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This experiment was carried out in order to determine whether the chronic administration of low doses of cadmium resulted in an alteration of the haversian bone remodeling system in dogs. Two pairs of littermate beagles were administered 25 ppm cadmium chloride in their drinking water for 6 months. Four beagles matched for age and sex from the same colony served as controls. By means of fluorescent labeling, we measured haversian bone remodeling parameters according to the techniques described by Frost. Statistical analysis of the results showed significant changes at the 0.01 level in: activation frequency, appositional rates, and number of osteoid seams. At the 0.05 level, significant differences were found in the number of resorption spaces and the bone formation rate. In the absence of other evidence indicative of an alteration in the internal milieu of the dogs, it is concluded that a direct toxic action of cadmium on the mechanisms of activation of cells responsible for the creation and formation of new haversian systems cannot be excluded.

The effect of phosphate supplementation on bone remodeling was assessed in six mature, healthy beagle dogs. The phosphate supplement was given in divided doses orally, daily for 12 weeks in the form of a neutral potassium phosphate preparation. The dose averaged 108 mg P/kg per day, which is double the normal canine phosphorus intake. Bone remodeling was assessed by measurement, at sacrifice, of areas of cortical bone containing different color-coded tetracyclines which had been continuously administered during 12-week control and treatment periods; remodeling was assessed kinetically during the control and treatment periods by replicate studies employing 47Ca intravenously. Both techniques demonstrated that the principal effect of phosphate supplementation was a significant stimulation of bone formation. Within cortical bone, formation was doubled, from an average of 2.7% to 5.3% per year. The major location of new bone deposits was endosteal. Whole skeletal mineral accretion, measured kinetically, increased 45% above an average control value of 0.154 g/day. These studies suggest that, in the adult dog, "normal" plasma phosphate levels are suboptimal for new bone formation. Even with this short duration of administration, phosphate produced microscopic calcification of the renal parenchyma. However, there was no biochemical evidence of renal functional impairment.

Male Charles River rats, 31-days-old, received i.p. injection of sodium diphenylhydantoin (DPH), 100 mg/kg in 0.9% NaCl, once daily for 26–27 days before death. Male Syrian hamsters, 40-days-old, received similar injections of DPH, 25 mg/kg for 46 days, no treatment for 39 days, then DPH for a further 17 days before sacrifice. All rats receiving DPH gained less weight than the controls, and more than 50% displayed acute neurotoxic reactions to the drug; hamsters were not so affected. Morphology and composition of caudal vertebrae, teeth, and jaws from control and DPH-rats were compared on the basis of measurements on radiographs and gross specimens, histological investigation, and determination of % dry volumes of ash, volatile inorganic component, lipid, and organic matrix. DPH-vertebrae were smaller and showed impaired osteogenesis, but chondrogenesis was similar to controls. Overall tail length was similar in both animal groups because caudal intervertebral spaces were wider in DPH-rats, compensating for reduced longitudinal growth of corresponding vertebrae. Incisors were smaller and third molar roots shorter in DPH-rats. In DPH-hamsters the attachment of the periodontal ligament to maxillary incisors was deranged. DPH administration did not change the composition of rat bone or teeth. Densities of dry bones and teeth were in accord with their composition. Possible modes of action of DPH are discussed. Species differences in response of mineralized tissues to DPH administration are emphasized in relation to reports of rickets and osteomalacia in patients on long term DPH therapy.

A histomorphometric evaluation of the iliac crest trabecular bone remodeling was performed after tetracycline double-labeling in 41 normal Danes (12 males and 29 females) aged 19 to 56 years. The fraction of formative (osteoid covered) and resorptive surfaces was unrelated to age but higher in males than in females (P less than 0.02 and P less than 0.05, respectively). The appositional rate (0.65 +/- 0.12 micrometer/day) was unrelated to age and sex, whereas the fractional labeled surfaces were higher (P less than 0.01) in the males (0.18 +/- 0.08 micrometer2/micrometer2) than in the females (0.12 +/- 0.05 micrometer2/micrometer2), and among the females inversely related to age (R = -0.38, P less than 0.05). The bone formation rate at BMU level (0.50 +/- 0.20 micrometer3/micrometer2/day) was unrelated to sex, but among the females inversely related to age R = -0.49, P less than 0.01). The bone formation rate at tissue level was higher (P less than 0.02) in the males (0.13 +/- 0.07 micrometer3/micrometer2/day) than in the females (0.07 +/- 0.03 micrometer3/micrometer2/day) and among the females inversely correlated to age (R = -0.43, P less than 0.05). the age- and sex-dependent variations in the dynamic parameters underline the importance of a more elaborated normal material.

