ARTICLE |
Correspondence to: A. Nanci, Lab. for the Study of Calcified Tissues and Biomaterials, Faculty of Dentistry, Université de Montréal, Montreal, Quebec, Canada H3C 3J7.
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Summary |
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The tooth organ is extensively used in developmental biology to investigate organogenesis and cell differentiation. It also represents an advantageous system for the study of the various cellular and extracellular matrix events that regulate the formation of both collagenous and noncollagenous calcified tissues. This article describes an in vivo surgical approach to access and experimentally manipulate the tooth organ and supporting tissues of the rat incisor. By use of a dental drill, a "window" was created through the alveolar bone on the buccal aspect of the hemimandible at the apical end of the incisor. It is at this site that epithelial and mesenchymal precursors are situated and undergo cellular differentiation to give rise to cells of the odontogenic organ. Active bone remodeling is also observed in this area to accommodate posterior growth of the tooth. An osmotic minipump connected to the bony window through an outlet catheter was used for controlled and continuous administration of experimental agents over a predetermined period of time. To validate the model, vinblastine sulfate, fetuingold, and dinitrophenylated albumin were thus infused. The animals were then sacrificed and the hemimandibles were processed for histological and immunocytochemical analyses. The effects of the drug and the presence of tracers were restricted to the treated hemimandible and were found in the enamel organ and pulp, as well as in the tooth supporting tissues. Cellular changes typically associated with the administration of vinblastine were obtained, and tracers were localized both in the extracellular milieu and within the endosomal/lysosomal elements of cells. These results suggest that this new surgical approach could serve as an advantageous in vivo model in which various chemical agents, therapeutic drugs, molecular probes are locally administered to study the molecular events that regulate calcified tissue formation. (J Histochem Cytochem 47:323336, 1999)
Key Words: osmotic minipump, experimental manipulation, tracers, vinblastine sulfate, calcified tissues, odontogenic organ, incisor, rat
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Introduction |
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Tooth development is mediated by reciprocal inductive interactions between neural crest-derived ectomesenchyme cells and the oral epithelium (reviewed in
The continuously erupting rat incisor has been extensively used to study the cellular and extracellular matrix events involved in odontogenesis because all stages of development can be found in a single tooth and it exhibits many similarities to human tooth formation (
Despite the advantages offered by the rat incisor, there have been only limited attempts to develop experimental approaches for direct manipulation of the cellular and matrix events in this tooth. Some experiments have involved removal of blocks of bone and apical portions of the incisor to study cell activity during bone remodeling and tooth eruption (
In this study we investigated whether the rat mandibular incisor could be exploited as an experimental model for local and selective targeting of the odontogenic organ and its associated periodontal tissues. A surgical technique was developed to create a "window" in the alveolar bone overlying the apex of the rat incisor, and an osmotic minipump was utilized to deliver specific experimental agents. Vinblastine sulfate and two tracer molecules, fetuingold and albumin tagged with dinitrophenol, were utilized to validate the efficiency of the surgical approach in targeting cells of the tooth organ. Minipumps have previously been used in dogs to deliver bafilomycin A1, an inhibitor of vacuolar H+-ATPases in osteoclasts, to block alveolar bone resorption and tooth eruption (
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Materials and Methods |
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Surgical Procedure
Male Wistar rats weighing 100 ± 10 g (Charles River Canada; St-Constant, QC, Canada) were anesthetized with an IP injection of 0.06 ml Somnotol (sodium pentobarbital; MTC Pharmaceuticals, Cambridge, ON, Canada). The vestibular surface of the right mandibular ramus was surgically exposed as follows (Figure 1). An incision about 8 mm long was made through the skin with fine scissors to access the muscle layer underneath, according to an imaginary line joining the auditory meatus and the lip commissure. The fibers of the masseter were separated along their longitudinal axis with a scalpel blade. A periosteal separator was then used to elevate the periosteum and expose the underlying bony surface of the ramus. The musculature was retracted with a plastic ring made from an embedding BEEM