The age factor in the response of bone tissue to alizarin dyes and the mechanism of dye fixation.код для вставкиСкачать
THE AGE FACTOR I N T H E RESPONSE OF BONE TISSUE TO ALIZARIN DYES AND T H E MECHANISM O F DYE FIXATION N. ERCOLI AND M. N. LEWIS Research Laboratories, Hoffmaiut-La Roche, Inc., Nutley, New Jersey INTRODUCTION The coloring of growing bone with madder, first experimentally studied by Belchier (1736), came into general use in ossification studies after the work of Du Hamel (1739). Within the last two centuries numerous authors have studied the mechanism of dye fixation in bone first using madder (Bazano, 1738 ; Haller, 1747 ; MacDdnald, 1799 ; Paolini, 1841 ; Serrhs and Doyhre, 1842; Brull6 and Hugueny, 1845), or, later, one of its coloring principals, alizarin (Flourens, 1847 ; Lieberkuhn, 1864), and, finally, a synthetic soluble derivative, sodium alizarin sulfonate (Gottlieb, ’14 ; Proell, ’26 ; Cameron, ’30 ; Proell and Diener, ’33; Schour, ’41). The conclusion drawn from the early studies was that the dye becomes fixed in the calcium salts which are deposited during treatment. This hypothesis was presented for the first time by Rutherford, before 1798. Some authors, Paolini, Serr6s-Doyhre, and Flourens, considered the coloration due to the staining of calcium phosphate. Coloration of the inorganic substrate by madder during the calcification process was accepted by all authors, with the exception of Strelzoff (1873) and Katschenko (1882), who considered the fixation as taking place in the organic matrix of bone. Most of the recent authors ascribe the fixation of alizarin to its affinity for “newly formed’’ calcium salts or “free calcium”. Under this assumption, the extent of coloration served 67 68 N . ERCOLI A N D M. N. LEWIS to explain the process of calcification. According to Gottlieb (’14)the variation in coloration intensity in different parts of bone is due to progressive chemical stages of calcification. According to Macklin ( ’17) the areas more deeply stained are composed of freshly deposited calcium salts. According to von Mollendorf (’20) the coloration is due to the formation of calcium alizarate in bone. Proell ( ’26) considered alizarin as an indicator of “free” calcium in bone. Cameron (’30) suggested that “the nature of the arrangement of the calcium complexes” may be different in young and old bone. Proell and Diener (’33) affirmed that the colored zone consists of freshly deposited calcium alizarate. Schour ( ’41) interprets the coloration as being due to a selective staining response of the calcifying or calcio-receptive zone of the collagenous matrix. Previous in vitro studies have shown that the fixation of alizarin, or its synthetic analogues, is a process of adsorption taking place between bone calcium phosphate and dye. The dye anion is adsorbed, while PO1 anion passes into solution (Ercoli, ’38). I n vivo the reaction is considered to take place with the calcium phosphate previously laid down and it may be assumed that the reaction will occur not only in growing but also in adult bone, irrespective of age. It was the purpose of this study to investigate the conditions of fixation of osteotropic substances with relation to the age of the animal and to analyze the mechanism of dye fixation in bone. EXPERIMENTAT, Material and methods Albino mice of different strains were used in the experiment. Adult animals were more than 7 months of age and weighed from 24 to 31.5 gm. These animals had not increased in weight for a period of 10 days preceding the treatment with dye. Growing animals were chosen from a group of mice 1P21 days old and weighing 8 to 11gm. OSTEOTROPISM 69 Alizarin Red S (sodium 1.2.dihydroxyanthraquinone-3-sulfonate) was used as the osteotropic dye. The concentrations were so adjusted that the dosage could be administered in 0.3-0.4 cc. per mouse. Injections were made into the tail vein except in those