Long term, low dose administration of adriamycin (ADR) to young growing rabbits resulted in significant alterations in bone structure and chemistry. Morphologic changes were most pronounced at epiphyseal and metaphyseal areas of long bones. Epiphyseal cartilage plates were thin and there was derangement of growth zones. Areas of primary and secondary spongiosa were deficient in trabeculae, osteoblasts and osteoclasts. Analysis of femora, humeri and lumbar vertebrae from ADR-treated rabbits revealed increased water and fat content and significant decreases in bone density compared to age-matched controls. Cortices of long bones were roentgenographically thin and contained large irregular spaces evident microscopically. Evaluation of bone ash from ADR treated rabbits revealed significant increases in the percentage of calcium and phosphorus, although Ca/P ratios were not different from controls. Results of in vitro studies indicate that ADR binds readily to nondemineralized, but not demineralized, fresh cortical bone powder. The findings of decreased bone density, histopathologic alterations, and a paucity of osteogenic cells in ADR treated rabbits are interpreted as retardation of bone maturation. It is suggested that ADR affects adversely both the organic and inorganic fractions of bone. Due to its unique characteristics of cytostatic action, binding to metal cations and orange-red fluorescence, ADR is a novel chemical agent that may be useful in experimental bone studies.

125I-labeled rat albumin injected intravenously into rats was taken up by growing bone. Some of this radioactive albumin could be removed from bone by washing with saline, the proportion so removed decreasing from 82.5% at 1 day to 7.4% at 8 days. Both the total radioactivity, and that remaining in bone after saline wash, were reduced when growth was slowed by alteration or restriction of the diet. Although the amount of 125I albumin in rachitic bone was reduced, autoradiography showed that radioactivity was present in rachitic osteoid. Immunoprecipitation using anti-rat-albumin serum showed that about half of the radioactivity released from normal bone by EDTA was still attached to albumin. These results suggest that albumin plays some part in the growth of bone.

Mucopolysaccharides (MPS) and collagen have been analyzed in different types of cartilage and some other tissues at various stages of prenatal and postnatal human development. The concentration of the chondroitin sulfate-type MPS increased from the 38th embryonal day, the 6-sulfate exceeding in amount the 4-sulfate. The increase in collagen concentration was accompanied by a decrease in the proportion of neutral salt-soluble collagen. There was no accumulation of chondroitin sulfates in the calvaria. A maximal concentration of MPS of a low molecular weight occurred in the skin and in the ear pinna after the 10th prenatal week. Postnatally, the MPS-concentration in the iliac crest and in the tibial articular cartilages decreased gradually, the highest values being reached in the newborn; the collagen-concentration showed an approximately reciprocal course.

We identified the cellular sites of glycosaminoglycan hydrolysis by localizing a key enzyme, N-acetyl-beta-glucosaminidase, in the rat periodonatal ligament. Reaction product was seen in osteoclasts, osteoblasts, fibroblasts, and in macrophages situated perivascularly in areas of bone remodelling. The preferential location of macrophages in areas of both bone formation and bone resorption, and their intense NAGase activity identified the macrophage as a significant participant in the total process of bone remodelling. We considered the presence of reaction product in the Golgi zone and the rough endoplasmic reticulum of osteoblasts and fibroblasts as possible localization of a NAG-transferase.

Bone remodeling in pathologic conditions was studied with the scanning electron microscope (SEM). Benign and malignant ossification were examined in cases of myositis ossificans, ossifying fibroma, osteoid osteoma, and osteosarcoma, Resorption of bone due to invasion by non-ossifying tumors was found in cases of squamous cell carcinoma, adenocarcinoma, ameloblastoma, and multiple myeloma. Bone formation due to excessive production of growth hormone was studied in a case of acromegaly. Resorption of bone due to pathologic processes resembled the pattern found in surfaces which were undergoing resorption by osteoclasts. Lamelar-cortical bone formation in acromegally was similar in nature to normal bone. The deformities were rleated to the excessive continuous osteogenesis that occurs in these instances. Neoplastic ossification was characterized by calcifying globules, the diameters of which ranged from 1 to 3 micron. The surfaces of these globules were constructed of minute calcospherites with diameters ranging from 0.1 to 0.3 micron. It is suggested that the pattern of globular calcification is similar to the type that was found with the SEM in fetal bone and cartilage, during healing of fractured bone, and also with the TEM in normal and pathologic calcification.