capsule size 1 (Marivac; Halifax, NS, Canada). The surgical area was kept moist with rinses of physiological saline. A slow-speed dental drill equipped with a carbide round burr size 6 was used to create a hole through the alveolar bone (Figure 1A). The bony window was placed approximately 2 mm anterior to the posterior border of the ramus and slightly superior to the bony elevation at the apical end of the incisor. Penetration through the alveolar bone into the periodontal space around the apex was established by slight bleed-ing on breakthrough. The burr was then removed and a cotton swab was placed over the hole until the bleeding stopped. A third incision through the skin in the neck area was made to accommodate an Alzet 1003D or 2001D osmotic minipump (Alza Corporation; Palo Alto, CA) (Figure 1B). The 1003D model has a capacity of 90 µl and a flow rate of 1 µl/hr for 3 days, and the 2001D has a capacity of 234 µl and a flow rate of 8 µl/hr for 24 hr. The pump was tunneled into a subcutaneous pouch on the back of the animal and connected to the bony hole using a vinyl tubing and a metal catheter made from a 20G1 syringe needle (BectonDickinson; Rutherford, NJ). The tubing was passed underneath the masseter muscle and through the neck area. Histoacryl glue (B. Braun Melsungen AG, Germany/Sherwood DG, Dorval, QC, Canada) and bone cement (Zimmer; Warsaw, IN) were used to immobilize the metal catheter against the bone surface and maintain its tip in the bony hole. The animals were then sutured and the surgical site was cleaned and disinfected with 70% ethanol. After surgery, X-ray photographs of the rat mandibles were taken to verify that the catheter was well in place (Figure 1C) and the rats were allowed to recover under observation. Some of the animals received 0.01 ml of buprenorphine HCl 0.3 mg/ml (Temgesic; Reckitt & Colman, Hull, UK) as analgesic immediately after surgery. All animal procedures and experimental protocols described above were in strict accordance with guidelines of the Comité de Déontologie de l'Expérimentation sur les Animaux of Université de Montréal.
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Administration of Vinblastine Sulfate
Six rats were implanted with Alzet osmotic minipumps model 1003D filled with a solution of 0.17 mg/ml of vinblastine sulfate in physiological saline (Sigma Chemical; St Louis, MO). The minipumps were connected to the vinyl tubing, also filled with the drug, and incubated in sterile saline at 37C for 3 hr before placement as described above.
Administration of Tracers
Groups of two rats were implanted with saline-preincubated Alzet 2001D minipumps filled with fetuingold or DNP-tagged albumin for 24-hr infusion. The fetuingold complex (particles of ~15 nm in diameter; 25 µg/ml) was purchased from EY Laboratories (San Mateo, CA). Bovine serum albumin (Sigma) was tagged with dinitrophenol (DNP) using the method of
Tissue Processing
On the third day of infusion of vinblastine sulfate and at 24 hr for the tracers, the animals were anesthetized with an IP injection of 0.25 ml of 20% chloral hydrate (Sigma) and sacrificed by intravascular perfusion through the left ventricle. The vasculature was prerinsed with lactated Ringer's solution (Abbott Laboratories; Montreal, QC, Canada) for about 30 sec (until the liver blanched), followed by perfusion with a fixative solution consisting of 1% glutaraldehyde in 0.08 M sodium cacodylate buffer containing 0.05% CaCl2, pH 7.3, for 20 min. Both hemimandibles were dissected out and immersed in the fixative overnight at 4C. They were then washed in 0.1 M sodium cacodylate buffer containing 0.05% CaCl2, pH 7.3, and decalcified in 4.13% EDTA for 14 days at 4C (solution was changed every 2 days) (
Each tooth segment was oriented for sectioning along its longitudinal axis (see schematic illustration in Figure 3). One-µm-thick sections were cut with glass knives on a ReichertJung Ultracut E ultramicrotome and stained with toluidine blue. Thin sections were cut with a diamond knife and mounted on 200-mesh nickel grids having a carbon-coated Formvar film. Selected sections were processed for postembedding colloidal gold immunocytochemistry (reviewed in
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Immunolocalization of Dinitrophenol-tagged Albumin and Amelogenin
Sections from osmicated samples were first treated with sodium metaperiodate (
Scanning Electron Microscopy
Two animals were sacrificed by anesthetic overdose after creation of the bony window. The hemimandibles were dissected and cleaned of soft tissue, fixed overnight by immersion with 1% glutaraldehyde in 0.08 M cacodylate buffer containing 0.05% CaCl2, pH 7.3, and washed in 0.1 M sodium cacodylate buffer. Residual soft tissue was removed by digestion with 6% sodium hypochlorite. They were then washed and kept in distilled water until observation in the humid state with an Hitachi S-3500N variable-pressure scanning electron microscope operated in the backscattered mode at 20 kV and 40 Pa pressure.