groups which received subcutaneous injections (the latter are tabulated separately). The experiments on the young and adult mice were done simultaneously. Fortyeight to 72 hours after injection, when elimination of the dye was complete, the animals were sacrificed by jugular bleeding. The bones were cleaned as much as possible and placed in saline for 3-4 hours, then under running tap water for 18-20 hours. After this they were put into a 10% solution of sodium hydroxide. The adhering tissue was removed by agitating in alkaline water (pH 8-8.5). After rinsing in tap water, the bones were placed in 95% alcohol for 10 minutes and then dried. Since gross observation shows the coloration more definitely than microscopic examination of sections, the observations here reported were made with the dissecting microscope. GROWING ANIMALS Ilztravenozcs ilzjectiolz Mice were arranged in several groups and each mouse was given one intravenous injection of Alizarin Red S. The indiand 80 mg./lcg. respectively. vidual doses were 20,30,40,50,60, The results are reported in table 1A. The following zones were found to be colored in the skull: the condyloid process of the mandible; the crest between the condyli; the zygomatic process ;the alveolar part of the maxillary ; the premaxillary ; the laminae of the posterior part of the ethmoid bone (there was no coloration in the cribrosa) ; in-the pelvis, the crest of the ilium, the tuberosity of the ischium, the descending ramus of the pubis ;also the articular processes of the vertebrae (the bodies showed only diffuse coloration) ; the sternal part of the ribs; the epiphyses of long bones; the deltoid tubercle; the olecranon ; the linea aspera ; and the junctions of the lamellar bones. 70 R’. ERCOLI A N D M . N. LEWIS TABLE 1 A. Fixation of AZizarin Red S in bone of growing mice. Intravenous injection. I NO. MICE DOSE MCI./KQ. ( 0 ) control 20 30 40 50 60 80 . AVERAGE WEIGHT GM. NO. COLORED NO. FAINTLY 11 9.1 10.2 8.5 9 10.5 10 .. .. 4 2 6 6 3 3 2 COLORED NO. UNCOLORED .. 4 3 4 1 .. .. .. * . .. .. .. R. Fixation of Alizarin Red S in bone of adult mice. Intravenous in,jection. 0 27 27 27 30 25 25 24 28.5 28 20 30 40 50 60 80 120 200 ;! 40 60 .. .. 1 1 2 2 2 4 1 2* 1 1 1 1 ; .. .. C. Fixation o f Alizarin Red S in bone of growing mice. Subcutaneous injection. ~ ~ I:.* 9.2 10.5 10.5 ~ .. 4 1 2 1 1 .. .. .. .. ______~---- * - One died 2 minutes after injection. Subcutaneous injection A number of mice were arranged in different groups and each mouse received one subcutaneous injection of Alizarin Red S. The individual doses were 20, 30, 40, and 60 mg/kg respectively. The same zones were colored as by intravenous injection. The results are reported in table 1C. According to the results summarized in tables 1A and C, a single intravenous or subcutaneous injection of 20-30 mg./kg. of Alizarin Red S causes coloration of bone in 50% of growing OSTEOTROPISM 71 mice injected, and 50 mg./kg. gives coloration in all mice. I n order to ascertain the time of occurrence of coloring after injection, 3 mice were injected with 60 mg./kg. of the dye and killed 2, 3, and 5 minues, respectively, after injection. The bones of these mice were found to be intensely colored. It was concluded that coloring takes place immediately after injection or resorption of the dye administered. ADULT ANIMALS Intravenows injection A single intravenous injection of Alizarin Red S was given to different groups of mice in doses of 2 4 30, 40, 50, 60, 80, 120, and 200 mg./kg., respectively. The results are reported in table 1B. 