In vivo experimentation on rat incisor enamel organs through a surgical window.код для вставкиСкачать
THE ANATOMICAL RECORD 210:693-705 (1984) In Vivo Experimentation on Rat Incisor Enamel Organs Through a Surgical Window M.D. McKEE AND H. WARSHAWSKY Department of Anatomy, McGill Uniuersity, Montreal, Quebec H3A 2B2 Canada ABSTRACT Experimental agents administered systemically are costly and often toxic to animals. An in vivo technique has been developed whereby a surgical window in the alveolar bone allows selected areas of the rat incisor enamel organ and underlying enamel to be exposed to various drugs, radiolabeled molecules, and molecular weight markers. Sherman rats weighing 100 gm were anesthetized and the inferior surface of each hemimandible was surgically exposed. A slow-speed dental hand drill was used to drill a small hole through the alveolar bone overlying the secretion or maturation zones of the enamel organ. The wound was closed and during recovery the mechanical trauma to the underlying tissue moved away from the hole due to the continuous eruption of the tooth. Two to 5 days later the hole was reexposed and microinjections of 3H-proline, 1251-salmoncalcitonin, vinblastine sulphate, and normal saline (as control) were administered through the hole with a micromanipulator and a microliter syringe. Radioautographic detection of 3H-proline incorporation in secretory ameloblasts and enamel a t 10 minutes, 30 minutes, 1 hour, 4 hours, 1day, and 2 days after microinjection was identical to that obtained previously by systemic injection. Two hours after microinjection of vinblastine sulphate the cellular response was again identical to that following systemic injection; 1251-salmoncalcitonin (M.W. - 3,600D) was used as a molecular weight marker and was seen to diffuse into the enamel of the maturation zone at 10 minutes after microinjection. This study has demonstrated the feasibility of this new technique for experimentation on rat incisor enamel organs. The incisor of the rat has been shown to be a n excellent model system in which to study the complex phenomena associated with amelogenesis (see reviews in Reith, 1960, 1961, 1963; Watson, 1960; Fearnhead, 1960, 1961a,b; Kallenbach et al., 1963; Warshawsky, 1968, 1971, 1978, 1979; Warshawsky and Smith, 1971, 1974; Weinstock and Leblond, 1971; Kallenbach, 1973; Skobe, 1976; Warshawsky and Vugman, 1977; Smith, 1979; Leblond and Warshawsky, 1979). Previously, many methods of in vivo experimentation on rat incisors have been devised. Surgical manipulations such as root resections and transections (Herzberg and Schour, 1941; Massler and Schour, 1941; Bryer, 1957; Ness, 1957; Kostlan et al., 1960; Berkovitz and Thomas, 1969; Berkovitz, 1971) have convincingly implicated the periodental ligament to be directly or indirectly 0 1984 ALAN R.LISS, INC. associated with the mechanism of eruption of this tooth. These procedures involved surgically removing a portion of the mandibular alveolar bone and wounding or removing the underlying dental tissues. Other ways of penetrating the alveolar bone have been developed (Melcher, 1970; Gould et al., 1977). These procedures consisted of drilling a hole through the alveolar bone to the level of the periodontal ligament in mouse molars in order to stimulate and locate progenitor cells of the periodontal ligament. This study was undertaken in order to determine the feasibility of approaching the enamel organ of the rat incisor through the alveolar bone overlying the labial surface of the tooth. A technique using a slow-speed Received May 16,1984;accepted July 16,1984 694 M.D. McKEE AND H. WARSHAWSKY Fig. 1. Enlargement of the right hemimandible of a Sherman-strain rat killed 24 days after drilling. The drill used for the surgery is shown above the hole in the labial alveolar bone (black arrow). The alveolar bone may be drilled anywhere along its inferior border where it overlies the secretion or maturation zones of the incisor. The enamel lesion has moved incisally away from the hole due to the continuous eruption of the tooth and appears as a shallow pit on the erupted surface of enamel (white arrow). x 6 . 