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Results |
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Surgical Procedure
Initial studies aimed at establishing the appropriate position of the bony window identified the posterior border of the ramus and the bony elevation overlying the apical end of the incisor as reliable reference points for drilling. Passing the minipump catheter below the masseter muscle and immobilization of its metal tip with Histoacryl glue and bone cement resulted in firm anchorage. The positioning and stability of the catheters in the bony window were confirmed on X-rays (Figure 1C). Visual inspection at dissection time was also used to confirm that the catheter was still in position (Figure 1D) and to rule out any blockage by tissue debris and blood clotting. The reference points allowed proper positioning of the bony window and no damage to the enamel organ (Figure 2, Figure 4A, and Figure B) in eight of 10 animals used to validate the surgical procedure before the present drug and tracer study. Inaccurate anterior positioning resulted either in compression by bone debris (Figure 4C and Figure 4D) or focal destruction (Figure 4E) of the enamel organ, while complete perforation of the thin alveolar bony walls occurred posteriorly. Inflammatory cells infiltrated the damaged enamel organ and a cementum-like substance was deposited in the forming extracellular matrix (Figure 4E, inset) (
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Morphological Alterations Induced by Vinblastine Sulfate
Continuous exposure of the dental organ to vinblastine sulfate for 3 days affected tooth eruption such that the treated incisor was about 1 mm shorter than the contralateral tooth (Figure 5A). It is noteworthy that none of the rats used in initial studies aimed at validating the surgical procedure (no minipumps placed), as well as those used for tracer studies, showed a difference in length between the treated and the contralateral untreated incisor. Cell organization and function were also altered (Figure 5BE). The Golgi apparatus of secretory stage ameloblasts was fragmented, clusters of secretory granules were found throughout the cell body (Figure 6A), and endosomal/lysosomal elements were abundant (Figure 6B). These cell compartments were all immunoreactive for amelogenin (Figure 6). Tomes' processes showed very few or no secretion granules (Figure 7). These were abundant and intensely immunoreactive for amelogenin in contralateral incisor ameloblasts (Figure 7B). The organization and shape of early secretory stage ameloblasts were disrupted and there was ectopic release of enamel proteins along their basolateral surfaces (Figure 6A). Groups of odontoblasts showed signs of degeneration, even though the appearance of the overlying dentin was normal (Figure 5D). There were abundant mitotic cells in the pulp and periodontal tissue (Figure 5, inset). In one case, focal alteration of dentin production and mineralization on the root analogue surface were also observed (Figure 5E). Tissues of the contralateral hemimandible appeared normal and showed none of the above cell and matrix alterations (Figure 5F and Figure 7B). The duodenum, kidney, and parotid cells also revealed no structural changes (data not illustrated).
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Distribution of Tracers
Dinitrophenol-tagged albumin was found in endosomal/lysosomal elements of periodontal fibroblasts and in the interstitial fluid surrounding them (Figure 8), and less frequently in pulp cells and odontoblasts (Figure 9). There was also abundant immunoreactivity around osteoblasts and in osteoid near the site of infusion (Figure 10), but the density of labeling associated with bone diminished significantly away from the bony window. DNPalbumin was also detected in multivesicular bodies of early secretory stage ameloblasts (Figure 11). Although some sporadic labeling was found in liver cells, only a few randomly distributed gold particles were observed on tissue sections of the contralateral tooth incubated with anti-DNP antibody. Control incubations with protein Agold alone resulted in a few gold particles randomly distributed throughout the tissue section.