'These results show that the dose causing coloration in 50% of adult mice is 50-60 mg./kg. The same zones were colored as in the young animals, except that the coloration of the vertebrae was not diffused. The follpwing protocol is characteristic for an adult mouse injected with 120 mg./kg. of the dye. Coloration was found in the following zones : the premaxillaries ; the malar process and around the alveoli of the maxillary (the alveolar wall was more intensely colored) ; the ethmoid; the bulla tympanica; the condyloid proces of the mandible (intensely colored) the mandibular notch; the mandibular symphysis ;the alveolar part of the incisors ;the occipital condyles ; the crests and marginal parts of the parietal, interparietal, and squamous ; the vertebrae - the bodies and the posterior articular, transverse and tubercular processes ; the distal part of the caudal vertebrae ; the coxal bone - the iliac crest, the inferior ventral spine, the articular surface of the sacrum, the acetabulum, and the ascending ramus pubis on the medial margin ;the scapula - the vertebral border and the coracoid process (less intensively) ; the femur - the linea aspera, the third trochanter and the epiphyses around the condyle ;the humerus - the epiphyses and the deltoid tuberosity; the ulna - the olecranon and the epiphyses ; the epiphyses on 72 N . ERCOLI AND M. N. LEWIS the tibia, fibula and radius; and the ribs-the sternal third part. The pattern of coloration most frequently found corresponds to this description. Subcutaneous injectiorz Six adult mice have been injected with doses of 80-120 mg./kg. Three showed slight coloration of the zones indicated. Toxicological observations During these experiments the possible toxic effects of Alizarin Red S (i.v.) were also watched. The reactions (dyspnea, jumping convulsions) were definitely not the same as the tetanic symptoms produced on injection of oxalate, which is known to reduce the concentration of circulating calcium ions. Three adult mice injected i.p. with doses of 500 mg./kg. of Alizarin Red S gave no tetany. They died 8-10 hours after injection, showing muscular weakness and dyspnea. It is concluded that Alizarin Red S does not bind circulatory calcium. DISCUSSION AND CONCLUSIONS The experimental results reported here demonstrate that bones of adult animals take up the alizarin dye if the injected dose, i.e., the concentration reached in the blood, is sufficiently high. Fixation can be considered to follow the absorption immediately since it occurs in less than 2 minutes after intravenous injection. To obtain 50% coloration adult mice require two to three times as great a dose as growing mice. Further, it was found that a dose of 80-120 mg./kg. given to adult mice by subcutaneous injection has only a slight coloring effect, but produces definite coloration by intravenous injection. Thus, in order to obtain visible fixation in adult bone, the intravenous administration is more adequate since it results in a higher concentration of the dye in the blood stream. In order to obtain coloration of adult bone a higher dye concentration is required, presumably since in the older ani- OSTEOTROPISM 73 ma1 certain conditions would tend to diminish the quantity of dye which could reach the inorganic material ; viz. : 1. Lower vascularization -According to Strelzoff, in pigeons, the diameter of the Haversian canals diminishes from 0.114-0.027 mm. in the 2-day-old animal, to 0.015-0.003 mm. in the adult. According to Lexer ( '03) upon aging the vessels of bone become relatively thinner and their number diminish. 2. Lower permeability of adult bone-The increase of the relative mineral content at the expense of organic material and water (Weidenreich, '30) evidently results in more compactness and less premeability. The percentage solubility of calcium alizarate (0.02% ), higher than that of calcium carbonate (0.0013%), speaks against the assumption that the coloring may be due to calcium alizarate formed by reaction between dye and circulating calcium. An anion forming a more soluble calcium salt than carbonate could not possibly precipitate the calcium present in serum. I n fact, the toxicological data show that Alizarin Red S, unlike oxalate, does not reduce the concentration of Ca