroid exlract aff~ct the para- meters under discussion, namely to stimulate precursor cell division and to induce osteoclast production ~ vivo and ~ vitro (Chase et a1" '69; Deguchi and Mori, 169; Gaillard, '61; Heller et al., 150; Jones, '70; Talmage, 166; Toft and Talmage, 160, Weinmann and Schour, '55, Young, 163). The theory continues that higher doses of cortisol may inhibit parathyroid gland secretion altogether, or may prevai lover the parathyroid hormone, thus producing the "true" effect of corticosteroids on bone cell kinetics; that is to say, inhibition of all cell functions: ant i - mit os is, ant i-a nabo 1 ism, and ant i - cat abo 1 ism (E lie let a 1 ., I 71) , The support for cortisol-induced secondary hyperparathyroidism is very convincing. There can be no doubt in fact that the adrenal cortex is intimately involved in the regulation of calcium metabol ism. Recently the 1 iterature has become replete with testimonials of the effect of adrenalectomy and corticosteroid administration on blood calcium levels (Aldred et al., '67; El iel et al., 171; Laron et al., '58; Myers and Lawrence, '61; Nishino and Munson, '69; Pincus et al., 151; Stoerk et al., '63). I t should not be overlooked, however, that as 12 early as the 1920ls increased serum calcium after adrenalectomy was reported in the rabbit (Kisch, 124), in the rat (Rohdenburg and Krehbiel, '25), in the dog (Rogoff and Stewart, 128), in the cat (Taylor and Caven, 127) and even in the human in Addisonian crisis (Loeb, 132). In addition, Taylor and Caven (127) demonstrated depressed serum calcium in rabbits as much as 30% normal due to extracts prepared from the cortex of ox adrenals. Numerous investigations have been conducted to determine the means by which a corticosteroid is able to reduce blood calcium levels. Reason for parathyroid hormone secretion is given a priori with the cl inical finding of increased blood nitrogen levels in corticosteroidtreated animals. McLean and Hastings ((35) reported that the degree of calcium ionization is determined chiefly by the protein content of the fluids. The relationship between calcium and protein can be described by a simple mass law equation. Therefore protein catabol ism, as in cortisol-induced lymphoid destruction (Dougherty and White, '43), is in itself grounds for virtual hypocalcemia. Beyond changes in ionized calcium, corticosteroids have direct effects on calcium transporting systems in the kidney, intestine, and bone. A human subject given 200-600 mg cortisone acetate per day had increased excretion of urinary and fecal calcium (El ie) and Heaney, '54). Laake (160) studied renal calcium clearance in 16 patients with non-endocrine diseases during long-term therapy with small doses of corticosteroids o Renal tubular reabsorption of calcium was reduced in seven of these patients; he attributed this phenomenon to a general anti-anabol ic action of the steroids on the cells of the renal tubules. 13 There is additional evidence that some of the calcium loss induced by corticosteroids is due to an effect on renal tubular reabsorption of diffusible calcium. Eisenberg (168) infused high levels of cortisol (from 11/2 to 3 times normal blood levels) into one renal artery of dogs that had bilaterally equal urine flow and that had been injected with 45Ca 1 hr before control collections were started. At the 1 1/2 x normal dose there was no change, at the 2 x normal dose there was an ipsilateral increase, and at the 3 x normal dose there was a bilateral increase in urine calcium within 20-40 minutes. One hour after stopping corticoid infusion, the effect diminished. The ratio of 4SCa to 40Ca of urine was greater than that of serum and was not affected by the steroids. Intestinal absorption of calcium was originally thought to be unimpaired by adrenal cortical steroids (Clark et al., 159). These investigators treated rats with 2 mg cortisol/100 gm/day for seven days and then measured the blood levels of 45Ca after the isotope had been introduced into the stomach. One explanation for their failure to detect a steroid effect on calcium absorption may be the fact that they maintained the rats on a calcium-free diet for the extent of the treatment period. This in itself would have stimulated the parathyroids. Parathyroid hormone, in return, stimulates calcium absorption in the intestine (Care and Keynes, 164; Cramer, 163; Rasmussen, 159). . . ( I 60) use d a 4 Sc a .In d·Icator .In everted .rntestlna . 1 Harrison an d Harrlson loops ~ vitro. Cortisol treatment antagonizes the vitamin D effect on both diffusion and active transport of calcium. 14 The effects of corticosteroids on calcium transport have also been measured in parathyroidectomized rats (Stoerk and Arison, 161). Twenty mg cortisol/kg/day strikingly delayed the appearance in blood and urine of orally fed 45 Ca • Surprisingly, similar animals exhibited the same rate of disappearance of intravenously injected 45 Ca from the blood as untreated controls. Adrenal cortical steroids lower blood calcium by effecting the physiology of the skeleton. Gordan et ale (16~ studied ACTH and nine glucocorticoids in parathyroidectomized rats which had been injected with 45 Ca five weeks previously. Both urine and feces radioactivities were depressed by all compounds tested. For this reason, they considered corticosteroids to be anti-osteolytic or calcium-retaining and they proposed that the calcium wasting caused by steroids in intact animals must be mediated through the parathyroids. Talmage et ale (170) also found cortisol (5 mg/kg/day for five days) to decrease the rate of 45 Ca removal from rat bone. Both Schafer et al. (169) and Stern (169) demonstrated a dose-response depression by cortisol of release of previously-incorporated 45 Ca from bone maintained in organ culture. Cortisol produces hypocalcemia by effecting a variety of systems. The fact cortisol effects the intestine, kidneys and skeleton in vitro would argue against mere antagonism of the parathyroid effect. Reidenberg et al. (168) administered methyl-prednisolone to thyroparathyroidectomized rats whose blood calcium level showed a doseresponse to varied doses of parathyroid hormone. At every dose of parathyroid hormone, methyl-prednisolone markedly inhibited the 15 hypercalcemic response. A statistical analysis of the dose-response curve showed that the inhibition of parathyroid hormone by methylprednisolone was not competitive. adrenal Furthermore, the hypercalcemia of insufficiency does not always depend upon intact parathyroids (Myers and Lawrence, '61). from adrenal In seven studies of four dogs suffering insufficiency (presumably due to sub-total 1 igations of the adrenal blood vessels), parathyroidectomy failed to abol ish the consequent hypercalcemia. Little is known of the effect of corticosteroids on the parathyroid glands themselves. Rogoff ('34) reported the appearance of the parathyroid glands after animal studies on adrenalectomy and steroid treatment. three days. Dogs subjected to adrenalectomy died in less than One of the most striking pathologic findings at autopsy was gross enlargement of the parathyroids with no detectable microscopic changes. While he admitted that such evidence does not warrant definite functional interpretation, the possibil ity exists that corticoids act to dampen parathyroid secretion. Eli e 1 eta 1. (' 65) are the 0 n 1yin v est i gat 0 r s tor e po r t the effects of adrenal cortical steroids on secretion of parathyroid hormone. Using the parathyroidectomized rat bioassay for parathyroid hormone concentrations, they studied the urinary excretion of parathyroid hormone in humans who had been treated with corticosteroids. One hundred mg cortisol/day and 30 mg prednisolone/day, both for four days, did not effect parathyroid excretion. On the other hand, prednisolone in sufficiently high doses (50 mg/day) resulted in the total disappearance of parathyroid hormone from the urine. Effects 16 of corticosteroids on the parathyroid glands, both direct and indirect, demand further attention. BIOTRANSFORMATION OF ADRENAL CORTICAL STEROIDS It is becoming increasingly evident that secondary hyperparathyroidism accompanying low doses of cortisol does not account for all aspects of the dichotomy of adrenal cortical steroid effects on the s ke 1e ton. Wit h the p ion e e r i ng wo r k 0 f Be r 1 i nera nd Do ugh e r t y (I 6 1 ) , such a dichotomy can be seen in a different 1 ight. It appears that different cell types have different capacities to respond to steroid molecules. Aside from possible resistance to the steroid due to a selective capacity to exclude it, individual cells, such as fibroblasts, may have the capacity to transform corticosteroid molecules in a characteristic manner (Sweat et al., 158). Berliner and Dougherty use the term "biotransformation ll to designate such a molecular change produced by a nonendocrine cell. Cortisone does not inhibit growth, enhance destruction or affect maturation of normal lymphocytes (Dougherty et al., 161). cortisol that produces these catabol ic effects. It is Furthermore, cortisone actually stimulates fibroblast mitosis (Ruhmann and Berliner, 167). In two of the i!:!. vitro experiments reported, a dose of cortisol of 0.01 ]Jg/ml increased fibroblast numbers above those of control cultures, while increasingly higher doses correspondingly decreased the populations. It might be postulated that at concentrations of 0.01 ]Jg cortisol/ml, the fibroblasts were able to transform all molecules of inhibitory steroid to molecules of growth. Thus it may 17 be possible that biotransformation plays a role in hormonal effectiven e s sat the c ell u 1a r 1eve 1. A no rma 1 c ell mig h tin fa c t con t r 0 1 its own fate by keeping the attacking molecules in a biologically productive state. Additional reports have been publ ished of bimodal responses of connective tissue cells in culture to corticosteroids, depending on the concentration present. phagocytic Rasche and Ulmer (170) monitored the abi1 ity of L929 fibrocytes under the influence of prednisolone. Low concentrations (2 x 10- 9%) increased, while higher 2 concentrations (2 x 10- %) inhibited the phagocytic activity of the L-cells. Maximal/activation of phagocytosis was measured 1/2 to 1 hr after hormone app1 ication. The increased rate in phagocytosis was reflected in an increased rate of mitosis. Likewise Achenbach et a1. ('70) showed a bimodal effect of prednisolone on thymidine incorporation into L-ce1 ls ~ vitro. At ]0- 4 and 10- 5 M, a pronounced inhibition of thymidine incorporation was found (38% of control). At 10- 7 and 10- 8 M, however, thymidine incorporation was stimulated up to 180% of control 0 The degree of inhibition and of enhancement became more pronounced after 24 hr. An indication of a direct stimulatory effect of corticosteroids on bone fibroblasts (170). ~~ is presented in a report by Ju1 ian et al. Using the periodontal 1 igament model in parathyroidectomized rats, 0.5 mg cortisol/kg/day inhibited the uptake of tritiatedthymidine into DNA of PDL fibroblasts 50% when compared to the uptake in parathyroidectomized - sham injected animals. This dose had no fect on the amount of tritiated-thymidine incorporated into fibroblast DNA 18 in intact animals (Roberts. 