Tetracycline-based bone remodeling was studied in multiple rib samples from 12 control beagles and from 27 perinatally irradiated beagles with different stages of renal disease, when biopsied or sacrificed at 2 and 4 years of age. In haversian bone, the 10 dogs with marked uremia showed, as a group, a trend (P

One hundred gram male Sprague-Dawley rats were divided into groups which were injected daily for 10 or 30 days with vehicle (control group), 0.2, 0.4, 2.0, or 10.0 mg ethane-1-hydroxy-1,1-diphosphonate (EHDP)/kg/day. The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation factors. Knowing these, information about hard tissue resorption was deduced. After ten or thirty days treatment with 2.0 or 10.0 mg EHDP/kg/day, there was an increase in percentage of hard tissue. This was due to a decrease in bone formation with a greater decrease in hard tissue resorption. Furthermore, after thirty days treatment with 0.2 and 0.4 mg EHDP/kg/day, there was an increase in percentage of hard tissue, which was due to a decrease in resorption with no change in formation. Ultrastructural studies on osteoclasts from EHDP-treated rats showed a general decrease in their vacuolization and amount of organelles as dosage of EHDP increased. Histologic findings suggest that EHDP is similar to fluoride in the way in which it depresses hard tissue resorption.

Hypocalcemia previously reported in rats and dogs following oral administration of ethanol may have been caused by a movement of calcium from blood to bone. This present study was undertaken to determine whether ethanol also causes hypocalcemia in chicks and to investigate the direct effects of ethanol on mineral accretion, glucose metabolism and growth of embryonic chick tibiae in an organ culture system. A high dose of ethanol (6 g/kg body wt) produced hypocalcemia, hypermagnesemia and an elevated hematocrit in chicks. Results in vitro were as follows: 1) 5 to 30 mul ethanol/ml medium produced dose-related increases in bone mineral from 58-440%; 2) lactate production was inhibited at all ethanol levels; 3) increased mineral accretion did not occur in ethanol-treated tibiae when iodoacetate was in the medium, but did occur in mechanically disrupted bones exposed to ethanol; and 4) the ethanol response in bone was directly related to the medium phosphate concentration. The results lead to the following conclusions: 1) ethanol has a direct stimulatory effect on bone mineral accretion and an inhibitory effect on bone glucose metabolism in vitro; 2) viable bone cells and an adequate phosphate supply are necessary for the ethanol response, but tissue integrity is not; and 3) the hypocalcemic effect of ethanol in vivo may at least partially result from ethanol-stimulated bone mineral deposition.

Adult female rats were subjected to a calcium-depriving regimen (calcium-deficient diet containing oxalate + pregnancy + lactation) to obtain maximum bone mineral mobilization in as short a time as possible. The femur and tibia were investigated by histological, microradiographic and fluorescent microscopic methods. The regimen caused osteoporosis, which varied in severity with the degree of calcium deprivation. Most of the bone tissue removed was taken from the metaphyseal trabeculae and from the endosteal surface of the diaphysis. The remaining bone tissue seemed unchanged. The cells responsible for the bone removal did not seem to be multinucleated osteoclasts, but mononuclear, hypertrophied, elongated cells, possibly derived from the "resting" osteoblasts normally lining the bony surfaces. No signs of osteocytic osteolysis were observed. Bone formation was reduced, but present even in the most calcium-deprived animals. No increase in the amount of osteoid was observed.