ions in circulation. Previously reported experiments and the results of this study suggest the following hypothesis concerning the mechanism of fixation of alizarin dye in bone. The circulating dye in contact with calcium phosphate is adsorbed with liberation of phosphate ion. Consequently, the coloration is localized mainly around the Haversian canals (Strelzoff, 1873) where contact occurs between inorganic material and dye. The onsorption reaction is irreversible ; therefore, once fixed, the dye remains in bone tissue until the latter is resorbed (Flourens, 1847). The higher vascularity and permeability of growing bone leads to an uptake of the alizarin dye with lower doses than is the case in the adult. The zones where the coloration is maximum are the same in young and adult animals. I n these zones, vascularization is greatest and metabolic processes, no doubt, are most intense. Emphasis is placed on the consideration that the higher fixation of alizarin in young 74 N. ERCOLI AND M . N . LEWIS bone is due to the anatomical setup and not to any specifio chemical condition of the calcium. A high degree of diffusibility, electronegativity, and the irreversibility of the adsorption product have been reported as necessary for the fixation of a substance in the inorganic material of bone (Ercoli, '38, '41). In the light of the aforementioned hypothesis, the role of these factors becomes obvious : a. Diffusibility facilitates the penetration of the dye into the compact material of bone. b. Electronegativity allows its adsorption, since bone calcium phosphate behaves as an electro-positive adsorbent. c. Irreversibility of the (dye) -Ca-phosphate adsorption product makes the fisstion permanent. Trypan blue, an electronegative dye which has been found to produce temporary coloring of growing bone (Blotevogel, '26), forms a reversible adsorption product. The small solubility of the calcium salt of alizarin led to the belief that the coloration is due to the formation of Ca salt. The relative insolubility of the Ca salt, however, is an indirect factor since only anions giving slightly soluble calcium salts arc irreversibly adsorbed by calcium phosphate. The following experimental data confirm the hypothesis that coloring of bone takes place by polar adsorption between preexisting calcium phosphate and circulating dye, rather than by formation of calcium alizarate or any reaction with Ca ions : 1. I n vitro the reaction between inorganic bone material and alizarin is a polar adsorption process, closely following the isotherm of Freundlich (Ercoli, '38) ; 2. As reported in the experimental part, adult bone may be stained with the dye ; 3. The fast rate at which coloring sets in; 4.The absence of toxic reactions, such as would be indicative of the disappearance of circulating Ca after administration of alizarin sulfonate, shows that no precipitation of Ca ions occurs; 5. The disappearance of coloration after decalcification proves that the dye is attached to the inorganic material ; while OSTEOTROPISM 75 6. The permanence of coloration after treatment with alkali and destruction of the organic matrix also shows that the dye is fixed in the inorganic material. I n conclusion, the fixation of alizarin in bone in the form of a product of adsorption with pre-existing calcium phosphate, precludes its use as an indicator of the age of the deposited calcium salt or of the biochemical conditions underlying calcification. Nevertheless, the value of alizarin for studies of ossification remains unchanged, if its use is limited to determining the addition of new substance to that existing at the moment of the injection, or to the measurement of zones lying between those colored by dye administered at intervals. SUMMARY The influence of age on the fixation of Alizarin Red S was studied. It was found that a single intravenous injection of 20-30 mg./kg. of dye caused bone coloration in