169). Jul ian's finding supports the hypothesis that a low dose of cortisol induces a secondary hyperparathyroidism. On the other hand. maximal label ing of the fibroblasts occurred 11 hours sooner in the cortisol-treated group when compared to controls. This suggests a stimulatory effect of cortisol independent of the parathyroid glands. It is tempting to speculate what the effect of still lower doses of cortisol would be on fib rob 1a s t pro 1 i fer a t ion i nth iss y stem. EFFECTS OF ADRENAL CORTICAL STEROIDS ON BONE IN VITRO It is the purpose of this dissertation to determine dosedependent effects of cortisol on bone cell populations independent of parathyroid hormone. An ~ vitro system was developed to isolate these direct effects of the steroid. The organ culture method is being increasingly used in the analysis of complex physiologic interactions whichare difficult to disentangle by 1 ive animal experiments alone. A closed system, in which there are few homeostatic mechanisms at play, has definite advantages in selecting out and controll ing en vir 0 nme n ta 1 fa c tor s • 0 nth e 0 the r ha nd • i tis d iff i c u 1t tor e 1ate in vitro responses to actual phenomena in the whole animal. Few morphologic studies have been conducted on the effects of corticosteroids on bone in culture. Of those that exist, all are expressed in gross, deductive terms and none are quantitative at the cellular level. Buno and Goyena ('55) reported that cortisol and cortisone, both at 25 ~g/ml concentrations, delayed the growth in length of embryonic chick cartilage. They found no difference between 19 the effects of the two compounds on this parameter. When treated femurs were transplanted to fresh medium without hormone, growth began again at a very high rate, the final dimensions being in some cases greater than those of controls. They felt that the inhibition of growth by the steroid was not due to a decrease in the size of cells or in the amount of fundamental matrix substance, but rather to a diminished prol iferation of the former. This work was corroborated (Sobel and Freund, '58), but in addition differentiation was retarded in terms of number of osteoblasts and in the deposition of bone. Fell and Thomas (161) first explanted the more differentiated bones of near-term mouse embryos. In this more complex system cortisol sodium succinate (7.5-75.0 ~g/ml) arrested cartilage excavation and showed a general retardation in normal osteolytic sequences on histologic examination of the bone. In order to characterize the in vitro system, a historical review will also be given of biochemical responses to corticosteroids in culture. The first work conducted on the subject concerns the synthesis of chondroitin sulphate in cartilage (Clark and Umbreit, 154). In 6 hr incubations of adult male rat xiphoid processes, cortisone and cortisone acetate inhibited the uptake of 35S-sulphate into the acid insoluble portion of the matrix, while cortisol and cortisol acetate stimulated uptake. was not given. A complete account of the dosages Since then the dosage problem has received special attention. In a second investigation using costal cartilages in culture for 24 hr (Daughaday and Mariz, 162), cortisol inhibited S04 uptake only 20 in concentrations greater than 5 ~g/m1. Whitehouse and Bostrom ('61, '62) reported that several corticosteroids (corticosterone, cortisol, cortisone, prednisolone, dexamethasone, and triamcinolone, at 2.5 x 10- 4 M) inhibited sulphate metabol ism after a time lag of 2 hr. The magnitude of the inhibitory effect of anyone drug tended to increase with the length of the incubation period, with the notable exception of cortisone. Only in short-term incubations was cortisone a more effective inhibitor than cortisol and other ll-B-hydroxysteroids. This may explain the confl icting report by Clark and Umbreit (154). Whitehouse and Bostrom substantiated their own claim that polysaccharide synthesis was depressed by cortisol by also monitoring "f t he .Incorporation 0 g 1ucose- l4C an d acetate- l4C .Into po 1ysacc h"d arl e sulphates (161, '62). Besides the steroid inhibition of the incorporation of these substances, they also found depression of 4 glucose-1 C oxidation to CO , indicating that the hormones inhibited 2 the util ization of glucose for respiration. In all experiments, the inhibitory actions of the drugs (even of the slow-acting steroids) were readily reversed with their removal. The possibility that cortisol altered the type of mucopolysaccharide being produced has been investigated ~ vitro (Barrett et a1., '66). There was no change in the ratio of sulphate to uronic acid in the newly synthesized polysaccharide of steroid-treated bone cultures. Also the ratio of sulphur atoms to residues of glucuronic acid was close to unity, as would be predicted for chondroitin sulphate. The ratio of glucosamine to galactosamine was not altered, which suggested that the polysaccharide was almost exclusively 21 chondroitin sulphate. Therefore, neither the suiphation nor the type of polysaccharide formed was altered; its synthesis was merely depressed in general. Concerned with the breakdown of the polysaccharide sulphates, Whitehouse and Bostrom (161, '62) prelabe1ed cartilage by incubation .In a me d .la conta . . 35 5-su 1pate. h Inlng Th e ra d . . loactlve cartl. 1age was treated for 4 hr with the six hormones and concentrations previously described. There was no significant change in release of 35 5 into the media in the presence of any steroid. of 1 ~g/ml At the higher concentration and after the longer cultivation period of six days, cortisol does appear to decrease the breakdown of polysaccharide (Dingle et a1., 166). The steroid depressed the release of hexosamines from chick cartilage rudiments into the media. This did not result in an increased quantity of hexosamines in the rudiments since total synthesis of hexosamines was also depressed. Reynolds ('66) found no effect of cortisol on polysaccharide breakdown at 1 ~g/ml, in agreement with Whitehouse and Bostrom. He considered the report of Dingle et a1. (166) to be valid, however, for the following reason. Dingle and his co-workers used six-day-old chick rudiments, which as controls released a large quantity of hexosamine, as much hexosamine-containing material as was present in the rudiments at the end of the experiment. Reynolds used larger rudiments from seven-day-01d chick embryos, which had a higher initial ratio of collagen to hexosamine. These bones 1 iberated 1 itt1e polysaccharide as controls, and therefore made it difficult to detect any inhibition of release in the presence of cortisol. 22 Skeletal tissue cells are active not only in forming mucoprotein, but also in incorporating polypeptide precursors into collagen. Cortisol (0.9 l-lg/ml) inhibits..!.!2. vitro the conversion of l4C-prol ine into incorporated labeled hydroxyproline (Daughaday and Mariz, 162). EVen smaller doses (0.01 pg/ml) depress labeled glycine incorporation into rat metaphyseal bone chips (Vaes and Nichols, 162). A non- significant decrease in glycine decarboxylation to glucose was also observed. Dingle et al. ('66) found cortisol (1 l-lg/ml) to depress the total hydroxyprol ine content of chick rudiments. They mentioned at this time, and were one of the first to do so, stimulation of cellular function (in this case hydroxypro1 ine content of the matrix) with lower concentrations of cortisol. They felt that part of the increase was due to inhibition of the releaseof hydroxyprol ine into the culture medium, but they also detected a small rise in the total synthesis of the imino acid. Stimulation of a catabol ic activity had been reported earl ier (Goldhaber, 165), namely that cortisol in rather large concentrations (10 l-lg/ml) enhanced ..!.!2. vitro resorption as stimulated by parathyroid extract. This stimulatory action sometimes seen in low doses of cortisol was referred to earl ier in reference to biotransformation by fibroblast cultures (Achenbach, et a1., 170; Rasche and Ulmer, '70; Ruhman and Berl iner, 167). Ebert and Prockop (167) used a double label technique of 35S_ sulphate and 3H-prol ine to determine whether cortisol specifically inhibited the synthesis of sulphated mucopolysaccharides or the 23 synthesis of collagen in embryonic chick tibiae in vitro. With moderate concentrations of cortisol, the inhibition of sulphate incorporation and protein synthesis were comparable, but they were of relatively small magnitude. With tremendously high concentration (1000 ~g/ml) sulphate incorporation was inhibited to a greater extent than protein synthesis. This dose has proved to be completely toxic in most culture systems. A set of experiments has been publ ished by Strangeways Laboratories in which cortisol (0.1-1.0 ~g/ml) was used to stop the breakdown of the intercellular material of cartilage and bone grown in organ culture determined on histologic examination) (Fell t '65). (2S Vitamin A, antiserum, and high concentrations of 02 caused pathologic resorption. Resorption induced by these agents could be much retarded and sometimes abol ished by the addition of cortisol to the medium. Evidence has accumulated that cortisol acts directly on bone cells by stabil izing lysosomal membranes and preventing the release of proteases for digestion of the matrix. Cortisone delays the thermal release of enzymes from isolated lysosomes (DeDuve et al., '61). Lysosomes, isolated from 1 ivers of cortisol-treated rabbits are more resistant to ultraviolet irradiation in vitro than those obtained from control animals (Weismann and Dingle, 162). Reynolds (168) reported a reduction of total protease activity by cortisol, but found that 0.2 ~g/ml had 1 ittle effect on the bone resorption induced by vitamin A. Thus it seems that although most doses of cortisol inhibit bone resorption in vitro, the dose level required may be higher than that needed for the inhibition of release of lysosomal enzymeso 24 In the presence of sucrose and other poorly metabol ized sugars, chick embryo 1 imb-bones demonstrate an increased synthesis and large extracellular release of lysosomal enzymes (Dingle et al., 169). The sugars accumulate in large cytoplasmic vacuoles in the fibroblasts of the connective tissue capsule and in chondrocytes of the articular region. The sucrose taken up by these cells is 1 iberated when the rudiments are returned to normal medium. This release, unl ike the secretion of lysosomal enzymes, was unaffected by the presence of 0.5 ~g cortisol/ml. The investigators lt this indicated that the 1 iberation of the enzymes is not a phenomenon involving the fusion of vacuolar membranes with plasma membranes as occurs in the expulsion of the sugar-containing digestive vacuoles, and concomitantly that cortisol must not affect such activities involving fusions of membranes. This information is interesting in relation to other types of membrane fusion, as in the formation of multinucleated osteoclasts. One of the most recent parameters to be studied in an in vitro system has been the effect of cortisol on the nucleic acid content of boneo Cortisol (0.0001-1.0 ~g/ml) has no effect on the total DNA content of seven day chick embryo tibiotarsi which have been cultured for ten days (Schryver, 165). DNA incorporation into bone cells isolated from rat calvaria is not affected by 10- 5M cortisol and the total amount of DNA/culture does not decrease (Peck et al., 167). These findings suggest that the inhibition of protein synthesis by cortisol is independent of change in DNA metabol ism o They also 25 suggest, since there is no decrease in DNA, that glucocorticoids do not exert a lytic effect on bone cells. In contrast, cortisol produces consistent decreases in total RNA/ culture and inhibits the incorporation of uridine-2et a1. , 169). l4 C into RNA (Peck The effect of the steroid on precursor accumulation may be responsible for the decrease in RNA and protein label ing since all three changes appear simultaneously within three hours and are equivalent in degree. Uridine-2by cortisol. 