5 . dental hand drill equipped with dental burs has been developed whereby a surgical window in the labial alveolar bone allows selected areas of the rat incisor enamel organ and underlying enamel to be exposed to microinjections of various drugs, radiolabeled molecules, and molecular weight markers. Experimental agents administered systemcally are costly and often toxic to animals. The main objective of this study was to develop a microinjection technique in which minute amounts of experimental agents could be introduced through the surgical window in the alveolar bone and allowed to diffuse down and over selected areas of the enamel organ and underlying enamel. Subsequently, the fate and effects of microinjected experimental agents on the enamel organ and enamel of the rat incisor could be compared with the previously described ef- fects of systemic injections of the same substance. A similar response of the enamel organ and the enamel to the two different procedures would establish the feasibility of the microinjection technique. MATERIALS AND METHODS Drilling procedure Sherman and Sprague Dawley rats weighing approximately 100 gm were used in this study. The animals were anesthetized with an intraperitoneal injection of Nembutal and the inferior surface of each hemimandible was surgically exposed. Retractors were used to hold back the masculature and the area was kept moist with rinses of physiological saline. A slow-speed dental hand drill equipped with a straight handpiece and carbide dental burs was used to drill a small hole (0.75 mm in diameter) through the al- SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS veolar bone overlying either the secretion or maturation zones of the enamel organ (Fig. 1).Complete penetration through the alveolar bone into the vascular periodontal space overlying the enamel organ was determined by tactile sensation and immediate bleeding upon breakthrough. The bur was removed and gauze was placed over the hole for 1 minute to stop the bleeding and the area was again rinsed with physiological saline. In animals that were killed within 10 minutes after drilling, the wound was kept moist, but open. At longer time intervals the wound was closed, and during recovery the mechanical trauma to the underlying dental tissues moved away from the hole due to continuous eruption of the tooth. Microinjection technique Animals were drilled in the alveolar bone overlying the secretion and maturation zones of the enamel organ. Two to 5 days later the hole was reexposed to permit microinjections over the “healthy”ename1 organ and enamel that had moved underneath the drill site due to eruption. A vertical compact micromanipulator (Brinkman Instruments, Model MM 33) was mounted on a column stand and a micrometer (Scherr-Tumico, Inc.) was inserted into its clamping mechanism to allow for extremely fine vertical movements of a microsyringe plunger. A 100-plmicrosyringe obtained from the Hamilton Co. (Cat. No. 710) was fitted with a 33-gauge needle. A rubber band was used to keep the plunger of the syringe firmly applied to the plunger of the micrometer. In this way, small amounts of the solution to be injected were drawn into the syringe by the micrometer. Graduations on the micrometer were calibrated to the graduations on the syringe, which allowed dispensing of accurate, reproducible, minute volumes of solution. Due to the small bore size of the 33guage needle several precautions were taken to insure accurate and consistent microinjections. The needle tip was filed down from its original bevel to a flat surface using 600grade emory cloth. Both microsyringe and needle were thoroughly rinsed with physiological saline, rapidly plunged to remove any compressible air bubbles, and the plunger was slowly withdrawn so as to partially fill the syringe with a “buffer zone” of saline. The syringe was then mounted onto the micromanipulator. Solutions could now be drawn into the syringe of the micromanipulator assembly. The syringe was filled only 695 immediately prior to injection, and paraffin film was used to seal the tip of the needle between injections to prevent evaporation of the solution from the small-bore diameter at the tip of the needle. Microinjection of 3H-proline L(2,3-3H)-proline(specific activity 32.2 Ci/ mmole) was purchased in 0.01 N HC1 (New England Nuclear). Under a stream of nitrogen gas, 0.1 ml of solution containing 100 pCi of 3H-proline was evaporated to dryness. The amino acid was redissolved in 1 pl of physiological saline to provide for ten microinjections of 0.1 p1 containing 10 pCi of 3H-proline. The solution was slowly drawn into the microsyringe. Two days prior to microinjection, a minimum of 20 animals were drilled in the alveolar bone overlying the secretion zone of the enamel organ in both hemimandibles and allowed to recover. The holes were then reexposed and the tip of the needle was lowered approximately 0.5 mm into the connective tissue plug that had filled the lesion in the bone. Approximately 0.1 pl of solution