Fetal rat bones were cultured in either growth-inducing or resorption-inducing media to study mineral losses during bone growth and atrophy in vitro. Whole radii and ulnae from 19-day-old fetal rats, prelabeled with 45Ca and/or 3H-tetracycline, were cultured intact or cut, and then digested by collagenase to obtain the calcified portion of the bones. Three- to five-fold more 3H-tetracycline than 45Ca was lost from the calcified portion when the bones were cultured for 4 days in growth-inducing media. Similar small amounts of 45Ca were lost from live and killed bones, but more 3H-tetracycline was lost from live bones than from killed bones. More 3H-tetracycline was released into the growth medium with a low concentration of calcium (0.5 mM) than when the calcium concentration was high (1.0 mM); no significant difference was seen in the release of 45Ca into the medium at different calcium concentrations. Larger amounts of both isotopes were lost when the prelabeled bones were cultured in resorption-inducing media than in growth-inducing media. When parathyroid hormone stimulated bone resorption in a resorption-inducing medium, equal proportions of both isotopes and bone collagen were lost. Greater losses of 3H-tetracycline than of 45Ca suggest that 45Ca was conserved locally during the resorption that accompanies bone growth, but not during resorption that accompanies bone atrophy.

The secretory territories of rat osteoblasts on the parietal bone were measured directly using scanning electron microscopy. The mean territory of 4620 cells in 19 fields was 154 μm2 per osteoblast. The range for the fields was 136 to 177 μm2 per osteoblast. Four hundred cells were measured individually—for these the mean value per osteoblast was 143 μm2 with a standard deviation of 33. The daily rate of apposition over an 8 day period was 3.12 μm (standard deviation 0.22) measured by tetracycline marking of the mineral front. This gave a daily matrix production rate of approximately 470 μm3 per osteoblast.

Matrix vesicles were observed in femurs of 8-day-old chick embryos prepared by ultracryotomy. Some of the sections were subjected to ultramicroincineration. The unfixed tissues never came into contact with solutions, and thereby artifacts due to dissolution, redistribution, or rearrangement of the mineral constituents were avoided. In the osteoid, electron dense objects with the size and appearance of matrix vesicles were seen, although limiting membranes were not visible. After ultramicroincineration the vesicles were observed to contain small crystals and a less dense amorphous mineral material which may te the precursor of bone mineral. In addition, a ring of ash enclosed the crystalline and amorphous mineral and appeared to occupy the position of the vesicle membrane as seen in conventionally prepared material.

This paper reports studies of the epiphyseal cartilage in normal and vitamin A deficient chicks. The organic composition and the phosphatase activity in the resting cartilage, ossifying cartilage and new bone were measured. The ossifying cartilage and new bone had a higher content of inorganic material, phosphate and collagen than the resting cartilage. Vitamin A deficiency caused increase in the phospholipid content of all three tissues. The resting cartilage from vitamin A deficient tissue had, after homogenisation and centrifugation, a supernatant with an activity of alkaline phosphatase and glycerophosphatase higher than that in control samples. It is considered that effects of vitamin A deficiency on enzymes are related to defects of the lysosomal membrane with release of phosphatases, and that normal mineralisation also involves phosphatases activity.

A new method is presented by which an increase in cortical bone mass over time can be determined from morphometric analysis of sections of undemineralized bone under the optical microscope. The method depends on the long-duration, continuous labeling of new bone by tetracycline during the experimental period. In addition, a comparison of the rates of new bone formation in the control and experimental periods can be made by administering a different fluorescent molecule during the control period. All bone deposited during the control period is thus labeled with a different color. By knowing the differences in rates of formation between control and experimental periods, additional insight is gained into the mechanism by which the increase in bone mass is produced.

A method is presented for separating the proliferative, maturing and hypertrophic cell zones of the epiphyseal growth plate of the weanling rabbit, in such a manner that protein-polysaccharide metabolism could be studiedin vitro on a per-cell basis. Using the timed incorporation of35SO4 as an index of protein-polysaccharide synthesis, and hexosamine concentration as an index of protein-polysaccharide content per zone, it was found that sulfate uptake varied inversely with matrix protein-polysaccharide content; being highest in the hypertrophic cell layer where matrix content was least. The possible significance of this relationship is discussed.