14 C prelabel also shows enhanced degradation of RNA A decrease in both major fractions of ribosomal RNA (18S and 28s) appears after fifteen hours of cortisol treatment. The products of the RNA breakdown do not accumulate within cells, but are released into the media as the nucleosides, UMP and free uridine. The release of activity is enhanced even from isolated microsomes derived from cortisol-treated, previously-labeled cells, but not from nuclear fractions of the same cells. It is interesting to note that Peck showed no change in total bone cell ATP levels after five hours of cortisol treatment. Roger and Ka1bhen (168) found prednisolone 1 mg% to inhibit the ATP content of calf cartilage by 90%. This effect in no way correlated with the inhibition of polysaccharide synthesis since the same dose inhibited 35S-sulphate incorporation less than 10%. In the system of isolated bone cells used by Peck, cortisol did not decrease the label ing of nucleotide pools during simultaneous inhibition of protein synthesis with either puromycin or cyc1ohexamide. He suggested that continued protein synthesis is required for cortisol 26 action and that it may block transport by inducing the formation of a protein which inhibits the key transport processes. This view is untenable in view of the fact that nucleotide synthesis requires protein formation. Raisz (165) establ ished a system for monitoring the dissolution of bone mineral in the presence of a variety of substances. He reported that a dose of cortisol as large as 200 Wg/ml media had no direct effect on parathyroid hormone-induced release of 45 ca from previously-labeled rat bones. Schafer et al. ('69), however, reported that doses as low as 1.0 wg/ml inhibited the release of 45 Ca from previously-labeled mouse 1 imb-bones. Stern (169) verified this direct inhibition of 45 Ca transport and/or resorption in rat bone. Chapter 3 MATERIALS AND METHODS GENERAL INFORMATION In the initial phases of this work, a system was devised for monitoring the direct effects of corticosteroids on resorption of bone in vitro. It was found that graded doses of cortisol caused a depression in release of previously-incorporated 45Ca from 1 imb-bones into media. fashion. The steroid affected 4SCa release in a dose-response The next step was to determine whether or not a change had occurred in anyone bone cell type which could account for the apparent depression of "resorption!! by cortisol. An ~ vivo investigation was conducted as a prel iminary to the in vitro studies on bone cell populations. Untreated newborn mice were given a pulse label of tritiated-thymidine (3HTdR) and the times required for this label to appear in the more differentiated bone cells were noted. This information was used to characterize changes in cel 1 populations caused by explantation to control cultures. For in vitro studies on bone cell differentiation, animals were given a pulse label of 3HTdR one hour prior to sacrifice. The effects of cortisol and parathyroid extract on the morphology of the bone cells, both labeled and unlabeled, were tabulated. Fig. gives the chemical structure of the steroid used. Steroid compounds are derivatives of a four-ring (cyclopentanoperhydrophenanthrene) system. The important functional positions on the cortisol 27 28 ... .... CORTISONE CORTISOL CH OH I CH20H I 2 c=o c=o ... CORTICOSTERONE ... II-OEHYOROCORTI COSTERON E Fig. 1 Structure of cortisol and corticosterone and their ll-keto conversion products. 29 molecule are the ll-S-OH, 17-a-OH, the side chain C20-21, bond between c4 and C5, and the 3-keto group. the double The molecular structure of corticosterone is included because it is the major naturally occurring adrenal cortical steroid in the mouse (Southcott et a1., 156). It differs from cortisol in lacking the 17-a-OH group; this decreases its anti-inflammatory potency to one-half that of cortisol (Ruhmann and Berl iner, 167) and its osteoporotic potency to one-fourth that of cortisol (Bartley, 168). Both cortisol and corticosterone can be hydroxydehydrogenases to ll-keto compounds. converted by 11- Parathyroid hormone, as isolated and characterized from bovine tissue (Potts et al., 168), is a straight chain polypeptide consisting of 83 amino acid residues. The biologically active portion of the molecule consists of a sequence of 35 amino acids including the n-terminal ~ EXPERIMENTAL ANIMALS AND TREATMENTS Thg effect of cortisol on the release of previously-incorporated SCa from mouse 1 imb-bones in vitro. Matings were dated by the harem method in which the male was left in a cage of four females for 24 hr and then removed. Pregnant CBA mice were given a subcutaneous injection of 100 ~Ci 45Ca as the chloride (New England Nuclear Corp., sp. act. 12.3 mCi/mg Ca) on the nineteenth day of pregnancy. Two days later, the 1 imb-bones of the newborn pups were placed into culture. Eighteen animals were util ized, six for each of the following doses of cortisol: ~g/ml media. 0.01, 0.1 and 1.0 The steroid was brought into solution in propylene glycol by adding methanol. The alcohol was removed under nitrogen gas before 30 the addition of the media. Control media 1 ikewise contained propylene glycol, all to a final concentration of 1 ~l/ml media (Berl iner and Ruhmann, '67). For this experiment BGJ b media was used, supplemented with 500 mg% bovine serum albumin, fraction V. Cultures were gassed with 5% CO 2 -95% 02 and incubated at 37 0 C for four days. Half the cultures at each dose level were pulverized and dissolved in 1 ml 1 N HCl. This solute and the media were dried and the calcium radioactivity was counted in a Packard Tricarb Liquid Scintillation Counter with an efficiency of 92%. The rest of the bones were processed for histologic observation as described in the Plexiglas-methylmethacrylate Embedding section. The second part of this experiment util ized bones previouslylabeled with 45Ca and cultured in graded doses of cortisol as described. These bones, however, were "killed" before culture by subjecting them to four cycles of freezing and thawing, at -20 0 C and 37 0 C respectively. and 48 hr. Groups of cultures were terminated at 8 hr, 24 hr, Bone and media radioactivity were determined and bone histology observed as above. The time reguired for bone cell differentiation in infant mice in vivo. Sixty C3Hf newborn mice, averaging 1.3 grams body weight at birth, were kept with their mothers for the duration of the experiment. Animals were included in the study only if their stomachs contained milk on the day after birth, indicating that the mother had not rejected them and that they were healthy. 31 Time 0 was defined as the hour between 0930 and 1030 on the second day of 1 ife, arbitrarily restricted to not less than 16 hr nor more than 40 hr after birth. At time 0, each newborn was injected subcutaneously using a 111-27 gauge needle with 0.5 l-lCi 3HTdR per gram body weight in 0.02 ml sterile sal ine (New England Nuclear Corp., sp. act. 8.2 Ci/mM). Animals were weighed daily between the hours of 0930 and 1030 for a two week time period. Groups were sacrificed and tibial bone samples taken at twelve time periods after 3HTdR injection: 1, 4, 6, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hr. A description of the methods used for embedding, autoradiography and cell counting are given in the Analytical Methods section. The effect of cortisol on bone cell differentiation in vitro. Eighteen C3Hf stra i n newborn, mice, on the second day of 1 ife, were used in this study. They were injected subcutaneously with 1.0 l-lCi 3HTdR (New England Nuclear Corp., gram body weight 1 hr prior to sacrifice. SPa act. 21.8 Ci/mM) per The labeled radi i were explanted into organ culture using Fitton-Jackson modified BGJb media, supplemented with 10% pooled human serum. The media was changed daily and the cultures were gassed with 5% CO 2 -95% air and incubated at 37 0 C for four days. Groups of explants were exposed to varying concentrat ions of cort i sol: 0.0001, 0.01, 1.0, and 100.0 l-lg/lTll med ia. A fifth group of bones was fixed immediately upon sacrifice at one hour post 3HTdR. Descriptions of the methods used for fixation, embedding, sectioning, autoradiography, staining and cell counting are 32 found in the Analytical Methods section. These specimens were exposed to Eastman Kodak NTB3 photographic emulsion for 5 days. The effect of parathyroid extract on bone cell differentiation in vitro. Twelve C3Hf newborn mice were used in this study on the day after birth. They were labeled with 3HTdR 1 hr prior to sacrifice and cultured under the same conditions as the previous experiment, with the exception of the following additions to the media: Groups of exp1ants were exposed to varying concentrations of par a thy r 0 i d ex t r act, U. S • P. ( Eli L ill yeo. ): units/ml media. o. 1, 1. 0 and 10 • 0 Dilutions of the concentrated extract and control media were made with physiologic sal ine plus 1.6% glycerine and 0.2% phenol, with adjustment of pH to that of the extract (Raisz, '65). In addition, the Fitton-Jackson modified BGJ b media contained 10% pooled human serum, 0.1 mg% bovine serum albumin [charcoai-activated fraction V, Pentex Biochemicals (Paula Stern, personal communication)] and 100]Jg glutathione/ml media (Reynolds and Dingle, '70). The bones were prepared for autoradiography and cell analysis made as described under Analytical Methods. ANALYTICAL METHODS Method of organ culture. The organ culture method used was basically that devised by Trowell ('59). The chief characteristics of this system are the use of a completely 1 iquid media to facil itate diffusion and the position 33 of the explants on a raft (any structural support) between the airliquid interface. Commercial culture dishes for such a system are now available (Falcon Plastics #3010) (fig 2). The 60 x 15 mm polystyrene petri dish contains an outer ring of absorbent cotton and a central well with a capacity for I ml media. The culture was humidified by placing 5 ml sterile distilled water in the outer moat. The media used throughout this dissertation changed according to supplements; the formula of the chemically-defined 1 iquid is given in table 1 (Grand Island Biological Co.; Biggers et al., 161; Fitton-Jackson, unpubl ished mod ification). A nontoxic stainless steel cloth, 60 mesh, with a wire diameter of .0075 11 (Falcon Plastics #3014), has been devised to span the top of the petri dish well, and it is on this grid that the explant is placed. Contrary to the directions of Trowell, the tissue can be maintained on bare stainless steel (Raymond Kahn, personal communication). The animals were killed by decapitation and the 1 imbs were washed rapidly in 70% ethanol and more thoroughly in Earle's Basic Salt Solution (BSS) (in gm/l: NaCl, 6~80; KC1, 0.40; MgS04' 0.10; NaH2P04, 0.125; NaHC0 , 2.20; glucose, 1.00; Ca C1 2 , 0.2). 3 The tissue to be cultivated was dissected out in a petri dish with a second wash of BSS, using jewelers forceps and a dissecting microscope. The bones of both s ides of the an ima 1 (one s ide for treated, one s ide for contro 1 cultures) were left in BSS until all had been dissected out; they were then transferred in rapid succession to the prepared culture dishes. 