growing mice. Adult mice required two to three times this amount to produce coloration. Coloration was produced in bone immediately after intravenous injection. The anatomical zones in which coloration takes place have been described. It is concluded that fixation occurs by a process of adsorption between existing calcium phosphate and dye anions, rather than by the supposed formation of calcium alizarate or a reaction with free calcium ions. The higher dosage required for coloration in adult mice depends on the anatomical and physical, not on the chemical, Conditions of bone. I n adult mice, subcutaneous injection produced less coloring than intravenous injection. LITERATURE CITED BAZANO,M. 1738 De eoloratis animalium quorundam vivokum ossibus. De Bononiensi Seientiarum ot Artium Insitutio atque Academia Commentari., 1701. I, 1st part, p. 129 (1745). BELCHIER, J. 1736 An account of the bones of animals being changed to a red colour by aliment only. Philosophieal transactions, vol. 39, p. 287. 76 N . ERCOLI AND M. N. LEWIS BLOTEVOGEL, W. 1924 Der vitale Farbstofftransport wahrend der Zahnentwicklung. Ztschr. f . Zellen u. Gewebelehre, vol. 1, p. 601. BRULLE,AND HUGUENY1845 Sur le developpement des 0s dans les mammifares et les oiseaux. Ann. Science. Nat., Se'ries 111, vol. 4, p. 287. CAMERON, G. E. 1930 The staining of calcium. J. Bact. & Path., vol. 33, p. 929. DU HAMEL 1739 Sur une racine qui a la facult6 de teindre en Rouge les 08 des Animaux vivants. Histoire de l'Academie Royale des Sciences - Memoires de M a t h h a t i q u e s et de Physique, p. 1. ERCOLI,N. 1938 L'osteotropismo dei farmaci, 111, Ricerche in vitro con l'alizarin 3. sulfonato d i sodio. Boll. SOC.It. Biol. Sperim., vol. 13, p. 757. I fattori chimico-fisici dell 'osteotropismo farmacologico. Proc. XVI Internat. Meeting of Physiol., Zurich (Switzerland), 11,p. 20. 1941 A chemotherapeutic agent with osteot'ropie properties. Proc. SOC.Exp. Biol. Med., vol. 48, p. 672. FLOURENS, P. 1847 Theorie experimentelle de la formation des 0s: ChAz Baill i h e , Paris. GOTTLIEB,B. 1914 Die vitale Farbung der kalkhaltigen Gewebe. Anat. Anz., vol. 46, p. 179. HALLER, A. 1747 Opera anatomica minora., vol. 11, p. 460, Lausanne. N. 1882 Uber die Erappfarbung der Froschgewebe. Arch f. mikr. KATSCHENKO, Anat., vol. 21, p. 357. LEXER, E. 1903 Die Entstehung entziindlicher Knochenerde und ihre Beziehung zu den Arterienverzweigungen der Knochen. Arch f . Klin. Chirurgie, vol. 71, p. 1. LIEBERKUHN, N. 1864 Uber Knochenwachstum. Arch. f. Anat. Physiol. u. Wiss. Med., p. 598. MACDONALD,1799 Disputatio Inaguralis. De necrosi ac callo. Edinburgh. MACKLIN,C. C. 1917 Studies in calcification by the use of vital dyes. J. Med. Research, vol. 31, p. 493. vON MOLLENDORF, W. 1920 Vitale Farbungen a n tierischen Zell. Cap. IX. Knochen. Erg. Physiol., vol. 18, p. 297. PAOLINI, M. 1841 Quorundam experimentorum de vi Rubixe ad ossa, ovorumque Gallinarum putamina calcaria coloranda. Novi Commentilri Instituti Bononiensi, vol. 6, p. 469. PROEL, F. 1926 Beitrage zur vitalen Knochenfarbung. Ztschr. f. Zellforschung u. mikr. Anat., rol. 3, p. 461. PROELL, F., AND DIENER,A. 1933 Model1 u. Tierversuche zum Problem der Verkalkung von Knochen. Ztschr. f . Zellforschung u. mikr. Anatomie, vol. 18, p. 244. RUTHERFORD, D. before 1798 reported by R. Blake, An essay on the structure and formation of the teeth in man, Dublin, William Porter, 1801, p. 154. SCHOUR, I., AND M. M. HOFFMANN; B. G . SARNAT, AND M. B. ENGEL 1941 Vital staining of growing bones and teeth with Alizarine Red S., J. of Dent. Res., vol. 20, p. 411. SERRES, AND DOYERE 1842 Expos6 de quelque faits relatifs a la coloration des 08 chez les animaux soumis au regime de la garance. Comptes Rendus de 1'Acad6mie des Sciences. STRELZOFF, 1873 Uber Krappfiitterung. Ztrlbl. f . d. Med. Wissensch., p. 737. WEIDENREICH, F. 1930 Dxs Knochengewebe, Hdb. d. mikr. Anat., vol. 2 (part 2 ) .