containing 10 pCi of 3H-proline was microinjected under a dissecting microscope into the right hemimandible. The needle was withdrawn immediately, the solution was allowed to “sink in” for 2 mintues, and the wound was closed. The left hemimandibles were not injected. Animals were killed at 10 minutes, 30 minutes, 1hour, 4 hours, 1 day, and 2 days after microinjection. Microinjection of vinblastine sulphate A group of five rats was drilled in both hemimandibles as above, two days prior to microinjection. The right hemimandibles were microinjected with 0.1 pl of a stock solution of 5 mg/0.3 ml saline of vinblastine sulphate (Sigma). Injections of physiological saline into the left hemimandibles served as controls. All animals were killed 2 hours after microinjection. Microinjection of 1251-salrnoncalcitonin Synthetic salmon calcitonin (approximately 2500 mU/pg) was iodinated with sodium 1251(specific activity 17 pCi/mg; New England Nuclear), by the Chloramine T method described by Hunter and Greenwood (1962). Following these rocedures the super51-salmon calcitonin natant containing the ! (M.W. - 3,600D) was evaporated to dryness under a stream of nitrogen gas and redissolved in 2.5% (w/v) bovine serum albumin SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS in 25 mM TRIS-HC1buffer, pH 7.4. From this solution, 0.1 pl was microinjected into the right hemimandibles of two animals drilled in the alveolar bone overlying the maturation zone 2 days prior to microinjection, and the animals were killed 10 minutes thereafter. Tissue processing All animals used in this study were anesthetized with intraperitoneal injections of Nembutal and killed by perfusion through the left ventricle with lactated Ringer’s solution (Abbott) for 30 seconds followed by perfusion for 10 minutes with a n aldehyde mixture consisting of 2% acrolein and 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.3. The mandibles were dissected and immersed in the above fixative for 4 hours a t 4°C followed by washing in 0.1 M sodium cacodylate buffer containing 0.05% CaC12, pH 7.3. The mandibles were then decalcified in 4.13% isotonic neutral disodium EDTA (Warshawsky and Moore, 1967) and were cut into segments that were extensively washed in the above 0.1 M sodium cacodylate buffer. The incisor segments were subsequently postfixed in 2% osmium tetroxide for 4 hours a t 4”C, dehydrated in graded ace- Fig. 2. A longitudinal section of the right hemimandible showing the original drill site (B) in the alveolar bone (ab) and the trauma to the underlying tissues (E) that has moved incisally away from the drill site over 2 days (the large arrow indicates the direction of tooth eruption). “Healthy” enamel (en) and enamel organ (eo) have been passively carried underneath the drill site by the continuous eruption of the tooth. Bone fragments (small arrows) may be found near the drill site. PS, periodontal space. x 115. Fig. 3. Bone lesion (B) drilled over the enamel secretion zone. The animal was killed 2 days after drilling. “Healthy” enamel (en) and enamel organ (eo) are found below the bone lesion. Arrows, bone fragments. X89. Fig. 4. Enamel lesion at 2 days after drilling. The enamel (en) is completely stripped from the dentin (den), and cells appearing to belong to the enamel organ continue across the lesion. The enamel thickness is constant for part of the lesion and may be due to an inhibition of secretion in this area. Accumulations of red blood cells, singly stacked or packed in oval clusters not bounded by endothelium, are found between ameloblasts (black arrows). At the dentinoenamel junction where the enamel has been removed is a thin layer of dark-staining material (white arrows). c, capillaries; PS, periodontal space. x356. 697 tone, and embedded in Epon 812. Each segment was orientated for sectioning along the long axis of the incisor. One-micron-thick sections were cut with glass knives on a Reichert Om U2 ultramicrotome and stained with toluidine blue or prepared for radioautography (Kopriwa and Leblond, 1962). Thin sections (gold interference color) were cut with a diamond knife, mounted on copper grids, and stained with uranyl acetate and lead citrate. Sections were examined with either a Siemens Elmiskop 1A or a Siemens 101 a t 80Kev. The tissues from the ‘251-salmoncalcitonin experiment were processed as described above with the exception that the fixitive contained only 2.5% glutaraldehyde in sodium cacodylate buffer containing 0.05% CaC12, pH 7.3. RESULTS Drilling procedure Drilling through the alveolar bone of the rat mandible causes trauma to