34 Fig. 2 Polystyrene organ culture dish by Falcon Plastics. Tissue is explanted on stainless steel grid at 1 iquid-air interface. Absorbent disc holds water to humidify chamber. TABLE I BGJb media Component Calcium lactate Dihydrogen sodium ortho phosDhate Glucose Magnesium sulphate 7H20 Potassium Chloride Potassium dihydrogen phospate Sodium bicarbonate Sodium Chloride L-Lysine hydrochloride L-Lysine L-Histidine hydrochloride H20 L-Histidine roch1oride L-Arginine L-Arginine L-Threonine L-Val ine DL-Val ine L-Leucine L-Isoleucine L-Methionine L-Pheny1alanine L-Tryptophan L Tyrosine Original BGJ b (mg/L) FittonJackson 1'1od i fica t ion "pepped Up" (mg/L) 555.0 555.0 5,000.0 200.0 530.0 90.0 10,000.0 200.0 400.0 160.0 3,500.0 8,000.0 .0 160.0 3,500.00 5,300.0 240.0 150.0 150.0 7.5 75.0 65.0 50.0 30.0 50.0 50.0 40.0 .0 175.0 75.0 65.0 50.0 30.0 50.0 50.0 40.0 40.0 Component Or i gina BGJ FittonJackson Mod i fica t ion "pepped Up" (mg/E) (mg/L) L-Cysteine HC1-H 20 90.0 L-Cysteine HC1 90.0 L-G1utamine 200.0 200.0 L-Alanine 250.0 L-Aspartic acid 150.0 Glycine 800.0 L-Prol ine 400.0 L-Serine 200.0 Nicotinamide 20.0 20.0 Thiamin hydrochlori e 4.0 4.0 Calcium pantothenate 0.2 0.2 Riboflavin 0.2 0.2 Pyridoxal phosphate 0.2 0.2 Fol ic acid 0.2 0.2 Biotin 0.2 0.2 Para aminobenzoic acid 2.0 2.0 Alpha tocopherol phosphate 1.0 1.0 Chol ine Chloride 50.0 50.0 Inositol 0.2 0.2 Vitamin B 0.04 0.04 Sodium acetate 50.0 Ascorbic acid 50.0 Phenol Red 20.0 20.0 Streptomycin 50.0 50.0 Penicillin 00,000 units 100,000 units VJ V1 36 Gas chambers were made according to the method of D. J. Nachtwey (personal communication). Bags were made of mylar (Falcon Plastics #5020), sealed on three sides with an iron, and gassed with 5% C02 for 5 min before seal ing. Each bag contained at least one treated and its companion control culture. pressures for 48 hr. Such bags are said to maintain gas The media in these experiments was changed daily, in which case the chambers l'Jere egassed as frequently. The entire unit was then placed in an incubator with a constant temperature of 37+10 C (National Appl iance Incubator #3321). Plexiglas-methylmathacry1ate embedding. Bones were placed in I oz glass vials and treated as follows: 1. Fixation a. Carnoy's acetic alcohol (1:3) b. 109~ neutral buffered formalin • • • 15 min (37-40% formaldehyde solution, 100 cc; water, 900 cc; acid sodium phosphate, monohydrate, 4 gm, anhydrous disodium phosphate, 6.5 gm) 2. Decalcification a. b. 3. •• 48 hr 10% ethylenediaminetetraacetic acid (EDTA or versene) (pH 7.4) • • 3 hr tap water . • • • • • • • overnight Dehydrat ion a. 70% ethano 1 b. acetone x 3 • • • a t 1ea s t 10 h r 1 hr each 37 4. Ernbedd i ng (Ca they, 163) a. Solution I (0.8 gm benzoyl peroxide to 100 m1 methyl-methacrylate, dried with sodium su 1pha t e) • b. 2 hr Solution II (27 m1 Solution to 4 ml dibuty1 phthalate to 6 gm polyethylene glyco1disterate heated to 50 0 C and shaken periodically until dis sol v ed ). • • c. • • • • • • • • Solution III (3 ml Solution II to 2 hr gm plexiglas molding powder, added slowly, agitated briskly for 10 min, and shaken frequently at 50 0 C until solution is clear. This is the embedding matrix. specimens If the were larger than 2 x 2 mm in any two directions, they were vacuumed 1 hr in a negative pressure of 21 psi. Otherwise, the specimens were positioned in the vials immediately and placed in a 43 0 C oven until methacrylate hardened 5. • • • overnight Sec t ion i ng The blocks were cracked out of the glass vials, reduced to the desired size and shape on a plastic trimmer, and positioned on the microtome (Jung Model K). carbide steel knife. Sections were cut at 5 micra using an HK 3 Ethanol, 70-80%t was used as the wetting fluid in transferring the sections from the microtome knife to gelatinized sl ides. Bibulous paper was flattened over the sections with a rubber 38 roller. When the sl ides were ready for autoradiography or staining, the methacrylate was removed with three 10 min changes of acetone. Autoradiographic procedure 1. Dipping a. Emulsion (unless otherwise specified, Eastman Kodak NTB) warmed in 38-40 2. b. S 1 ide s dip ped • c. S 1 ide s d r i ed. • 0 C \.vater bath. 30-45 min 10 sec each .1 1/2-4 hr Exposing a. Sl ides (in bakel ite boxes without 1 ids) placed in coffee can with CaC1Z. Can 1 id taped on with Scotch Brand electrical tape #33. Stored at -20 0 C (unless otherwise specified). 3. 4. • 28 days Developing and fixing (Kodak Pamphlet No. P-64) a. Sl ides brought to room temperature. b. Eas tman Kodak D-19. c. Tap water d. Stop bath (dilute acetic acid). e. Tap wa ter f. Eastman Kodak "Acid Fixer". g. Cold, gently-running tap water. h. Dem i nera 1 i zed wa t e r, 40 Co. • • 30 min . ... . 4 min . · · 15 · · 15 sec · · 15 sec ..... 10 min or until the emulsion is clear ... S ta in i ng a. sec Filtered Mayer1s hemalum (1 gm hematoxyl in, 1 1 iter demineral ized H 0, 0.2 gm sodium 2 iodate, 50.0 gm potassium alum, 50.0 gm 1 hr store 39 chloralhydrate, 0.2 gm citric acid) • • • • b. • 20 min Demineral ized H 0, saturated with aluminum 2 ammonium sulfate. • • • • • • • • • • • • •• 5 min or until differentiated c. Demineral ized H 0 2 10 min d. 50%, 70% ethanol. 10 min each e. Eo sin Yin 70% e t ha no 1 • f. 95%. 100% ethanol x 3 10m i n each g. xylene x 3 • • • • 10 min each h. Coversl ipped with Fisher's Permount min 0 r until counterstained as desired Ce 11 count i ng Bone cells were classified according to the following characteristics: progenitor cell - an immature cell type with relatively clear, inconspicuous cytoplasm and large, oval, centrally-placed nucleus with dust-like chromatin and one or more prominent nucleol i. In these experiments, the bone cell progenitor (osteoprogenitor) was of primary concern. Therefore such a cell was counted only when it appeared on or near. within three cell layers, of the bone surface. Included in this category were cells known as spindle cells, fibroblasts,reticular cells,mesenchymal cells and, when adjacent to a bone surface, quiescent 1 in i ng ce 1 1s (f i g s. 3, 4, 5). osteoblast - a plump cell, adjacent to osteoid, with abundant and very basophil ic cytoplasm and an eccentrically disposed nucleus with one prominent nucleolus (fig. 3). 40 Fig. 3 Photomicrograph of proximal metaphysis of tibia of newborn rno use. 0 s teo b 1as t (0 B) i s 1a yin g dow nos teo i don prj rna r y t r abe c u 1a . Intertrabecular space is filled with progenitor cells: fibroblasts (F) and mesenchymal cells (MC). Fig. 4 Photomicrograph of lower edge of proximal growth plate of tibia of newborn mouse. Two types of cells are seen resorbing calcified carti lage: endothel ial cells (E) and an "old" osteoclast (OC) • ECCLES MEDICAL SCIENCES LIBRARY 41 4 Fig. 5 Autoradiograph of trabecular bone in tibia of newborn mouse 1 hr post 3HTdR. Two types of resorbing cells are seen: a macrophage (M) and a "youngll osteoclast (ac). 3HTdR label is in progenitor cells, endothel ial cells, and resorbing cells. .J:N osteocyte - an osteoblast which has completely surrounded itself with bone matrix. Its cytoplasm is only faintly basophil ic and its processes extend through apertures of the lacuna into canal icul i. resorbing cell - included three cell types: 1) osteoclast - a giant cell with up to twenty nuclei and acidophil ic, foamy cytoplasm. The younger clasts have nuclei similar to those of osteoblasts, i.e., large and ovoid with smooth nuclear membranes, fine, evenly-distributed chromatin granules and one or two prominent nucleol i. The older clasts have wrinkled nuclear membranes and small dark-staining, pyknotic-looking nuclei (figs. 4, 5). 2) macrophase - a mononuclear cell with very dark nucleus and withdrawn cytoplasm (fig. 3) 5). endothel ial cell - the 1 ining cell of capi llaries and sinusoids, with a dark, elongated nucleus and sieve-l Ike cytoplasm laying in a flattened plane (fig. 4). These cell types were considered to be resorbing through "guilt by association " , i.E., they were located on the bone surface within resorbing lacunae. A bone cel 1 census was taken for three areas of each bone: the endosteal surface of the metaphysis, the endosteal surface of the diaphysis and the periosteum. Fig. 6 illustrates the location of the microscopic fields considered in these measurements. Not less than 1000 cells were counted and characterized for each treatment. counts were made under oil immersion (lOOOX). Cell Fields of 0 12 mm 0 length in the diaphysis and 0.014 mm 2 in the metaphysis were sampled 45 Fig. 6 Location of microscopic fields considered in census of bone cells. A indicates the metaphysis; B, the diaphyseal endosteum; and C, the diaphyseal periosteum. 46 with an eyepiece reticle (American Optical Company). The accuracy of the field size was checked with a stage micrometer. The following tabulations were made for each bone cell population: 1) Unlabeled cells/field 2) Total cells/field 3) Label ing index (labeled cells/total cells x 100) 4) Grain count profile, referring to the number of silver grains over individual nuclei of a given population. Chapter 4 RESULTS THE EFFECT OF CORTISOL ON THE RELEASE OF PREVIOUSLYINCORPORATED 45Ca FROM MOUSE LIMB-BONES IN VITRO Increasing doses of cortisol caused increased depression in release of previously-incorporated 45Ca from bone to media. While 0.01 jJg cortisol/ml media did not alter the percent 45Ca released when compared to controls, 1.0 jJg cortisol/ml media produced a 40% reduction in the percent radioactivity released from bone after a four day culture period (fig. 7). The depression of 45Ca release by cortisol also occurred in bones kil led by freezing and thawing (table 2). The maximal depression of 45Ca release from bone to media occurred within an 8 hr time period; thereafter, the diffusion of radioactivity appeared to be returning to control values. THE TIME REQUIRED FOR BONE CELL DIFFERENTIATION IN INFANT MICE IN VIVO Infant mice used in this study averaged 1.5 gm on the second day of 1 ife, the time of tr i t iated-thymid i ne (3HTdR) inject ion. Fourteen days later, the time of the final sacrifice, the mice weighed an average of 7.6 gm, a 500% increase in weight during the experimental t j me pe r i od (f i g. 8). A census of labeled cells in the proximal metaphysis of the tibia revealed that populations of endothel ial cells, progenitor cells, 47 48 1.2 ..... 1.0 I -------- --------I 0 a: I- z 0 u ........ 0 La.J I- ........ .......... 0.8 cr a: La.J I- 0.6 0.4 01-22--,------------------------~1~----------------------~1 o 0.01 0.1 jlg CORTISOL Iml MEDIA Fig. 7 Effect of cortisol on release of previously-incorporated 45Ca from mouse limb-bones four days in culture. Data are expressed as treated to control ratios of percent 45Ca released to media. 1.0 49 TABLE 2 Effect of cortisol on %54Ca released from dead bones in vitro 1 fl9 S Cortiso1/ml media hr Time in culture 24 hr ~S hr 0.001 0.62 + 0.06 0.78 + 0.01 0.80 + 0.02 0.01 0.67 ~ 0.24 0.74~0.13 0.72 + 0.16 0.1 0.26 + 0.06 0.39 ~ 0.04 0.42 + 0.06 1.0 0.37 + 0.04 0.46 ~ 0.48 ~ 0.03 0.03 Data are expressed as treated to control ratios of percent 45Ca released. 