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Fetuingold was predominantly found at the site of drilling, but some complex was detected in the periodontal tissue along the incisor up to 5 mm away from the bony window. Gold particles accumulated within clotted matrix (Figure 12A) and in endosomal/lysosomal elements of macrophages and neutrophils in the bony defect. Periodontal fibroblasts in the proximity of the window (Figure 12 and Figure 13A) and, less frequently and intensely, pulp cells (Figure 13B) also showed the intracellular presence of tracer. No gold particles were detected in the enamel organ, in the contralateral tooth, or in other distant tissues sampled.
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Discussion |
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Despite efforts to elucidate the regulatory role of matrix molecules and growth factors in tooth and bone formation, there are still many questions regarding the molecular mechanisms that underlie their formation. To address these issues, we have developed an in vivo approach to experimentally access and manipulate the odontogenic organ of the rat incisor and its associated periodontal tissues. This tooth was chosen because it offers the possibility to investigate developmental processes and the deposition of both collagenous and noncollagenous mineralized matrices in a well-defined temporospatial sequence. An osmotic minipump, connected to a bony window in the alveolar bone overlying the apical end of the tooth, allowed controlled and continuous administration of experimental agents to the tooth organ and its surrounding tissues.
To avoid complications associated with previous experimental approaches for accessing the tooth organ (see Introduction), we positioned our surgical access on the buccal side of the alveolar bone, overlying the periodontal tissue just posterior to the apical end of the mandibular incisor. This site allows access to both the tooth organ and a zone of active bone remodeling along the posterior wall of the hemimandible. From this site, the experimental agents can diffuse to the enamel organ and its adjacent structures via the periodontal space separating the alveolar bone and the tooth. Damage to the odontogenic organ was observed in very few cases of operated animals and was, in general, due to anatomic variability. Most of the damage was confined to the apical end of the tooth and did not appear to significantly alter tooth formation and/or eruption. Damage to the enamel organ in some cases resulted in the production of a matrix resembling that found at the enamel-free area on the cusp tips of rodent molars, an observation relevant to the proposed epitheliomesenchymal transformation of enamel organ cells during odontogenesis (
In contrast to systemic injection or local microinjection, with minipumps it is possible to deliver relatively large volumes of an experimental agent over a precise period of time. Vinblastine sulfate was infused over a 3-day period to obtain local tissue alterations without any systemic effects. The changes obtained in the enamel organ are consistent with previously published data using local or systemic injection of vinblastine (
It is generally accepted that the presence of protein outside the cell stimulates the ingestion of solutes from the interstitial fluid (
In conclusion, the data presented herein validate our experimental approach and show that the various cells of the tooth, as well as those of the surrounding tissues, can be targeted. It can be advantageously applied to manipulate in vivo, using various therapeutic agents, the complex cellular and extracellular matrix events involved in the formation of both collagenous and noncollagenous calcified tissues. The ability to interfere with the genes responsible for the production of target proteins of the odontogenic organ and associated tissues may help advance our understanding of mineralized tissue formation and pathological alterations. Application of molecular probes through the bony window offers the possibility to activate/inactivate gene products locally and selectively for studies of function. Such an approach is potentially less time consuming and costly than the genetic engineering of knockout or transgenic animals, and may be particularly valuable in cases where genetic alterations result in a lethal phenotype. Enamel molecular probes have already been used in vitro (anti-sense;
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Acknowledgments |
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Supported by a grant from the Medical Research Council of Canada to AN. We are grateful to M. Fortin for excellent technical assistance, Dr L. Ghitescu for preparing and donating the dinitrophenylated albumin, and Nissei Sangyo Canada for use of the variable-pressure scanning electron microscope.
Received for publication August 19, 1998; accepted October 13, 1998.