the underlying dental tissues. The damaged tissues, however, are passively moved incisally away from the drill site due to the continuous eruption of the tooth. A gross skeletal preparation of a drilled right hemimandible and its incisor shows the drill hole in the alveolar bone, and the trauma to the enamel which is seen near the incisor tip 24 days after drilling (Fig. 1).Bone remodeling has occurred a t the drill site, and the trauma to the underlying enamel appears as a shallow pit on the erupted surface of enamel. The distance between the bone lesion and the enamel lesion (15 mm) corresponds to the normal eruption rate (651pndday) over a 24-day period (Smith and Warshawsky, 1975). The bone lesion is seen in histological sections as a prominent interruption in the alveolar bone that is filled with a plug of connective tissue (Figs. 2, 3). The bones were drilled over the enamel secretion zone and the animals were killed 2 days after drilling. The enamel lesion (Fig. 2) moved incisally and “healthy” enamel organ has been passively carried underneath the drill site by the continuous eruption of the tooth. The cut edges of bone are clearly visible, and occasional bone fragments may be observed (Figs. 2, 3). Bleeding often occurs into the connective tissue of the periodontal space and extends laterally from the drill site. Drilling of the labial alveolar bone in the rat mandible almost invariably causes le- 698 M.D. McKEE AND H. WARSHAWSKY sions in the enamel organ and the underlying enamel (McKee, 1984). The lesions presumably occur when the drill bur penetrates the alveolar bone and plunges a short distance into the periodontal space. Histologically, the dental tissue trauma appears as a n altered or ruptured enamel organ overlying a complete discontinuity in the enamel layer in which the enamel has been completely stripped from the dentin (Figs. 2, 4). In most cases, the dentin is unbroken. Figure 4 shows a lesion in which the animal was killed 2 days after drilling. The cells lining the dentinoenamel junction appear to be derived from the enamel organ as the capillary network of the papillary layer can be followed over the lesion. The cells resemble stratum spinosum or stratum intermedium cells. At the periphery of the lesion the enamel thickness is constant, indicating a n inhibition of enamel secretion due to the trauma. Ameloblasts have decreased in height but Tomes’ processes appear intact. Accumulations of red blood cells, singly stacked or packed in oval clusters not bounded by endothelium, are found between ameloblasts. These accumulations conform to the spatial limitations of the ameloblast layer. At the center of the lesion the enamel has been completely removed. At the dentinoenamel junction and extending only as wide as the lesion, is a thin layer of dark-staining material (Fig. 4). This layer completely covers the dentin and varies in thickness. With the electron microscope (Figs. 5-8), this layer is bounded by two zones of increased electron density. In all cases, the arrangement of collagen fibrils a t the dentinoenamel junction is unusual because the irregular interdigitations of initial enamel with collagen fibrils of the dentin are not present. Microinjection technique Microinjected 3H-proline over the enamel secretion zone is picked up by the secretory ameloblasts and incorporated into all proteins being synthesized a t that time, including enamel matrix proteins. At 10 minutes after microinjection, radioautographic silver grains are located over the supranuclear region of the ameloblasts (Fig. 9). By 30 minutes, the distribution of the label in the supranuclear zone is unchanged from above, but in addition, a weak reaction band is seen over Tomes’ processes of the ameloblasts (Fig. lo). At 1 hour after microinjection the reac- tion band over Tomes’ processes has greatly intensified (Fig. 11). Silver grains are still located over the supranuclear zone. By 4 hours, fewer grains are found over the supranuclear zone and the reaction band in the enamel extends toward the dentinoenamel junction (Fig. 12). Following the same cohort of labeled cells as they moved incisally, the reaction at 1 day is seen over the entire thickness of the enamel (Fig. 13). Some silver grains are still observed over the ameloblasts. By 2 days after microinjection, the cohort is now in outer enamel secretion, and silver grains are distributed over the entire enamel layer but very few are over ameloblasts (Fig. 14). Despite the minute quantity injected into the drill site, 3H-proline must have entered the bloodstream because a reaction