50 2 Fig.8 4 6 8 10 AGE, DAYS Daily total body weights of normal infant mice. 12 14 16 51 resorbing cells and osteoblasts were labeled 1 hr post 3HTdR injection (table 3). Maximal label ing in the endothel ial cells and progenitor cells occurred 1 hr post 3HTdR when data is expressed as number of labeled cells/field (5 and 11 labeled cells respectively). During the two-week period that followed, there was a decrease in the number of labeled endothel ial and resorbing cells/field, with no labeled progenitor cells detected at 336 hr. Three labeled resorbing cells/field were seen 1 hr post 3HTdR. This figure increased to a maximum of 5 labeled cells/field at 48 hr and had decreased to 0.3 labeled resorbing cells/field by 336 hr. Three labeled osteoblasts/metaphyseal field also were seen 1 hr post 3HTdR. This figure reached a maximum of 7 labeled osteoblasts/ field by 12 hr and no labeled osteoblasts were detected at 336 hr. The first labeled osteocytes (O.l/field) appeared in the metaphysis of the tibia at 24 hr post 3HTdR • This figure increased to a maximum of 0.9 labeled osteocytes/field at 120 hr, followed by a decrease in labeled osteocytes/field with time until none were detected in the proximal metaphysis at 336 hr. With the exception of progenitor cells, the total numbers of each cell type/field showed no such consistent increases or decreases in the metaphysis throughout the two week time period (table 4). mean population for each cell type/field was as follows: The endothel ial cell, 19; progenitor cell, 41; resorbing cell, 19; osteoblast, 26; and osteocyte, 1.5. The progenitor cells, in contrast to the other cell types, were more dense at 1 hr post 3HTdR (64 cells/field) than at 336 hr (22 cells/field). 52 TABLE 3 Labe 1ed ce 11 s per field in proximal metaphysis of tibia Hrs post 3HTdR Endothe1 ium Progenitor Resorbing ce 11 Osteoblast Osteocyte 5. O:;'~ 11 .4.,', 3.0 3.1 0 4 5.0 10.0 2.6 3.0 0 6 1.8 4.6 1.3 0.8 0 12 5.0 8.7 3.0 6.9'" 0 24 4.6 6.2 1.2 3.2 0.1 48 2.1 3.5 5.1,', 1.7 0.2 72 2.4 3.9 2.6 1 .8 0.3 96 1.4 4.4 3.3 1.8 0.4 120 2.6 5.2 1.2 5.3 0.9,1, 144 1.0 4.0 2.3 2.7 0.7 168 0.7 3.6 0.7 2.6 0.3 336 0.2 0 0.3 0 0 ,',Max imum number of labeled ce 11 s per field. 53 TABLE 4 Total cells per field in proximal metaphysis of tibia Endothe1 ium Progenitor Resorbing ce 11 28.2,', 63. l'k 30.0,'( 24.8 1.5 4 23.7 52. 1 28.3 25.2 0.2 6 14.1 26.1 14.1 17.0 1 .2 12 28.6 47.0 15.5 44.8:;\- 0.8 24 21.6 40.8 12.0 29.1 1.8 48 13.0 22.7 20.7 10.8 0.6 72 19.4 34.2 19.8 19.4 1.0 96 10.9 38.6 20.1 23.1 1 .4 120 16.4 40.9 12.5 32.1 3.0·': 144 12.7 58.0 21.3 19.6 3.0 168 17.5 40.0 14.6 41.3 1.9 336 25.0 22.0 21 .4 20.2 1.2 mean 19.3 40.5 19.2 25.6 1.5 Hours post 3HTdR ,':Max imum number of total ce 11 s per field. Osteoblast Osteocyte 54 Label ing indices of the cell populations in the proximal metaphysis of the tibia showed that 1 hr post 3HTdR 18% of the endothel ia1 cells and progenitor cells, 10% of the resorbing cells and 13% of the osteoblasts were labeled (table 5). The label ing indices of the resorbing cell population reached a maximum of 25% at 48 hr; osteoblasts, of 17% at 120 hr (although similarly high values were seen as early as 12 hr); and osteocytes, of 32% at 48 hr. A similar census of labeled cells in the diaphysis of the tibia (table 6) revealed that, as in the metaphysis, progenitor cells, resorbing cells and osteoblasts were labeled 1 hr post 3HTdRt although none of these cells in either the endosteum or periosteum were maximally labeled at this time period. In the periosteum, the maximum number of labeled progenitor cells/field was 3 at 72 hr. The labeled population did not deviate from this number at any other time period; 2 labeled progenitor cells/field were seen at both hr and 336 hr. Few resorbing cells, labeled or unlabeled, were seen in the periosteum; however, as many as 3 labeled resorbing cells/field were seen at 144 hr post 3HTdR. The maximum number of labeled osteoblasts in the periosteum (1.5/field) were seen at 144 hr. osteocytes were seen in the diaphysis at 12 hr. reached a maximum of 1.5/field at 144 hr. The first labeled Labeled osteocytes Osteoblasts in the periosteum and osteocytes paralleled each other in decrease of labeled cells/field to 0.6 labeled osteob1asts and 0.5 labeled osteocytes/field at 336 hr. The endosteum appeared to be more heavily labeled than the periosteum. A maximum of 6 labeled progenitor cells/field was seen 55 TABLE 5 Labe 1 i ng indices in proximal metaphysis of tibia Hrs post 3HTdR Endothe1 ium Progenitor Resorbing ce 11 Osteoblast 17.7 17.9 10.0 12.5 0 4 21.1,', 19.2;', 9.2 11.9 0 6 12.7 17.6 9.2 4.7 0 12 17.5 18.5 19.4 15.4 0 24 21.3 15.2 10.0 11 .0 5.5 48 16. 1 15.4 24.6,', 15.7 32.2,', 72 12.4 11.4 13.1 9.3 31.1 96 12.8 11.4 16.4 7.8 28.4 120 15.9 12.7 9.6 16.5'" 29.7 144 7.9 6.9 10.8 13.8 23.7 168 4.0 9.0 4.8 6.3 15.4 336 0.8 0 1. 4 0 ,';:Max i mum 1abe 1 i ng index. Osteocyte 0 TABLE 6 Labeled cells per field in diaphysis of tibia Hours post 3HTdR * Progenitor Periosteum Resorbing ce 11 Osteoblast Osteocyte Osteoblast Endosteum Resorbing cell Progenitor 1.8 0.1 0.5 0 0.5 0.2 4.0 4 0.5 0 0.8 0 0.3 0.3 6.0,;', 6 0.4 0 0 0 0 0.8,', 1.6 12 1.8 0 0.4 0 1.4 0.2 0.3 2.2 0 0.2 0 0.8 0.5 3.0 48 0.8 0.1 0.4 0 0.2 0 1 .4 72 2.6,;', 0 0.4 O. 1 0.2 0.2 0.9 96 2.4 0.1 0.4 0.6 0.8 0.1 1.6 120 1.3 0 1.1 0.9 1. 0 0.8 144 0.8 2.5,', 1 .5,', 1 .5,', 1.0 0 1.8 168 1.3 0 0.5 0.8 1 .0 0 1.0 336 1.9 o. 1 0.6 0.5 0.4 0 0.4 Maximum number of labeled cells per field. ., ...... - 0" 57 in the periosteum at 4 hr post 3HTdR; this value decreased to 0.4 labeled progenitor cells/field at 336 hr. One hr post 3HTdR 0.2 labeled resorbing cells/field were seeno This value increased to 0.8 labeled resorbing cells/field at 6 hr and decreased to no label after 96 hr. Osteoblasts in the endosteum reached a maximum label ing of 1.6 labeled cells/field at 120 hr. With the exception of the osteocyte, the densities of each cell type, i.e., labeled plus unlabeled cells/field, in the diaphysis did not change during the two-week experimental time period (table 7). In the periosteum, 19 progenitor cells/field were seen, 0.9 resorbing ce]ls, and 7.0 osteob1asts. In the endosteum of the diaphysis, 13 progenitor cells/field were seen, 2 resorbing cells, and 90steob1asts. Osteocytes increased in number with time from 3/fie1d at h r pos t 3HTdR to 15/field at 336 hr. Label ing indices (table 8) of cells in the periosteum indicate that hr post 3HTdR 7% of the progenitor cells were labeled, 25% of the resorbing cells and 9% of the osteob1asts. This value reached a maximum in the progenitor cells of 17% at 72 hr, in the resorbing cells of 25% at 96 hr, and in the osteoblasts of 25% at 144 hr. Label ing indices of the osteocyte reached a maximum of 24% at 144 hr. Label ing indices in the endosteum of the diaphysis indicate that 1 hr post 3HTdR 29% of the progenitor cells were labeled, 14% of the resorbing cells, and 6% of the osteoblasts. This value reached a maximum in the progenitor cells of 31% at 4 hr, in the resorbing cells of 17% at 12 hr and in the osteoblasts of 18% at 120 hr. TABLE 7 Total cells per field in diaphysis of tibia * Endosteum Resorbing ce 11 Progenitor Periosteum Resorbing cell Osteoblast Osteocyte Osteoblast 26. 5~': 0.4 5.4 2.9 8.6 1.4 13.7 4 18.5 0 11 .4;', 4.2 15. o~'~ 0.3 19.5;', 6 17.4 0 0 3.4 0 6.3;', 10.8 12 20.5 1.3 8.2 6.4 11.0 1.2 2.3 24 25.0 0.2 8.3 2.3 7.1 3.0 16.7 48 13.8 2.8;', 7.7 4.5 8.3 0 12.0 72 15.8 0.9 6.3 10.0 5.9 3.8 12.0 96 20.5 0.4 6.3 10.0 10.8 0.1 13.8 120 15.1 0.5 8.3 12.3 8.8 3.3 12.3 16.7 2.0 6.0 6.3 8.8 0 11.8 168 17.8 0.3 8.9 8.6 8.2 0.7 16.9 336 18.3 2.2 7.2 14.7-'''" 10.3 0.1 11.8 mean 18.8 1.9 7.0 8.6 1.7 12.0 Hours post 3HTdR Progenitor \.J1 Maximum number of total cells per field. ex:> TABLE 8 Label ing indices in diaphysis of tibia Progenitor Periosteum Resorbing cell Osteoblast Osteoblast Endosteum Resorbing cell Progenitor 6.8 25.0 9.2 0 5.8 14.3 29.3 4 2.7 0 7.0 0 2.0 6 2.3 0 0 0 0 12.7 14.8 12 8.8 0 4.9 0 12.7 16.6'k 13.0 24 8.8 0 2.4 0 11.2 16.4 18.0 48 5.8 3.6 5.2 0 2.4 0 11 .7 72 16.5~·' 0 6.3 1.0 3.4 5.2 7.5 96 11.7 6.4 6.0 7 "l (75.0) 11.6 120 8.6 0 13.3 7.3 18.2"', 0 6.5 144 4.8 12.5 25.0'1, 24.0"\' 11.4 0 15.2 168 7.3 0 5.6 9.3 12.2 0 5.9 336 10.4 4.5 8.3 3.4 3.9 0 3.4 Hours post 3HTdR * Maximum label ing index. 25.0,', Osteocyte (100.0) 30 . 8~', \.n \.D 60 THE EFFECT OF CULTURE ON BONE CELL POPULATIONS The radi i used in these experiments consisted of a shaft and metaphyses, all of finely trabeculated woven bone, surrounding a marrow cavity of scant hematopoetic tissue (fig. 9). These bones were actively engaged in processes directed towards enlargement of the marrow cavity. Most important of such processes was endochondral growth, i.e., invasion of the hypertrophic cartilaginous zone and continual removal of the intercellular partitions of the calcified cartilage. Bone apposition occurred on those cartilaginous trabeculae of the metaphysis which were not resorbed. in increased width of the marrow cavity. A second process resulted This involved endosteal resorption concomitant with osteoid production in the periosteum. On the periosteal surface of the metaphyseal regions of these bones, there was a characteristic region of resorption associated with maintaining the funnel shape of the bone (subperiosteal resorption). There were also isolated areas of resorption along the shaft, apparently associated with the formation of nutrient artery and vein foramina. Four days in the described control organ culture system resulted in several characteristic histologic transformations (fig. 10). Most dramatic was the disapparance of hematopoetic tissue, followed by a fibrinoid replacement within the marrow cavity. Cartilage interstitial growth proceeded at a rate greater than cartilage excavation within the metaphyseal growth plate, resulting in an increased epiphyseal cartilage mass. Nevertheless, osteoblast differentiation did occur and 61 Fig. 9 Photomicrograph of preculture bone. Bone of both diaphysis and metaphysis is composed of finely woven trabeculae. Scant hematopoetic tissue invades the metaphysis. Fig. 10 Photomicrograph of bone grown 4 days in control culture. Fibrinoid prol iferation has replaced former marrow elements. Numbers of total osteoblasts and resorbing cells have decreased when compared to preculture values. 62 ~) "1 63 osteoid was seen deposited on primary trabeculae. The numbers of labeled osteoblasts in all areas of the bone were either equa1 to preculture values or increased (tables 9,10,11,15,16,17). Changes in the diaphysis of cultured bone were not as marked as those in the metaphysis. The numbers of total osteoblasts and resorbing cells decreased in all areas of control bone when compared to precu1ture values. An increased number of undifferentiated, quiescent 1 ining cells occupied bone surfaces. THE EFFECT OF CORTISOL ON BONE CELL DIFFERENTIATION IN VITRO The addition of cortisol to the culture media in concentrations greater than 0.01 ~g/m1 resulted in a reduction in the invasion of fibroblasts in the marrow cavity and in a reduction in the interstitial growth of metaphyseal cartilage (fig. 11). Besides a general reduction in the necrotic processes that follow the initial metabol ic adjustment made by tissues placed in culture, 1 ittle change in the histology of the diaphysis could be noted. There appeared to be no change in total bone mass, although osteoid formation appeared reduced and scalloped surfaces appeared increa in the quiescent 1 ining cell. There wa s a genera 1 increase The descriptive term "protectivell would be wel l-app1 ied to the histologic effect of cortisol in culture, with the exception of the 100 ~g/ml media dose level which was apparently toxic. The direct effect of graded doses of cortisol on bone cell populations was studied in the distal metaphysis, diaphyseal endosteum, and periosteum of mouse radii maintained in organ culture for four 64 TABLE 9 Effect of culture on bone cell popUlations in metaphysis of radius l eell type/field Preculture Control culture Labeled cells/field: progenitor 7.50 (6.17-8.33) 2.33 (1.25-3.00) resorbing cell 1.94 (1.33-3.00) 0.92 (0.00 - 1.50) osteoblast 0.56 (0.00-1.00) 1.00 (0.75-1.25) progenitor 44.67 (42.33-47.67) 15.75 (14.25-18.00) resorbing cell 10.83 (7.50-13.67) 3.42 (1.50-5.00) osteoblast 25.83 (8.17-38.00) 21.00 (19.50-23.75) Total cells/field: lNumber out of parentheses is mean of three cultures. Numbers in parentheses are range of cell counts. 