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Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alberts B, Lewis J, Roberts K, Bray D, Raff M, Watson JD (1994) Vesicular traffic in the secretory and endocytic pathway. In Alberts B, Lewis J, Roberts K, Bray D, Raff M, Watson JD, eds. Molecular Biology of the Cell. New York, Garland, 600-651
Bendayan M (1995) Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 29:1-163[Medline]
Bendayan M, Zollinger M (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein Agold technique. J Histochem Cytochem 31:101-109[Abstract]
Berkovitz BKB (1971a) The effect of root transection and partial root resection on the unimpeded eruption rate of the rat incisor. Arch Oral Biol 16:1033-1043[Medline]
Berkovitz BKB (1971b) The healing process in the incisor tooth socket of the rat following root resection and exfoliation. Arch Oral Biol 16:1045-1054[Medline]
Berkovitz BKB, Thomas NR (1969) Unimpeded ereution in the root-resected lower incisor of the rat with a preliminary note on root transection. Arch Oral Biol 14:771-780[Medline]
Bosshardt DD, Zalzal S, McKee MD, Nanci A (1998) Developmental appearance and distribution of bone sialoprotein and osteopontin in human and rat cementum. Anat Rec 250:1-21[Medline]
Bosshardt DD, Nanci A (1998) Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells. J Histochem Cytochem 46:135-142
Butler WT (1998) Dentin matrix proteins. Eur J Oral Sci 106:204-210[Medline]
Eisenmann DR, Chen J, Lehman G, El-Moneim Zaki A (1989) Studies on the influx of [3H]-histidine and 45Ca through a surgical opening to rat incisor ameloblasts and adjacent enamel. Arch Oral Biol 34:93-102[Medline]
Freemont AJ (1993) Basic bone cell biology. Int J Exp Pathol 74:411-416[Medline]
Frens G (1973) Controlled nucleation for the regulation of particle size in monodispersed gold suspensions. Nature Phys Sci 241:20-22
Gehron Robey P (1996) Bone matrix proteoglycans and glycoproteins. In Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of Bone Biology. San Diego, Academic Press, 155-165
Ghitescu L, Bendayan M (1993) Transendothelial transport of serum albumin: a quantitative immunocytochemical study. J Cell Biol 117:745-755[Abstract]
Goldberg M, Septier D, Lecolle S, Chardin H, Quintana MA, Acevedo AC, Gafni G, Dillouya D, Vermelin L, Thonemann B, Schmalz G, Bissila-Mapahou P, Carreau JP (1995) Dental mineralization. Int J Dev Biol 39:93-110[Medline]
Leblond CP, Warshawsky H (1979) Dynamics of enamel formation in the rat incisor tooth. J Dent Res 58B:950-975[Medline]
Linde A, Goldberg M (1993) Dentinogenesis. Crit Rev Oral Biol Med 4(5):679-728[Abstract]
Little JR, Eisen HN (1967) Preparation of immunogenic 2,4-dinitrophenyl and 2,4,6-trinitrophenyl proteins. In Williams CA, Chase MW, eds. Methods in Immunology and Immunocytochemistry. Vol 1. New York, Academic Press, 128-133
Lyngstadaas SP, Risnes S, Sproat BS, Thrane PS, Prydz HP (1995) A synthetic, chemically modified ribozyme eliminates amelogenin, the major translation product in developing mouse enamel in vivo. EMBO J 14:5224-5229[Abstract]
Marks SCJ, Schroeder HE (1996) Tooth eruption: theories and facts. Anat Rec 245:374-393[Medline]
Marks SCJ, Sundquist KT (1995) Bafilomycin A1 in bone resorption and tooth eruption in dogs. Eur J Oral Sci 103:231-235[Medline]
McKee MD (1993) Effects of CO2 laser irradiation in vivo on rat alveolar bone and incisor enamel, dentin, and pulp. J Dent Res 72:1406-1417[Abstract]
McKee MD, Nanci A (1996a) Secretion of osteopontin by macrophages and its accumulation at tissue surfaces during wound healing in mineralized tissues: a potential requirement for macrophage adhesion and phagocytosis. Anat Rec 245:394-409[Medline]
McKee MD, Nanci A (1996b) Osteopontin at mineralized tissue interfaces in bone, teeth and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover and repair. Microsc Res Tech 33:141-164[Medline]