The changes in the tibial diaphysis as a result of feeding a high calcium diet to rats previously fed a calcium free diet were determined. The calcium free diet resulted in an increase in the medullary area, and the subsequent feeding of a high calcium diet caused a reduction in medullary area. However, the amount of endosteal bone lost during ten days of feeding a calcium free diet was not completely restored after 78 days of feeding a high calcium diet. The decrease in medullary area was brought about by decreased endosteal bone resorption and particularly by increased endosteal bone formation. Bone formation at the periosteum and at the epiphyses were unchanged, indicating that the high calcium diet did not cause a generalized increase in bone formation. The increase in endosteal bone formation was limited to those sites along endosteum where greatest loss of bone had occurred during the calcium depletion period. This indicates that a local factor is at least partially responsible for the stimulation of endosteal bone formation during calcium repletion. Mechanical stress, which stimulates bone formation, was increased in calcium deficient animals and returned to normal during calcium repletion. In addition, mechanical stress was probably greatest in those sites where the greatest amount of bone repletion occurred and may have been the factor which contributed to the increase in endosteal bone formation during calcium repletion.

A new mechanism is presented to explain how increased or decreased mechanical stresses applied to bone are translated into osteoblastic and/or osteoclastic activity. A mechano-chemical hypothesis for bone remodeling induced by mechanical stress is presented in an attempt to explain this phenomenon. Bone responds to mechanical stress by differential growth so as to resist the applied stress; therefore mechanically induced bone remodeling is probably regulated by a negative feedback system. The hypothesis is that a change in the loading of bone results in an altered straining of the hydroxyapatite crystals in bone. This in turn alters the solubility of the crystals, providing the required negative feedback message to the bone cells in the form of a mechanically induced chemical change. The cells then take appropriate action to compensate for the alteration in the localized calcium activity either by building up bone to redistribute an increased stress, or by removing bone which is surplus to the structural needs imposed by a reduced stress. In order to test the hypothesis, synthetic hydroxyapatite crystals were stressed and changes in calcium ion activity were recorded from a divalent cation activity electrode. The results show that a mechanochemical effect can be detected in hydroxyapatite crystals which, when stressed, generate a calcium activity of 9×10−5 moles/l compared to 7×10−5 moles/l when unstressed. The experimental results in this study and evidence from cellular physiology are consistent with the mechanochemical hypothesis proposed here.

Hydrated calcium monohydrogen phosphate is proposed as the logical precursor in the formation of hydroxyapatite and a unifying theory for the formation of low calcium, or defect apatites, is presented. Structural relationships between calcium monohydrogen phosphate dihydrate and hydroxyapatite indicate that either material can provide the atomic arrangment for the epitaxial growth of one on the other. The formation of apatite is presented as the summation of two rate processes: the initial fast formation of amorphous calcium monohydrogen phosphate dihydrate and the subsequent slow formation of crystalline hydroxyapatite from the initial precipitate. Ca/P ratios of calcium phosphates, formed from compositions in the phase region of hydroxyapatite as a function of time, suggest a varying composition of calcium monohydrogen phosphate dihydrate and hydroxyapatite. Hydrated calcium monohydrogen phosphate is proposed on the basis of rate and composition of calcium phosphate formed and on crystallographic data to be a necessary seed for growth of hydroxyapatite in bone and teeth at physiological pH.

This investigation was undertaken to determine if the metabolic activity of homotypic segments of dog fibulae could be reliably compared in a thirty day period. Activities analyzed were: cumulative formation, porosity, resorption and apposition. Analyses were performed on contiguous tissue sections using microradiographic and tetracycline techniques. Spatial arrangements of the various activities were analyzed by constructing three dimensional models. The data permitted the following conclusions: 1) The mean differences between dog homotypic fibular segments are much smaller than the mean differences between heterotypic sites. 2) The use of homotypic fibular sites as valid controls should be limited to investigations in which the differences worth detecting are at least greater than 4% (apposition), 2% (resorption), 0.6% (porosity), and 2% (cumulative formation). 3) Of the four parameters measured, porosity was the most constant, showing no significant differences between mirror segments. 4) Lag correlations emphasize the importance of utilizing all available contiguous sections in a given specimen. 5) Physiologic resorption was not exclusive in old bone. 6) The sites of metabolic activity were predominantly found among specific active osteons which were primarily distributed peripherally in a strikingly similar pattern between mirror segments.