65 TABLE 10 Effect of culture on bone cell populations in diaphyseal endosteum of radius 1 Preculture Control culture progenitor 3.17 (3.00-3.38) 0.71 (0.25-1.50) resorbing cell 0.06 ( 0 . 00 - o. 13) osteoblast 0.39 (0.13-0.55) 0.43 (0.17-0.75) progenitor 13.67 (11.37-15.75) 7.24 (4.88-9.83) resorb i ng ce 11 1. 28 (0.63-1.75) 0.31 (0. 17 - 0 . 50) osteoblast 9.00 (7.50-10.88) 5.48 (3.63-6.63) Cell type/field Labeled cells/field: o Total cells/field: 1Number out of parentheses is mean of three cultures. Numbers in parentheses are range of cell counts. 66 TABLE 11 Effect of culture on bone cell populations in diaphyseal periosteum of radius l Precu1ture Control culture progenitor 0.58 (0.35-0.88) 0.59 (0.38-1.00) resorbing cell 0.04 (0.00-0.13) Cell type/field Labeled cells/field: o (0.00-0.25) 0.28 (0.25-0.33) progenitor 17.86 ( 13. 70- 23. 13) 12.29 (12.00-12.50) resorb i ng ce 11 0.24 (0.10-0.38) 0.04 (0.00-0.13) osteoblast 8.63 (4.25-11.38) 7.56 (6.75-8.67) as teob 1as t 0.13 Total cells/field: lNumber out of parentheses is mean of three cultures. Numbers in parentheses are the range of cell counts. 67 Fig. 11 Photomicrograph of bone grown 4 days in culture with 1.0 wg cortisol/ml media. Number of fibroblasts is decreased. Although bone has maintained preculture mass, bone surfaces (particularly endosteal) are scalloped, i.e., resorbing. Fig. 12 Photomicrograph of bone grown 4 days in culture with 0.1 unit PTE/ml media. Although not evident in this field, number of fibroblasts is increased. Trabeculae have been resorbed and whole areas of diaphysis are gone (between arrows). 68 , ,. -, 69 days. \vhen radii were placed into culture (1 hr post 3 HTdR ), cells in the distal metaphysis were labeled as follows: eel Is, I 7% 0 f the res 0 r bin gee lIs, and 2% 0 f the 17% of the progenitor 0 s teo b 1a s t s (t a b 1e 9). Few osteocytes were present and were not tabulated. In the metaphysis of control radii, there were 16 progenitor cells/field (2 labeled). The numbers of both total and labeled progenitor cells were increased to 22 and 3 respectively at 0.0001 119/ml dose. The number of labeled progenitor cells was decreased to l/field at the 0.01 11g cortisol/ml media dose level, but the number of total progenitor cells was increased (20/field). At the two highest doses (1.0 and 100.0 119/ml). the numbers of both labeled and total progenitor cells were decreased from control values (0.3 labeled/ field and 10 total/field at 100.0 119/ml) (table 12). Three resorbing cells/field (0.9 labeled) were seen in the metaphysis of control bones. The numbers of both labeled and total resorbing cells were increased at all dose levels of cortisol in a dose-response fashion. The maximum number of total resorbing cells/ field was seen at 100.0 119 cortisol/ml media. In contrast, total osteoblast numbers were decreased at all dose levels of cortisol. Control cultures contained 21 osteoblasts/ field (1 labeled) in the metaphysis of the radii. All doses of cortisol caused an approximate 40% decrease in total osteoblast numbers. The numbers of labeled osteoblasts were decreased in relation to controls at the three highest dose levels. At 0.0001 11g cortisol/m1 media, the number of labeled osteob1asts was increased to 2/field. TABLE 12 Effect of cortisol on bone cell populations in distal metaphysis of radius in vitrol Cortisol/m1 media 1.0 0.01 ~g Control culture Cell type 0.0001 100.0 Doseresponse 2 Labeled cells/field: progenitor 3.35 (2.00-4.25) (0.33-2.25) 0.36 .13-0.70) 0.92 (0.00-1 .50) 1. 93 (0.80-3.00) 1. 08 .17-1.33) 2.15 (1.25-3.70) 1. 81 (0.80-2.88 (0.75-1.25) 2.27 (2.00-2.80) 1.05 (0.00-1.65) 1.25 (1.00-1.50) 0.67 (0.00-1.75) progenitor 15.75 (14.25-18. 22.45 (21 .20-23.75) 19.58 (16.50-25.25) 12.75 (9.50-16.50) 10.02 (7.50-12.30) I', ,,;'~ I'; resorb i ng ce 11 3.42 (1 .50- 5.00) 8.28 (6.20-9.40) 8.92 (7.25-11.00) 11.07 (8.75-13.20) 14.68 ( 12.80 - 16.25) if, ../~ osteoblast 21.00 (19.50-23.75) 13.78 (11.75-15.40) 13.00 (10.50-16.50) 14.18 (12.75-15.50) 9.40 (8.25-10.25) resorbing cell osteoblast 1.00 1. 25 0.30 (0.00-0.50) 2.33 (1.25-3. *1; '";t, Total cells/field: lNumber out of parentheses is mean of three cultures. Numbers in parentheses are range of cell counts. 2,'~ 0 . 0 1< P< 0 . 05; ,h', 0 . 00 1< P< 0 . 0 1; ,bb', P< 0 . 00 1 . ""-.J o 71 In the endosteum of the diaphysis 1 hr post 3HTdR , 21% of the progenitor cells were labeled and 4% of the osteob1asts. resorbing cells were seen in this area {table 10}. Few The diaphyseal endosteum of control radii contained 7 progenitor cells/field {0.7 labeled}. Cortisol decreased the number of labeled progenitor cells/ field at 100.0 ~g/ml media. but at no dose level was the number of total progenitor cells in the endosteum decreased. 0.0001 ~g/ml To the contrary, nearly doubled the number of total progenitor cells in the endosteum {table 13}. The number of resorbing cells in the endosteum of control radii was very small, 0.3/fie1d; none of these were labeled. No effect of cortisol on endosteal resorbing cells could be detected. At no dose level were labeled resorbing cells seen in the diaphyseal endosteum. Five osteoblasts/field (0.4 labeled) were seen in the endosteum of control radi i. The three highest doses of cortisol decreased the numbers of labeled osteoblasts to almost 0 at 100.0 ~g/ml media. The numbers of total osteoblasts in the endosteum was not consistently effected by cortisol. Doses of 0.0001 and 1.0 ~g/ml appeared to increase the total number of osteoblasts to a maximum of 9/field at 0.000 1 ~g/m 1 • Few cells were labeled in the diaphyseal periosteum 1 hr post 3HTdR • Three percent of the progenitor cells and 1% of the osteoblasts were labeled at this time (table 11). In the periosteum of control cultures, there were 12 total progenitor cells/field {0.6 labeled}. Increasing doses of cortisol decreased the numbers of both labeled and total progenitor cells. The maximum depression in the number of TABLE J 3 Effect of cortisol on bone cell populations in diaphyseal endosteum of radius ~. vitro l Cell type Control culture ~g 0.0001 Cortisol/m1 media 0.01 1.0 100.0 Doseresponse 2 Labeled cells/field: progenitor resorb i ng ce 11 osteoblast 0.71 (0.25-1.50) 0 0.43 (0.17-0.75) 1. 50 (1.25-1.83) 0.56 (0.00-1.00) 0 0 0.68 (0.29-1.25) o. 19 0.75 (0.25-1.29) 0 o. 19 "k;t~ (0.00-0.43) 0 (0.00-0.44) 0.30 (0.14-0.50) 0.05 (0.00-0.14) 12.62 (10.38-16.33) 7.10 (4.71-9.38) 9.39 (5.88-11.43) 7.18 (3.17-12.00) if, Total cells/field: progenitor 7.24 .88-9.83) resorb i ng ce 11 0.31 (0.17-0.50) 0.48 (0.00-1.43) 0.37 (0.00-1.00) 0.36 (0.00-0.71) 0.27 (0.00-0.43) osteoblast 5.48 (3.63-6.63) 9.03 (7.71-10.00) 5.88 (3.75-7.00) 7. 19 (6.07-8.86) 4.27 (2.17-7.00) lNumber out of parentheses is mean of three cultures. Numbers in parentheses are range of cell counts. 2*0.01 P<0.05; **O.OOl<P<O.Ol. ----Not significant. "-J N 73 progenitor cells was produced by 100.0 lowest dose level, 0.0001 ~g/ml. ~g cortiso1/m1 media. The increased the numbers of both labeled and total progenitor cells above control values (table 14). Few resorbing cells were seen in the periosteum of control cultures. All doses of cortisol appeared to increase the resorbing cell populations in the periosteum, but at no dose level were labeled resorbing cells seen in this area. Eight osteob1asts/field (0.3 labeled) were seen in the periosteum of control cultures. Doses of cortisol greater than 0.01 ~g/ml decreased the numbers of both labeled and total osteoblasts. The maximum decrease in the number of osteoblasts occurred at 100.0 cortisol/ml media (2 osteoblasts/field J 0.05 labeled). 0.0001 ~g ~g The dose of cortisol/ml media more than doubled the number of labeled osteob1asts, with no change in number of total osteoblasts. THE EFFECT OF PARATHYROID EXTRACT ON BONE CELL DIFFERENTIATION IN VITRO In contrast to the effect of cortisol, the addition of parathyroid extract (PTE) to culture media resulted in an obvious acceleration of resorption to the exclusion of osteoid production (fig. 12). The greatest resorption occurred in regions which were sites of normal resorption, i.e., metaphyseal trabeculae and subperiosteal metaphysis. Particularly at the lowest dose used, 0.1 unit PTE/ml media, entire metaphyses were removed, resulting in detachment and complete resorption of trabeculae and complete perforation of the shaft. Although multinucleated osteoclasts were seen an increase in their numbers above control values could not be detected. The described TABLE 14 Effect of cortisol on bone cell populations in diaphyseal periosteum of radius in vitro l Cell type Control culture 0.0001 0.59 .38-1 .00) 0.78 (0.43-1.25) Wg Cortisol/m1 media 0.01 1 .0 100.0 Doseresponse 2 Labeled cells/field: progenitor resorbing cell osteoblast 0 0.35 (0.00-0.75) 0 0 0.99 (0.71-1.50) 0 0.10 (0.00-0.29) 0 0.28 (0.25-0.33) 0.76 (0.50-1.14) 0.36 (0.00-0.79) 0.25 (0.22-0.29) 0.05 (0.00-0.1 progenitor 12.29 (12.00-12.50) 14.57 (14.00-1 5.71 ) 11.66 (8.29-16.25) 11 .37 (9.00-13.36) 10.43 (6.33-17.71) resorb i ng ce 11 0.04 (0.00-0.13) O. 19 (0.00-0.57) 0.40 (0.00-0.88) 0.10 (0.00-0.29) 0.10 (0.00-0.29) osteoblast 7.56 (6.75-8.67) 7.62 (6.63-8.67) 5.88 (5.63-6.14) 6.58 (6.13-7.33) 2.27 (0.83-4.86) "';',~/: Total cells/field: -/:; ../:: lNumber out of parentheses is mean of three cultures. Numbers in parentheses are range of cell counts. 2** 0.001 <P<O.Ol. ----Not significant. "-....l ...t:- 75 resorption was apparently produced by mononuclear cells. Within the diaphysis, osteocytes could be seen in enlarged lacunae and, in fact, could be seen leaving their lacunae altogether. The fibrinoid prol iferation of the marrow cavity obviously exceeded control levels. The direct effect of graded doses of PTE on bone cell populations was studied in three areas of mouse radii maintained in organ culture for four days. These areas were the distal metaphysis, the diaphyseal endosteum and the periosteum. post 3HTdR. Radii were placed into culture 1 hr At this time in the distal metaphysis, 13% of the progenitor cells were labeled, 4% of the resorbing cells and 5% of the osteoblasts (table 15). After four days in culture, 15 progenitor cells/field (0.9 labeled) were seen. Doses of 0.1 and 1.0 unit PTE/ml media increased the numbers of both total and labeled progenitor cells. One unit PTE/ml media produced 25 progenitor cells/field (2 e 8 labeled). Ten units PTE/ml media decreased the numbers of total and labeled progenitor cells/field. Eleven resorbing cells/field (1.4 labeled) were seen in the metaphysis of control radii. The two lowest doses of PTE increased the numbers of both labeled and total resorbing cells. Ten units PTE/ml media depressed the numbers of both labeled and total resorbing cells to 0.9 and 10/field respectively. Although the number of osteoblasts in the metaphysis was increased in control radii when compared to radii before culture, at no dose of PTE were either labeled or unlabeled osteoblasts seeno One hr post 3HTdR 11% of the progenitor cells and 3% of the resorbing cells were labeled in the endosteum of the diaphysis {table TABLE 15 Effect of parathyroid extract on bone cell populations in distal meta Ce 11 type Preculture is of rad i us in vi t ro 1 Units PTE/ml media Control culture 0.1 1.0 10.0 Doseresponse 2 Labeled cells/field: progenitor 5.17 (3.00-7.33) 0.93 (0.20-2.33) 1.70 (0.50-3.60) 5.07 (1.50-9.50) 0.88 (0.14-2.33) resorb i ng ce 11 I. 17 (0.33-2.00) 1. 