band is seen in the predentin at 4 hours in the uninjected contralateral left hemimandible (Fig. 15) from the same animal as the injected right hemimandible shown in Figure 12. Ten minutes after microinjection (Fig. 16) 1251-salmoncalcitonin microinjected into the right hemimandible over the early maturation zone was localized by radioautography. Most striking is the reaction observed over the entire thickness of the enamel. Silver grains are found in greater density over the outermost enamel but nevertheless extend completely through the enamel up to the dentinoenamel junction. The dentin only shows background labeling. Labeling aIso is seen in the ameloblasts and in the periodontal space, and to a lesser extent in the papillary layer. Microinjection of vinblastine sulphate into the right hemimandible over the enamel secretion zone causes disorganization of organelles within the ameloblasts (Figs. 17-20). In the supranuclear zone (Fig. 17),marked accumulations of dense-content granules are seen, rER appears fenestrated or fragmented, and patches of a granular, dense material are seen between ameloblasts. These are presumed to be ectopic secretion sites of enamellike material (Fig. 18). The infranuclear zone contains mitochondria, rER, and numerous densecontent granules (Fig. 19). Tomes’ processes also show significant alterations in normal morphology. The interdigitating portion of Tomes’ process is completely devoid of organelles (Fig. 20). Although a few membrane infoldings are found, they are not as frequent as in controls. Microinjection of physiological saline served as controls, and these animals show normal ameloblast morphology. SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS Figs. 5-8. Electron micrographs taken a t the center of enamel lesions. The enamel has been completely removed, and an amorphous layer of material is seen at the dentinoenamel junction (asterisks).This layer completely covers the dentin (Den), may vary in thickness, and is bounded by two zones of increased density (arrows, Fig. 8). The irregular interdigitations of collagen fibrils normally seen at the dentinoenamel junction are not present. Leu, leucocyte. Figures 57, X 10,500; Figure 8, ~52,000. 699 Figs. 9-14. Light microscope radioautographs of secretory ameloblasts (am) at various time intervals after microinjection of 3H-proline (Pro). At 10 minutes after microinjection (Fig. 9, x712) grains are present mainly over the supranuclear cytoplasm. At 30 minutes (Fig. 10, ~ 7 1 2 the ) reaction is seen over Tomes’ processes as well as the supranuclear zone of the ameloblasts. At 1 hour (Fig. 11, ~ 5 3 4 Tomes’ ) processes and part of the enamel matrix (en) are heavily labeled, and at 4 hours ) reaction band extends deeper into the (Fig. 12, ~ 7 1 2the enamel and the ameloblasts are still labeled. At 1 day ) 2 days (Fig. 14, X534) the reaction is (Fig. 13, ~ 7 1 2and seen over the entire enamel matrix but stops at the dentinoenamel junction. en, enamel matrix. SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS 701 DISCUSSION A new technique has been developed whereby the formation of a surgical window in the labial alveolar bone of the rat hemimandible allows the underlying enamel organ of the rat incisor to be exposed to manipulation and microinjection of various experimental agents. The validity of such a n approach was tested by the application of several previously published procedures so that comparisons could be made. The enamel organ lesion Early in this study it was noted that complete penetration of the labial alveolar bone over the zone of enamel secretion by a dental drill bur invariably produced trauma to the underlying enamel organ and caused complete removal of enamel underlying the drill site (Figs. 2-8). Immature enamel from this zone contains about 30% organic matrix by weight (see review in Leblond and Warshawsky, 1979) and is referred to as “cheesy enamel” because of its consistency. The lesion occurs immediately after drilling of the bone and indicates that the relatively soft enamel in this zone can be disturbed by nearby turbulence such a s that caused by the bur (McKee, 1984). The more rigid matrix of the dentin remains intact along the entire length of the lesion. During the process of repair a layer of amorphous material coated the surface of the dentin previously covered by enamel. Part of the mantle dentin seems to be resorbed along with the enamel, and the electron-dense lines resemble lamina limitans seen in regions of functional bone reversals. Microinjection of drugs and radiolabeled precursors Vinblastine