The demonstration of centers of osteoblastic activity by use of vital dyes of the benzidene series.код для вставкиСкачать
THE DEMONSTRATION OF CENTERS OF OSTEOBLASTIC ACTIVITY BY USE OF VITAL DYES OF THE BENZIDENE SERIES P. G. SHIPLEY Fi A ~ C. D C . hIhCI<LIS om the Dcpoi tment of d n a t o n i ? ~.Johns , Hopkinc Univei ~ i / y Ilnltim , v e , lltl. The sites of osteoblastic activity have hitherto been studictl in two ways: by use of microscopic, sections, and by the examination of gros? specimens in which the areasshowing bone formation have been revealed by rnakirig the surrounding tissues transparent. Of the socalled ‘clearing methods,’ the best known and most used are those of Schultzc arid Spaltcholz, the first of which depends for its success on the relativc opacity of accumulations of calcium salts, and the latter on the post iriortem staining of bone by solutions of alizarin. Therefore they are deficient since they shed no light on the beginning of the ossifi(~atioriprocess, and they (30 not make visible the areas in which it is t:tking place until extensive calcification of thc locus has occurred. If mi zzo dyc (c.g., trypan blue) he atitninisterc4 t o a very young :ttiitiinl the k)ones are stained quickly arid very intensely with the vital color. The staining is so rapid and sclective that thc growing osscoiis system is colored blue black throughout its entire extent, whereas the organs (the liver, spleen, etc.), which are so deeply staiiied :tfter the same treatment in the norrnal adult, are in thvse young animals in a grcat nieasure excliitletl from participation in the storage of the dyc. The bones, if tlisscctctl out arid cleared, show distinct epiphyses and apophysrs sharply outlined within their cartilaginous capsules, and separated by intervening cartilage from the diaphyses. In preparations of this kind, the various cerit,ers of osscous developnicnt may he readily seen and studied. The method pursued to attain this end is hiefly as follows: Young animals are given a single large dose of a sterile 1 per cent solution of trypan blue. The site of election for the exhibition of the dye is the peritoneal cavity, because the superficial veins are very tiny, and the subcutaneous injection of large amounts of fluid favors pressure necro . sis. That the different stages in the process of ossification may he as sharply cut as possible, it is best t o administer a large quantity of tryp a n blue solution a t once, rather than t o give the same amount of dye in dividcd doses. Concentrated solutions should be given to avoid the mechanical difficulties involved in the administration of large quantities of fluid. The animal is sacrificed forty-eight hours after staining and the tissues are fixed by injection of a 10 per cent solution of neu597 598 P. G. SHIPLEY AND C . C. MACXLIN t r d formalin through the hlootl vessels, followed by immersion in t,hc sanw solution for a period of from tweiity-four to forty-eight hours. Tlic bones are then washed thoroughly, and hardened in ascending grades of alcohol, after which the soft parts are dissected away. Thc boncs are now dehydrated in absolute alcohol, and, after immersion in henzol for twenty-four h o u d , are cleared in oil of wintergreen according to the method of Spalteholz. Oil of wintergreen is an excellent clenring agent and causes little or no shrinkage in the tissues even after long immersion in the fluid. These preparations are cotivenicntly studied with the aid of the dissecting or binocular microscope. Bones which have been tlecalcificd previous t o clearing may be used for study in the sanic way, and d rward embcddcd in paraffin directly from oil of wintclrgl ccn and sectioned; or bones previously cleared withOUL dedcification may be retmnrd through absolute to 95 per cent alcohol, decal(4k.l and redehytliated, embedded and cut. In hones which have been decalcified before clearing, the osteogciictic foci are as distinct though not as opaque as in bones which have iiot bceii treated with acid. ?‘he loss in opacity is due t o the cxtractioii of a sinall aniount of &in which goes hand in hand with the solution of the calcium salts. I t is sonietiirnes of advantage in these specirnens t o introduce as :L contrast a very li7ht eosin stain. The pictures seen in examining these bones are very beautiful. The cyiphyscal and apophyseal centers, colored deep blue, are seen em1 ccltlctl in their transparent cartilaginous matrices, separated from the growing end of the diaphysis by a disc of modified cartilage which is readily recognized by the striatelcl appearance which is given it hy thc :trrangenient of its cells in parallel columns. The growing ends of the (liaphysis are much more darklg stained than the remainder of the ]_)one’sshaft; this phenomenon can be readily seen in the advancing ventral ends of the ribs and in thc ends of the digital diaphyses adjacent t o the epiphyses. I t may be well to emphasize here the fact that stained areas in these hones are not necessarily the so-called primary ossification centers. The stain is deposited in the sites, where, at the t h e of the exhibition of the t1yc1, active ossification is in progress, and tliesc loci of osteo11I:istic activity may or niay not be identical with the ossification ccnter o f thc. tiorics or epiphysis under ohservation. Nowhere is the difference hetween the primary ossification center and the focus of active growth I)rouglit so iharply to the attention of the observer as in the membrane t)oncs of thcslcull. If oneof these bones isrxamined, i t is apparent even to t h e iinaidrtl eye that the vital stain is deposited in a heavy line along the rapidly growing edge of the bone, while the primary ossification wntcrs are sceri t o be alrriost entirely without color. Thc unique feature of this method is that it demonstrates clearly the tliffercnt ossification areas even before the process of deposition of calcium salts has occurred in any marked degree. I n centers where, by the older methods, the new bone is almost or quite invisible (on account CENTERS O F OSTEOBLASTIC ACTIVITY 599 of the slight amount of calcium salt present) this method brings out the locus of ossific activity very distinctly. A large field for the application of this method is suggested by the appearance of the bones which have been examined. In the skull, for instance, graphic demonstrations of the growth of membrane bones and closure of the sutures are afforded. I n the base of the skull the various anlagen are easily separated through the sharpness of the contrast between the stained new bone and the intervening masses of cartilage. In the long bones, the epiphyses and apophyses stand out with diagrammatic clearness. The physiological and histological aspects of the vital staining of bone with trypan blue are now being considered, and will be published in the near future.