39 .25-2.33) 2.30 (1.40-3.00) 2.83 (2.00-4.00) 0.92 (0.17-1.43) osteoblast 0.30 .00-0.60) o 0.33 (0.00-1 . o o Total cells/field: progenitor 39.17 (36.00-42.33) 14.87 (8.00-19.80) 18.90 (10.00-35. 24.97 (16.40-42.00) 6.92 (1.33-17.30) resorbing cell 23.10 (16.20-30.00) 11.03 (7.00-15.80) 28.10 (22.00-39.80) 17.10 (5.50-30.00) 9.57 (5.50-13.20) osteoblast 6.20 (0.00-12.40) 6.87 (2.60-10.00) l Number out of parentheses is mean of three cultures. Numbers in parentheses are range of cell counts. 2*0.01<P<0.05. ----Not significant. o o o -/\ 77 16). No labeled osteoblasts were seen. After four days in culture, 10 progenitor cells/field (1.2 labeled) were seen in control bones. Increasing doses of PTE decreased the numbers of both labeled and total progenitor cells/field. Ten units PTE/ml media reduced the number of progenitor cells to a minimum of 6/field (0.7 labeled). Three resorbing cells/field (0.3 labeled) were seen in the endosteum of the diaphysis of control radii. All three doses of PTE increased the number of total resorbing cells in this area, but only at the lowest dose (0.1 unit/ml) was the number of labeled resorbing cells increased. The number of osteoblasts in the endosteum was increased in control cultures when compared with preculture values. PTE decreased the number of osteoblasts. All doses of No labeled osteoblasts were seen in the diaphyseal endosteum of PTE-treated bones. Only 2% of the progenitor cells were labeled in the periosteum 1 hr post 3HTdR (table 17). No other cell types were labeled. Twelve progenitor cells per field (0.3 labeled) were seen in the periosteum of control cultures. No dose of PTE changed the number of progenitor cells in the periosteum, although 10 units/ml decreased the number of labeled progenitor cells to nearly one-half control values. Three resorbing cells/field (0.2 labeled) were seen in the periosteum of control cultures. All doses of PTE increased the number of total resorbing cells/field in the periosteum, but the number of labeled resorbing cells did not appear changed. Although labeled osteoblasts were seen in the periosteum of control cultures, none were seen in PTE-treated bones. Likewise, all TABLE 16 ea1 endosteum of radius in vitro 1 Effect of parathyroid extract on bone cell populations in dia Cell type Precu1ture Control culture Un its 1 med ia ------:-::--::---10.0 0.1 Doseresponse 2 Labeled cells/field: progenitor resorbing cell 1.00 0.20 (0.00-0.40) o osteoblast 1. 24 (0.63-1.60) 1. 35 (0.40-2.33) 0.31 (0.00-0.67) (0.17-0.83) o. o 0.47 (0.00-0.75) 1. 13 .50-1.88) 0.66 (0.38-0.89) o. 19 (0.00-0.38) 0.21 .00-0.33) o o Total cells/field: progenitor 10.27 (8.33-12.10) 10.57 (8.20-12.70) 8.77 (6.17-10.80) 5.51 (4.00-8.11) (4.70-6. 3.22 (1.40-5.25) 4.90 (2.17-7.20) 4.38 (2.00-7.3 4. (3.57-5.25) 2.90 (0.80-5.00) (4.00-5. 1 .56 (0.00-2.50) 0.78 (0.00-2.33) 9.35 (7 . 40-11 .30) 5. resorb i ng ce 11 osteoblast 4. 0.45 .14-1.00) 1Number out of parentheses is mean of three cultures. Numbers in parentheses are range of cell counts. 2*0.01 <P<0.05. ---- Not significant. -.........J co TABLE 17 Effect of parathyroid extract on bone cell populations in diaphyseal periosteum of radius In vitrol Cell type Preculture Control culture Units PTE/ml media 0.1 1.0 10.0 Doseresponse 2 Labeled cells/field: progenitor 0.27 (0.20-0.33) 0.30 (0.00-0.50) 0.22 (0.00-0.67) 0.52 (0.00-0.88) o. 18 (0.04-0.29) 0.06 (0.00-0.33) 0.19 (0.00-0.33) 0.16 (0.00-0.25) resorb i ng ce 1 1 o o. 18 (0.00-0.33) osteoblast o 0.12 (0.00-0.20) o o o Total cells/field: progenitor 14.05 (10.80-17.30) 11.73 (8.20-13.50) 7.77 (0.00-14.30) 13.33 (10.50-18.50) 10.54 (7.29-12.20) resorb i ng cell 3.75 (1.80-5.70) 2.96 (2.00-4.38) 7.27 (5.17-8.83) 5.81 (4.33-8.60) 3.77 (2.57-6.00) osteoblast 2.25 (1.70-2.80) 1.94 (0.33-4.00) 0.50 (0.00-1.50) 0.67 (0.00-2.00) 0.81 (0.00-1.29) lacunae 3.37 (3.33-3.40) 2.70 (2.50-3.00) 1.60 (1.00-2.00) 1.97 (1.75-2.33) 2.27 (1.14-3.00) Length (ern) 3.07 3.39 (3.36-3.42) 3.50 (3.19-3.81) 3.61 (3.47-3.76) 3.87 (3.81-3.93) INLJmber out pa rentheses Numbers in parentheses are 2----Not significant. ree cultLJ-res-.--· cell counts. ~ 80 doses of PTE produced a decrease in the number of total osteoblasts, from 1.9/field in controls to a minimum of 0.5/fie1d at 0.1 unit PTE/ml media. A tabulation of the number of osteocyte lacunae/field revealed that all doses of PTE decreased the number of lacunae. More than half the lacunae placed in culture (3.4/field) were resorbed by 0.1 unit PTE/ml media. In contrast, the length of the total bone increased with increasing doses of PTE. Ten units/ml increased the length of the radii an average of 26% of the pre-culture value, while control bones increased only 10% in length. Chapter 5 DISCUSSION THE EFFECT OF CORTISOL ON THE RELEASE OF PREVIOUSLYINCORPORATED 45Ca FROM MOUSE LIMB-BONES IN VITRO Mineral removal from bone to media was the result of a combination of three influences. The first is passive exchange which occurs in the absence of cellular activity. The second is an active process referred to as a "calcium pump" and believed to be located in the osteocyte (Belanger et al., 163; H. M. Frost and J. S. Arnold, personal communication) and the osteoblast (Talmage, 169, 170). The third influence on calcium release is the conventional process of resorption by osteoclasts. Because 45Ca was incorporated into these bones two days prior to culture, the radioactivity was buried by new bone and was probably out of reach of the osteoclasts. Calcium release in the experiment reported here was depressed by 1.0 ~g cortisol/ml media. At this dose, the number of resorbing cells was increased; which supports the contention that bone cells other than osteoclasts were controll ing 45Ca removal. If the net flux of 45Ca from bone to blood is primarily the result of an active process (as it must be for the ion to move against a concentration grad'ient), the cortisol-induced suppression of 45Ca release from bone to blood in this experiment is understandable. Cortisol has been shown to inhibit bone cell respiration in vi ro (Roger and Kalbhen, '68; Whitehouse and Bostrom, '62) as well as to 81 82 cause connective tissue cells, such as fibroblasts, to become inactive (Schneebel i and Dougherty, 1(,2). Cortisol depression of 45 Ca release also occurred in bones killed by freezing and thawing. This phenomenon lends support to the idea that the mechanism of steroid actionmay bea physical-chemical one. Cortisol appears to induce changes in skeletal tissue matrix not mediated by 1 ive cells. It has, for instance, been shown to increase polymerization of connective tissue extracellular substances (Asadi et al., 156). The effect of parathyroid extract on calcium metabol ism in this organ culture system was not studied. It should be mentioned, however, that both parathyroid hormone and parathyroid extract are known to stimulate resorption ~ vitro (Gaillard, '61). A dose of 1 unit PTE/ml media doubled the amount of 45 Ca found in the media when compared to controls after 72 hr in culture (Raisz, 165). This move- ment of calcium out of the bone in response to parathyroid hormone is due to a stimulation of the calcium pump and to an induction of osteoclast differentiation (Talmage, '69). Parathyroid hormone therefore has an effect opposite to cortisol on the calcium metabol ism of the skeleton and on the activity of the calcium pump. THE TIME REQUIRED FOR BONE CELL DIFFERENTIATION IN INFANT MICE IN VIVO Various dogmatic theories have been postulated which force the osteoprogenitor cell through a series of irreversible steps on its way to becoming a differentiated bone cell (Owen, 170; Young, '62a). 83 These steps toward differentiation are represented in fig. 13 by sol id arrows. The cell is given no alternative route once it has begun to differentiate. These theories also insist that only the osteoprogenitor cell is capable of division, i.e., would be the only significantly labeled cell at 1 hr post 3 HTdR • Young and Owen, using simpl ified models, have suppl ied data which support such a theory. They postulate the existence of a smooth transition of cells from the undifferentiated form to the fully-differentiated one, actively engaged in bone formation or resorption. Such conclusions cannot be drawn from the studies on 3HTdR labeled cell populations in the tibia of infant mice reported here. This data suggests that cell morphology gives 1 ittle indication of the activity of a cell. A problem inherent in this study is the absence of critical means by which to identify cells by morphology alone. Histochemical techniques, used in connection with kinetic studies, may give more definitive information. In addition to the problem of cell identification, it is evident that all bone cell types possess the same genetic material and that all have the potential to divide, to form bone or to resorb it, as the microenvironment may dictate. Bone cell morphologists who bel ieve in this concept refer to the abil ity of cells to vacillate between special ized activities as "modulation". The concept of modulation is supported by the fact that the labeled cells in this study did not follow a simple sequence of steps toward differentiation. The shortest time required by an osteoblast to completely surround itself with bone matrix was 24 hr. The metaphyseal bone was 84 FIBROBLAST (OSTEOPROGENITOR CELL) ......... Y OSTEOBLAST LINE) tl / / (PRE-OSTEOBLAST) ~' '"' """", \ OSTEOCLAST LINE \ (PRE-OSTEOCLAST) I ~ i OSTEOBLAST ~--:::-.OSTEOCLAST t~ ~,~, ,,/ ~~" , "1IIfIII"~ " ""."". OSTEOCyTE ............ /' ,/ Fig. 13 Possible inter-relationships between rlifferent bone cells. The sol id 1 ines represent "irreversible ll steps toward differentiation., The dotted 1 ines represent proposed alternative path\'1ays in the process termed '!modulation"~ 85 remodeled within five days after it was laid down, i.e., the number of labeled osteocytes/fie1d diminished after 120 hr post 3HTdR • Labe1ed osteocytes appeared in the diaphysis at 72 hr post 3HTdR , two days after they had appeared in the metaphysis. Bone matrix turnover in the diaphysis occurred at the same rate as in the metaphysis; the number of labeled osteocytes/fie1d diminished after a period of five days. Simmons ('63) found labeled osteocytes in the metaphysis of older mice as early as 12 hr post 3HTdR • No labeled osteocytes appear in any region of the infant rat tibia prior to 50 hr post 3HTdR (Young, '62a). There is no report in the 1 iterature concerning the disappearance rate of labeled osteocytes in the whole bone with time. Fibroblastic progenitor cells comprised the largest cell pool in both the diaphysis and metaphysis of the tibia in this study. It was usually at least twice as large as any other bone cell pool. It was also consistently the pool with the largest number of labeled cells/field. It seems probable therefore that the fibroblastic progenitor cell is the most important, i.e. the most frequent, source of special ized bone cells. The prol iferative activity of these cells (as indicated by the percent of the population incorporating 3HTdR into DNA at 1 hr post 3HTdR ) varied in the bone according to regions. The metaphysis was the most active, the endosteum was intermediate and the periosteum was the least active area. This corroborates the regional differences reported by Young ('62b)in the infant rat tibia. Fewer progenitor cells were present per mataphyseal field at 336 hr post 3HTdR than at 86 earl ier time periods for two reasons: the primary trabeculae widened by the process of osteoid apposition and secondly, blood elements from the central marrow cavity began to invade the intertrabecular spaces. Cell division in these young bones was not always the function of less highly special ized cells. Both bone formation and resorption require the synthesis of compl icated proteins and enzymes. Yet cells that were involved in these processes were also seen labeled 1 hr post 3HTdR . The 1 iterature contains references to the abil ity of osteoblasts to resupply their own population (Ham and Leeson, '61). Mitotic activity of osteoblasts has been described (Hel ler et al., 150; Bloom, 137) and histochemical data has suggested that a certain portion of these cells form a potentially dividing population (Pritchard, 152). In addition, label ing of osteoblasts 1 hr post 3HTdR has also been reported in the tibial metaphysis of the youngadult rat (Simmons and Kunin, 167; Kember, 160) and in the distal metaphys is of the young mouse femur (S i mmons, 163). Little information is available on the importance of the endothel ial or perivascular cells in the origin of bone cells. This author bel ieves that osteoprogenitor cells may be derived from the capillary wall in much the same manner as hematopoetic precursors. Such an idea is not new, but was first presented by Maximow (124). There have been attempts to re-establ ish this bone cell origin (Allgower, 156; Sandblom, '44; Trueta, 163), but the definitive experiment remains to be performed. 87 The majority of cells within resorbing lacunae of these bones were mononuclear. Those labeled at 1 hr post 3HTdR were either macrophagic and endothel ial in appearance. Because there is 1 ittle agreement about the cellular component of resorption, it is necessary to discuss this subject here. Many investigators feel that the osteoclast is the only active, resorbing cell. Some consider the osteocyte also to have the potential to dissolve both organic and mineral bone matrix (Belanger, 163; Talmage, 169). Trueta ('63) proposes that osteolytic osteocytes, once freed of their lacunae, coalesce to form the classical osteoclast. At the opposite end of the spectrum is the idea that naked bone is treated by the animal as a foreign body and that it is attacked by macrophages arriving from the blood stream (Hay thorn, 129; Jee and Nolan, '63). Such macrophages are said to fuse and form multi- nucleated giant cells, thus an osteoclast is nothing more in this concept than a fully-formed foreign body giant cell. Proponents of this theory consider the foreign body giant cell, once it has formed to be inactive. The main reason for dissatisfaction with the relationship of the osteoclast to resorption is that many times its numbers are insufficient to account for the amount of bone that is being removed. Mononuclear cells seem especially important in the removal of calcified cartilage (Addison, '27; Dodds, '32; Fell, 125). In addition, the endothel ial cell may have the abil ity to produce histolytic elements, particularly during the invasion of the growth plate (Cameron, '61). 88 THE EFFECT OF CULTURE ON BONE CELL POPULATIONS Culture decreased the size of all bone cell pools. The numbers of osteoprogenitor and resorbing cells were decreased more than osteoblasts. Both progenitor and resorbing cell types decreased by a factor of three in relation to precu1ture values, while the number of osteob1asts decreased only 20% and the number of labeled osteoblasts doubled. THE EFFECT OF CORTISOL ON BONE CELL DIFFERENTIATION IN VITRO The anatomical position of cells within the whole radius determined the response of cells to the treatments in question. Real differences may have occurred in cellular response to the drugs according to their anatomical positions for the following reasons: 1) drug availabi1 ity may have differed according to the efficiency of transport in each area 2) regional differences in cell pool sizes may have changed the ratio of drug molecules to individual cells, and 3) basic differences in metabol ic activity of cells may have made one area more responsive to a drug than another. The numbers of progenitor cells, both labeled and total, show a highly significant dose-response relationship to graded doses of cortisol in the metaphysis. A dose-response of number of progenitor cells is suggested in the data from the endosteum and periosteum. In all three areas, the lowest dose of cortisol, 0.0001 ~g/m1, increased the numbers of progenitor cells when compared to control values. Low doses of corticosteroids in bone cultures may dupl icate the stimulatory phenomenon seen in fibroblasts (Achenbach, et al., '70; Rasche and Ulmer, 170; Ruhmann and Berl iner, '67). It cannot be said at this point whether or not the stimulatory phenomenon is due to biotransformation. One study has been conducted concerning the metabol ites of cortisol in a skeletal tissue culture system (Murota, et al., 166, 167). These invest igators confirmed that enzymic conversion of cortisol occurs in cultured embryonic chick femora, but they did not find evidence for enzymes such as 20a, 20B or 11 B-hydroxysteroid dehydrogenases. The dose level 0.01 ~g cortisol/ml did not change progenitor cell populations in any area from control values. doses to 100.0 ~g/ml With increasing the size of the progenitor pool was decreased. Cortisol decreases the number of osteoprogenitor cells ~ vivo (Deguchi and Mori, '69; Roberts, 169; Simmons and Kunin, 167). In the metaphysis, endosteum and periosteum of the radii, the numbers of total osteoblasts as well as labeled osteoblasts were decreased to a simi lar degree by the three lowest doses of cort isol. Dose-response relationships were present in the number of labeled osteoblasts/field of all three bone areas, but the number of total osteoblasts/field showed a dose-response only in the periosteum. It is possible that a population of osteoblasts exists which are resistant to all but the toxic levels of the corticosteroids. Simmons and Kunin ('67) observed such a phenomenon in young rats given 50 mg of cortisone/kg/day. Cortisone reduced the total numbers of osteoblasts, but it did not lower the percentage of the differentiated osteoblasts which could synthesize DNA. 90 The effect of cortisol on resorbing cell populations varied according to the area of bone. In the distal metaphysis of the radii, cortisol increased the numbers of resorbing cells in a dose-response fashion. Since the numbers of labeled resorbing cells/field did not exceed preculture values at any dose level. there is no evidence for increased differentiation of resorbing cells. The effect of cortisol was primarily to lengthen the 1 ife-span of the average cell and/or to induce the modulation of osteoblasts to resorbing cells. In contrast to the response of resorbing cells to cortisol in the metaphysis, resorbing cell numbers in the diaphyseal endosteum and periosteum decreased with increasing doses of cortisol. 0.0001 ~g/ml and 0.01 ~g/ml increased the number of total resorbing cells above those in control cultures. control values at 100.0 ~g Doses of cortisol/ml. This number decreased to When this data is viewed in relation to the stabil ization of lysosomal membranes by cortisol (DeDuve et a1. 161, Weissman and Dinqle 162) and to inhibition by cortisol of the release of lysosomal enzymes (Dingle et al., 169; Reynolds, 168) it seems probable that the resorbing cell, despite its increased numbers, is not effective in breaking down bone matrix when in the presence of cortisol. THE EFFECT OF PARATHYROID EXTRACT ON BONE CELL DIFFERENTIATION IN VITRO The lowest dose of PTE (0.1 uni t/m1 med ia) increased both labeled and total progenitor cells in all areas of the radii, with the exception of total progenitor cells in the periosteum. Increasing 91 doses of PTE, decreased the numbers of progen i tor ce 11 sin a doseresponse fashion until they were below control values at the 10 unit/ ml dose level. Little information is available concerning the physiologic level of parathyroid hormone. The normal concentration in bovine plasma is 1 mllg/m1 (Rasmussen, 168) and the biological activity is 2500 USP units/mg (Potts et al.~ 168). For this reason, the decrease in numbers of progenitor cell below control values at 10 units/ml is probably a toxic effect of the hormone. The stimulatory effect of PTE on progenitor cell mitosis is well documented in the 1 iterature. Although control bone grown in organ cultures produced osteoblast differentiation when compared to precu1ture values, parathyroid extract at all doses used in this experiment el iminated the osteoblast population in all three areas of the radii examined. In contrast both labeled and unlabeled resorbing cells increased at all dose levels to more than double the numbers seen at 0.1 unit PTE/m1 media in the metaphysis and periosteum. to PTE. The endosteum was the least responsive The numbers of resorbing cells were greater than precu1ture values only in the metaphysis. It seems 1 ikely that parathyroid extract not only induces the differentiation of resorbing cells, but also increases the modulation of osteob1asts to resorbing cells o acute stimuation of resorbing cell differentiation by parathyroid extract in vitro has been reported (Gaillard, 161), but these kinetics have not been studied in vitro with a 3HTdR-label. The Chapter 6 SUMMARY Cortisol has been shown to have an effect on bone cell populations in an organ culture system. This effect has a bimodal nature dependent on the concentration of steroid in the media. Although interaction with the parathyroid glands must be considered, action of cortisol alone can account for the fact that skeletal tissues respond to steroids in a variety of ways. Low doses of cortisol (0.0001 ~g/ml) stimulate progenitor cell mitosis and induce bone cell remodel ing through increased differentiation of osteoblasts and osteoclasts. (above 1.0 ~g/ml) Higher doses of cortisol decrease progenitor cell mitosis, inhibit bone formation through decreased osteoblast differentiation and preferentially protect and increase resorbing cell populations. Parathyroid extract, except at toxic levels (10 units PTE/ml media) stimulates progenitor cell mitosis and preferentially increases resorbing cell differentiation to the exclusion of osteoblast production. Cortisol and parathyroid extract can be seen to oppose each other in most effects on bone cell dynamics. While both increase resorbing cell populations, 45 Ca metabol ism of the whole bone in culture indicates that the activity of the individual cell has been depressed by cortisol. 92 BIBLIOGRAPHY Achenbach, C., R. Suess, V. Kinzel, O. Wieser and H. A. Sturm 1970 Time and dose dependent inhibition and enhancement of thymidine incorporation into fibroblasts by prednisolone. Experientia 26: 405-406. Addison, W. H. F. 1927 Bone. 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