sulphate and 3H-proline were selected as representatives of a drug and a radiolabeled molecule, respectively, to assess the feasibility of the microinjection technique. Microinjection of vinblastine sulphate caused disruptions in normal ameloblast morphology and functional activity identical to those resulting from systemic injection of vinblastine sulphate (Moe and Mikkelsen, 1977; Takuma et al., 1982; Nanci, 1982) and colcemid (Karim and Warshawsky, 1979; Nishikawa and Kitamura, 1982).Microinjected 3H-proline was utilized by the secretory ameloblast and incorporated into the organic matrix of enamel in the same way and a t the Fig. 15. Light microscope radioautograph of odontoblasts and dentin at 4 hours after microinjection of 3Hproline. Silver grains are seen in the predentin (pd) of the uninjected left hemimandible from the same animal as the injected contralateral right hemimandible shown in Figure 12. d, dentin; en, enamel. x510. Fig. 16. Light microscope radioautograph of the enamel maturation zone 10 minutes after microinjection of ‘251-salmon calcitonin. Grains are found over the entire thickness of enamel (en).Labeling also is seen in the ruffle-ended ameloblasts (r-am) and in the periodontal space (ps), but to a lesser extent in the papillary layer (pl). d, dentin. X340. 702 M.D. McKEE A N D €1. W A R S H A W S K Y SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS same time intervals as systemic injections of 'H-proline (Warshawsky, 1966, 1979; Leblond and Warshawsky, 1979). These results demonstrate the feasibility of administering drugs and radiolabeled molecules by the microinjection route. In some circumstances, the local microinjection technique has advantages over systemic injection. First, experimental agents that are toxic when administered systemically may be microinjected over selected areas of the enamel organ and the animal killed a t any time thereafter. Second, in the continuously growing incisor of the rat, a cohort of cells in the enamel organ and the underlying enamel matrix, a t any specific developmental stage of amelogenesis, may be selectively exposed to various experimental agents. Furthermore, due to the incisal eruption of the tooth, the evolution of this same cohort and its underlying enamel, and the fate and effects of the previously administered experimental agents, may be followed as they pass through the different developmental zones of amelogenesis. Third, intravascular administration of radiolabeled molecules requires that they be injected in quantities large enough to reach suitably high concentrations in all the tissues of the body. Most systemic studies use dosages of 1,000 pCi, costing about $100 per 100-gm animal. Using the microinjection technique, only 10 pCi of label, lilOOth of that necessary for systemic injection, is introduced to a se- 703 Fig. 19. Infranuclear region of secretory ameloblasts 2 hours after microinjection of vinblastine sulphate. This region contains mitochrondria im), rER, and numerous dense-content granules (dg). Dense-content granules are occasionally linked by continuous membrane (arrow). cw, cell web. x 15,000. lected volume of cells, thereby eliminating unneccesary distribution of radioactivity to all tissues of the body, and eliminating the risk of radiation damage to sensitive cells and tissues. When a single injection of 'H-proline is administered intravenously, the labeled amino acid circulates to capillaries overlying the enamel organ and rapidly appears in the dental tissues. When the animal is fixed by perfusion and the tissues processed for radioautography, the free labeled amino acid is washed out, while the labeled protein is retained. Presumably, a similar route is followed after microinjection. When 3H-proline is microinjected into the connective tissue plug that results from healing after the drilling procedure, the labeled proline rapidly diffuses to the ameloblasts of the enamel organ. Once the amino acid has reached the level of the connective tissue surrounding the capillary network of the enamel organ, presumably it behaves and is utilized by the enamel organ in the same manner as amino acids diffusing from the capillary lumen following intravenous injection. The only difference is the continuous high specific activity available after local injection. Concurrent with the diffusion of 'H-proline into the enamel organ after microinjection, there exists a similar rapid diffusion of 3H-proline into the bloodstream as demonstrated by radioautographic reactions over odontoblasts and predentin of the contralateral, uninjected