Sections of the tibias from both intact and gonadectomized male and female rats were subjected to various histological and histochemical methods to determine the changes in the bone following gonadectomy. It was observed that both in castrated males and in ovariectomized females, the proximal epiphyseal cartilage became “sealed off” by bone after the fifth post-surgical month. In addition osteoporosis of the shaft of the bone was also observed. The epiphyseal cartilage of the gonadectomized rats exhibited intense PAS positive intercellular matrix in contrast to the alcianophilic ature of the matrix from control animals. The diaphyseal trabeculae from gonadectomized rats stained red with PAS and contained wide regions of calcified cartilage cores. In addition, the epiphyseal cartilage and bony trabeculae from these animals stained faintly in the ninhydrin-Schiff reaction. The staining reactions indicated that in gonadectomy there was an alteration in the state of aggregation of protein-mucopolysaccharide component of the organic matrix, a situation which would result in an interference in the mineralization of the bone.

Different coloured, fluorescent bone-seeking chemicals, viz., tetracycline, Alizarin Red S, and DCAF, have been administered sequentially to weanling rats and the rate of formation and resorption of bone measured from hard-ground cross sections of the upper third of the diaphysis of the femur. On a meat diet, bodily growth is significantly restricted for the first week and then recovery occurs. While bones grow they fail to mineralize normally and rapidly become fragile and rarefied. Resorption of bone is at first slow, then accelerates for a period of 2–3 weeks to about 15μ/day and then slows again. While the rate of bone formation is reduced relative to normal bone, resorption proceeds at approximately two to three times the rate of bone growth. Microradiographic studies confirm tht while resorption occurs on the endosteal margin and formation proceeds on the periosteal aspect of meat fed Ca-deficient rats, new bone is less calcified than that in control animals.

Heparin was administered to kittens, to intact cats, and to cats whose thyroids, or parathyroids, or both thyroids and parathyroids had been removed. Over 2 or more months, the serum calcium levels rose in all animals. This result, occurring both in the animals lacking thyroids and parathyroids and in those still having them, suggests that the calcium mobilizing effect of heparin was a direct one.

Guinier diagrams of pulverized medullary bone samples of one-year-old layers (HNL) showed, apart from apatite-like lines typical for bone, other reflexions which are characteristic of calcite and thus confirm the presence of calcium carbonate as a separate phase. The role that calcite possibly plays in mobilization of minerals during egg shell formation is discussed. It is emphasized that quantitative determination of calcite turnover in laying hens is still required.

An investigation involving chicks fed diets with 0.5% Ca and 0.5% Sr supplemented with various levels of vitamin D3 suggests that one-half of the Ca requirement of body growth in the chick is met by Sr as long as the vitamin D3 level was maintained at 500 to 1000 ICU/kg foodstuff. The entire Ca requirement for bone growth could not, however, be met by Sr. Even though Sr was incorporated into the bone when low levels of vitamin D3 were fed in the diet, the total ash content, as well as Ca content, was reduced.

The microradiographic-photometric method of studying the X-ray absorption, and the microhardness testing technique were concurrently applied to investigate the rate of secondary mineralization of bone of known age in the osteons of young immature and adult dogs.

Chick embryo frontal bones at 12 and 13 days of development cultivatedin vitro exhibit different patterns of glucose utilization, periosteal cellular density and calcium and hydroxyproline content. When dehydroepiandrosterone sulfate is added to the medium at a concentration 1 mM, 12-day frontals engage primarily in osteoid tissue synthesis while 13-day frontals calcify at a significantly greater rate than controls.

FL or WISH cells, originating from the human amnion, were grafted intramuscularly into various strains of mice. 1. When grafted, cells of both lines evoked cartilage formation in their vicinity, provided that the animals were pretreated with cortisone (5 mg) or ACTH (36 u). 2. No host strain differences were found in respect to the cartilage induction. 3. Cartilage was replaced by bone, tissue, which was gradually resorbed within two months. 4. Grafting the FL cells into animals treated with hydrocortisone or imuran and into newborn or X-irradiated mice failed to induce cartilage or bone tissue.

A serial longitudinal study was undertaken to determine the activities of the alkaline and acid phosphatases in the long bones and mandibles. The optimum pH of the two enzymes was recorded at 10.2 and 5.4 for alkaline and acid phosphatase, respectively. Synchronized and randomized litters of rats were killed, 1 litter daily, starting at day 1 to day 25 post partum. Samples were analyzed for protein concentration and activity of the phosphatases. A pattern of low and high activity was observed in both bony tissues, as well as a pattern of low alkaline phosphatase activity during acid phosphatase activity peaks, and vice versa.