McKee MD, Warshawsky H (1984) In vivo experimentation on rat incisor enamel organs through a surgical window. Anat Rec 210:693-705[Medline]
Miake Y, Yanagisawa T, Takuma S (1982) Electron microscopic study on the effects of vinblastine on young odontoblasts in rat incisor. J Biol Buccale 10:319-330[Medline]
Moe H, Mikkelsen H (1977) Light microscopical and ultrastructural observations on the effect of vinblastine on ameloblasts of rat incisors in vivo. Acta Pathol Microbiol Immunol Scand [A] 85:73-88
Nanci A, Fortin M, Ghitescu L (1996a) Endocytotic functions of ameloblasts and odontoblasts: Immunocytochemical and tracer studies on the uptake of plasma proteins. Anat Rec 245:219-234[Medline]
Nanci A, Hashimoto J, Zalzal S, Smith CE (1996b) Transient accumulation of proteins at interrod and rod enamel growth sites. Adv Dent Res 10:135-149[Medline]
Nanci A, Smith CE (1992) Development and calcification of enamel. In Bonucci E, ed. Calcification in Biological Systems. Boca Raton, FL, CRC Press, 313-343
Nanci A, Uchida T, Warshawsky H (1987) The effects of vinblastine on the secretory ameloblasts: an ultrastructural, cytochemical, and immunocytochemical study in the rat incisor. Anat Rec 219:113-126[Medline]
Nanci A, Warshawsky H (1984) Characterization of putative secretory sites on ameloblasts of the rat incisor. Am J Anat 171:163-189[Medline]
Neiss WF (1984) Electron staining of the cell surface coat by osmium-low ferrocyanide. Histochemistry 80:231-242[Medline]
Redondo LM, Cantera JMG, Hernandez AV, Puerta CV (1995) Effect of particulate porous hydroxyapatite on osteoinduction of demineralized bone autografts in experimental reconstruction of the rat mandible. Int J Oral Maxillofac Surg 24:445-448[Medline]
Robinson C, Brookes SJ, Shore RC, Kirkham J (1998) The developing enamel matrix: nature and function. Eur J Oral Sci 106:282-291[Medline]
Ruch JV, Lesot H, Begue-Kirn C (1995) Odontoblast differentiation. Int J Dev Biol 39:51-68[Medline]
Slavkin HC (1990) Molecular determinants of tooth development: a review. Crit Rev Oral Biol Med 1:1-16[Medline]
Slavkin HC (1995) Antisense oligonucleotides: an experimental strategy to advance a causal analysis of development. Int J Dev Biol 39:123-126[Medline]
Smith CE, Nanci A (1989) A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection. Anat Rec 225:257-266[Medline]
Smith CE, Nanci A (1995) Overview of morphological changes in enamel organ cells associated with major events in amelogenesis. Int J Dev Biol 39:153-161[Medline]
Smith CE, Warshawsky H (1975a) Histological and thee dimensional organization of the odontogenic organ in the lower incisor of 100 gram rats. Am J Anat 142:403-430[Medline]
Smith CE, Warshawsky H (1975b) Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine. Anat Rec 183:523-562[Medline]
Sundquist KT, Marks SCJ (1994) Bafilomycin A1 inhibits bone resorption and tooth eruption in vivo. J Bone Miner Res 9:1575-1582[Medline]
Thesleff I, Vaahtokari A, Vainio S, Jowett A (1996) Molecular mechanisms of cell and tissue interactions during early tooth development. Anat Rec 245:151-161[Medline]
Triffitt JT, Joyner CJ, Oreffo ROC, Virdi AS (1998) Osteogenesis: bone development from primitive progenitors. Biochem Soc Trans 26:21-27[Medline]
Warshawsky H, Josephsen K, Thylstrup A, Fejerskov O (1981) The development of enamel structure in rat incisors as compared to the teeth of monkey and man. Anat Rec 200:371-399[Medline]
Warshawsky H, Moore G (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J Histochem Cytochem 15:542-549[Medline]
ZeichnerDavid M, Diekwisch TGH, Fincham AG, Lau EC, MacDougall M, MoradianOldak J, Simmer JP, Snead ML, Slavkin HC (1995) Control of ameloblast differentiation. Int J Dev Biol 39:69-72[Medline]