hemimandible. Because of the appositional formation of dentin by odontoblasts, it becomes a continuous and stable record of the availability of labeled amino acids for incorporation into protein (Josephsen and Warshawsky, 1982). Therefore, following microin'ection of minute quantities ( - 10 pCi) of 3'H-proline into a 100-gm animal, some of the labeled amino acid rapidly diffuses into the local vasculature, is removed from this site by the circulation, and is incorporated by other tissues, as for example the dentin of the contralateral incisor as a secretory product of the odotoblasts. A similar amount of radioactivity may have recirculated back to the experimental incisor. However, this small amount of radioactivity did not obscure the boundaries of the originally labeled cohort of cells following microinjection. Fig. 20. Tomes' processes of secretory ameloblasts 2 hours after microinjection of vinblastine sulphate. Tomes' processes (TP) are completely devoid of organelles, but some membrane infoldings (arrow) can be found. en, enamel. x 12,400. Microinjection of permeability tracers Several studies have investigated the permeability of the enamel organ and the enamel matrix to relatively large macromo- Fig. 17. Supranuclear region of secretory amcloblasts 2 hours after microinjection of vinblastine sulphate. Accumulations of dense-contentgranules (dg) are seen, rER appears fenestrated or fragmented, and patches of dense material (arrow)are seen between ameloblasts. x 12,800. Fig. 18. Higher magnification of the dense material seen between ameloblasts similar to that shown in Figure 17. Presumably the location of this material represents ectopic secretion sites of enamel matrix (en). dg, dense-content granules; cv, coated vesicles; arrows, coated pits. ~ 5 1 , 7 5 0 . 704 M.D. McKEE AND H. WARSHAWSKY lecules such as serum albumin (Kinoshita, 1979; Ogura and Kinoshita, 1983; Okamura, 1983). In the enamel maturation zone of the rabbit both endogenous and exogenous serum albumin were localized to the ruffled-and smooth-ended ameloblasts and to the intercellular spaces between them (Okamura, 1983). Serum albumin was not located in the enamel matrix of the maturation zone although in the secretion zone the surface layer of enamel was heavily stained by the fluorescent antibody to albumin (Okamura, 1983). Salmon calcitonin has a molecular weight of approximately 3,600D and was used in this study as a molecular weight marker to investigate the permeability of the enamel organ and the enamel matrix to proteins of that size. Radioautography revealed 1251salmon calcitonin over the entire thickness of the enamel as soon as 10 minutes after microinjection. The rapidity of appearance in the enamel suggests that the label is not a secretory product of the ameloblast and must represent diffusion either extracellularly or transcellularly through the enamel organ to reach the enamel matrix. The results presented here are in agreement with those of Okamura (1983)in which relatively large exogenous molecules may reach and enter the ameloblast layer of the enamel maturation zone. Furthermore, this study presents evidence that the enamel matrix of the maturation zone is completely permeable to a protein having a molecular weight of approximately 3,600D. In conclusion, this study has demonstrated the feasibility of a drilling procedure that penetrates into the periodontal space overlying the rat incisor enamel organ. In addition, this procedure shows that surgical penetration of the alveolar bone may cause trauma to the enamel organ and enamel. This study has demonstrated the feasibility and advantages of a microinjection technique for in vivo experimentation on the enamel organ and enamel of the rat incisor. ACKNOWLEDGMENTS The authors wish to thank Dr. P. Bai for his assistance during this study, and Dr. D. Goltzman for supplying the iodinated calcitonin. This work was supported by a grant from the Medical Research Council of Canada t o Dr. H. Warshawsky. LITERATURE CITED Berkovitz, B.K.B. (1971) The effect of root transection and partial root resection on the unimpeded eruption rate of the rat incisor. Arch. Oral Biol., 16:1033-1043. Berkovitz, B.K.B., and N.R. Thomas (1969) Unimpeded eruption in the root-resected lower incisor of the rat with a preliminary note on root transection. Arch. Oral Biol., 14:771-780. Bryer, L.W. (1957)An experimental evaluation of physiolom on tooth eruption. Int. Dent. J., 7432-478. Fearchead, R.W. (1‘460) Mineralization of rat enamel. Nature, 188509-510, Fearnhead, R.W. 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