This Classic Article is a reprint of the original work by Marshall R. Urist, A Morphogenetic Matrix for Differentiation of Bone Tissue. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1068-3; and a third Classic Article is available at DOI 10.1007/s11999-009-1069-2. The Classic Article is © 1970 by Springer and is reprinted with permission from Urist MR. A morphogenetic matrix for differentiation of bone tissue. Calc Tiss Res. 1970:4(Suppl);98–101.

Young donor rats of an isogenously related strain were injected with3H proline (1 μ Ci/g) and killed from 6 hours to 28 days later. The scapulae were removed, decalcified with EDTA and implanted subcutaneously into the backs of recipient rats. They were removed 14 days later with the surrounding tissue, sectioned, processed for autoradiography and stained with hematoxylin and eosin. In all cases there was a giant cell response round the bone. In bones removed from the donor animals 6 hours after proline injections, the label was on the edge of the appositional side of the bone and the giant cells did not remove it. By 28 days after proline administration when, due to apposition and resorption, the label was on the resorptive side of the bone, giant cells were seen removing the label, which however they did not ingest. It thus appears, as has been suggested in the literature, that recently formed bone collagen is removed with difficulty, but older collagen can be resorbed.

A quantitative method for the application of compressive forces to long bones in culture is described. Using this method it was found that a physiological pressure of 80 g/cm sq. applied to tibiae of 16-day-old chick embryos reduced glucose consumption to 50% of controls. Twenty four hours after the release of pressure glucose utilization again increased, approaching control levels. A pressure of 80 g/cm sq. also stimulated thymidine incorporation into DNA. The same pressure decreased the size of the extracellular fluid pool by 8%, but had no effect on the rate of equilibration of this pool with22Na.

Bone from rats sacrified at 2, 5, and 95 weeks of age following 45Ca and 3H-tetracycline labeling in utero or postnatally was placed in solutions of physiological saline, nonradioactive calcium, or tetracycline. This procedure was designed to determine the availability for exchange of 45Ca and 3H-tetracycline from bone mineral in vitro. With time after labeling a gradual decrease occurred in both the nonspecific loss and specific exchange of 45Ca and 3H-tetracycline. 45Ca and 3H-tetracycline in bone could be partially exchanged in vitro with their respective nonradioactive counterparts. Each isotope could be partially exchanged, independently of the other istope. Nonradioactive calcium increased the exchange of 45Ca while suppressing the release of 3H-tetracycline. Molecular 3H-tetracycline exchange occurred to a greater degree than ionic 45Ca exchange and was associated with a small loss of 45Ca. The molecular exchange of 3H-tetracycline with bulk tetracycline did not induce any significant loss of tritium from 3H-tetracycline.

Estrone, 0.4 mg administered daily for 7 days to developing chicks at 2 weeks post hatching, increased total mass and accelerated the rate of growth of the diaphyseal and proximal epiphyseal diameters of intact radii. Fractured radii of animals subjected to the same hormonal treatment displayed decreased bone weight but increased rate of growth of the proximal epiphyseal diameters and longer but thinner and lighter calluses. Two-week-old chicks, which had received no estrone, when exposed to a week-long 2g environment showed decreased intact bone weight and, with the exception of length of fracture callus, a reduction of all measured parameters of fractured bones. Estrone treatments of 0.4 mg administered daily to hypergravity-exposed animals over a week-long period resulted in greater length of fracture callus and increased rate of growth of the proximal epiphyseal diameter of both intact and fractured bones. The same treatments decreased weight and linear growth of fractured bones and width and weight of the fracture callus.

Male rats weighing 100 g were assigned to groups and injected daily for 10 days with vehicle (control), 0.4, 2.0, 4.0, 10.0, or 20.0 mg/kg/day of ethane-1-hydroxy-1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (C12MDP). The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation parameters. From the data, information about hard tissue resorption was deduced. All doses of C12MDP and doses of 2.0 mg EHDP/kg/day and greater caused significant increases in percentage of hard tissues with C12MDP being more effective than similar doses of EHDP in decreasing bone resorption. Osteoclast population parameters were increased with all doses of both C12MDP and EHDP with C12MDP having a greater effect than similar doses of EHDP. Decreases in the proliferation of the osteoprogenitor pool parallel the decreases in osteoblasts and bone formation parameters. These decreases in osteoprogenitor pool proliferation do not account for the increases with diphosphonates in osteoclast population parameters.

Microhardness was measured in sampling sites in the tibial diaphysis of control rats that received less than 1 ppm fluoride in the drinking water, and experimental rats that received 30, 90 and 120 ppm fluoride in the drinking water for 17 days. The latter dose was toxic, as evidenced by a decreased final body weight in this group. By means of tetracycline labelling, it was possible to measure bone hardness in four zones of increasing bone age: I) 3 days, II) 8 days, III) 13 days and IV) 22 days. Zones I through III represented bone formed during fluoride treatment, and Zone IV bone formed before fluoride treatment. In the control group, microhardness increased from Zone I to II, probably because mineral concentration was relatively low in Zone I, and remained constant thereafter. In the 90 and 120 ppm fluoride-treated groups, maximum microhardness was not achieved until Zone III. This delay was probably due to the fact that fluoride in large doses inhibits the rate of mineralization. In the 30 ppm fluoride-treated group, there was no delay in achievement of maximum microhardness; microhardness values in Zones I and III were greater than those in control animals, and microhardness in Zone III was higher than that in Zone IV. These results show that: 1) bone microhardness is increased in bone formed during fluoride treatment in rats given 30 ppm fluoride in the drinking water, 2) toxic doses of fluoride delay, but do not prevent achievement of normal maximum microhardness, and 3) changes in microhardness are seen only in bone formed during fluoride treatment.

The effects of varying the initial calcium and phosphate concentrations of the culture media on bone calcium and phosphate release were examined, using whole calvaria from 3-day-old mice in 48-hour cultures. The initial calcium and phosphate concentrations of the culture media were varied in the range 3-10 mg/100 ml; either calcium or phosphate alone was changed while the other ion was held constant, or the concentrations of both were varied while the Ca:P ratio was held constant. For all combinations, 3 treatment groups were used: i) control (no added hormone); ii) 0.5 U/ml PTH; iii) 50 mU/ml CT. The release of calcium and phosphate from the bones was greatest at low initial calcium or phosphate concentrations in the media, and least at high initial concentrations. High concentrations of both ions together abolished hormonal responses and resulted in extensive uptake of calcium and phosphate by the bones. The response to PTH was lost at a high concentration of either ion alone, while a response to CT was observed under all experimental conditions except simultaneously high calcium and phosphate concentrations.

Archaeological rib samples were subjected to quantitative histologic analysis to determine rates of cortical bone formation. Histologic features are usually well enough preserved to permit the determination of mean annual Haversian bone formation rate averaged over the life span of the individual. Moreover, gross estimates of aging archaeological bone correlate well with histologic parameters expected for particular ages. Age-associated changes in bone histomorphology in extinct populations have remained essentially unchanged for at least 1,600 years. Bone formation rates determined for these populations agree with age-matched values determined for extant Homo sapiens. A relatively high frequency of pathologic conditions reported by others for the Ledders population may be reflected by the wide range of histomorphometric parameters present in the ribs of these individuals. On the basis of morphophysiologic relationships in extant populations, it can be assumed that mean annual osteonal creation frequency, and mean annual Haversian bone formation rate can be reliably determined in extinct populations. To our knowledge, this is the first time a dynamic physiologic parameter has been measured in an extinct population of H. sapiens.

The passage of tissue fluid through cortical bone has been investigated using radioactively labelled macromolecules as markers. The results suggest that in the cortex of young rabbit femur the movement of tissue fluid is in the same net direction as blood, mainly from the endosteal to the periosteal surface. Some albumin is incorporated from extravascular tissue fluid into calcified matrix at sites of bone formation. Polyvinylpyrrolidone, average molecular weight 35,000, is able to pass through extravascular tissue fluid in bone but is not incorporated into calcified matrix. In rabbits made vitamin D deficient, much less alblmin is retained in regions of bone formation than is the case with controls. Albumin adsorbs to the surface of calcium phosphate precipitates and it is suggested that this mechanism may be mainly responsible for its incorporation into bone.