Histochemical and immunohistochemical examination of odontoblasts (petroblasts) in petrodentine formation of lungfish.

OBJECTIVE: Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques. METHODS: Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure. RESULTS: Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca2+-ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage. CONCLUSIONS: The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts.

Gene-expression profile and localization of Na+/K(+)-ATPase in rat enamel organ cells.

The sodium pump Na(+)/K(+)-ATPase, expressed in virtually all cells of higher organisms, is involved in establishing a resting membrane potential and in creating a sodium gradient to facilitate a number of membrane-associated transport activities. Na(+)/K(+)-ATPase is an oligomer of α, ß, and γ subunits. Four unique genes encode each of the α and ß subunits. In dental enamel cells, the spatiotemporal expression of Na(+)/K(+)-ATPase is poorly characterized. Using the rat incisor as a model, this study provides a comprehensive expression profile of all four α and all four ß Na(+)/K(+)-ATPase subunits throughout all stages of amelogenesis. Real-time PCR, western blot analysis, and immunolocalization revealed that α1, ß1, and ß3 are expressed in the enamel organ and that all three are most highly expressed during late-maturation-stage amelogenesis. Expression of ß3 was significantly higher than expression of ß1, suggesting that the dominant Na(+)/K(+)-ATPase consists of an α1ß3 dimer. Localization of α1, ß1, and ß3 subunits in ameloblasts was primarily to the cytoplasm and occasionally along the basolateral membranes. Weaker expression was also noted in papillary layer cells during early maturation. Our data support that Na(+)/K(+)-ATPase is functional in maturation-stage ameloblasts.

Compounded PHOSPHO1/ALPL deficiencies reduce dentin mineralization.

Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1(-/-) mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1(-/-);Alpl(+/-) ). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1(-/-) mice, and the almost complete absence of matrix vesicles in the Phospho1(-/-);Alpl(+/-) mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization.

Modulation of cell-cell junctional complexes by matrix metalloproteinases.

The ameloblast cell layer of the enamel organ is in contact with the forming enamel as it develops into the hardest substance in the body. Ameloblasts move in groups that slide by one another as the enamel layer thickens. Each ameloblast is responsible for the formation of one enamel rod, and the rods are the mineralized trail that moving ameloblasts leave behind. Matrix metalloproteinases (MMPs) facilitate cell movement in various tissues during development, and in this review we suggest that the tooth-specific MMP, enamelysin (MMP20), facilitates ameloblast movements during enamel development. Mmp20 null mice have thin brittle enamel with disrupted rod patterns that easily abrades from the underlying dentin. Strikingly, the Mmp20 null mouse enamel organ morphology is noticeably dysplastic during late-stage development, when MMP20 is no longer expressed. We suggest that in addition to its role of cleaving enamel matrix proteins, MMP20 also cleaves junctional complexes present on ameloblasts to foster the cell movement necessary for formation of the decussating enamel rod pattern. Therefore, inactivation of MMP20 would result in tight ameloblast cell-cell attachments that may cause maturation-stage enamel organ dysplasia. The tight ameloblast attachments would also preclude the ameloblast movement necessary to form decussating enamel rod patterns.

Assessment of dental fluorosis in Mmp20 +/- mice.

The molecular mechanisms that underlie dental fluorosis are poorly understood. The retention of enamel proteins hallmarking fluorotic enamel may result from impaired hydrolysis and/or removal of enamel proteins. Previous studies have suggested that partial inhibition of Mmp20 expression is involved in the etiology of dental fluorosis. Here we ask if mice expressing only one functional Mmp20 allele are more susceptible to fluorosis. We demonstrate that Mmp20 (+/-) mice express approximately half the amount of MMP20 as do wild-type mice. The Mmp20 heterozygous mice have normal-appearing enamel, with Vickers microhardness values similar to those of wild-type control enamel. Therefore, reduced MMP20 expression is not solely responsible for dental fluorosis. With 50-ppm-fluoride (F(-)) treatment ad libitum, the Mmp20 (+/-) mice had F(-) tissue levels similar to those of Mmp20 (+/+) mice. No significant difference in enamel hardness was observed between the F(-)-treated heterozygous and wild-type mice. Interestingly, we did find a small but significant difference in quantitative fluorescence between these two groups, which may be attributable to slightly higher protein content in the Mmp20 (+/-) mouse enamel. We conclude that MMP20 plays a nominal role in dental enamel fluorosis.

Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino-enamel junction, and a decussating enamel rod pattern.

Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.

Distribution pattern of cholinesterase enzymes in human tooth germs.

The two distinct molecular forms of cholinesterase (ChE) are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Our previous studies have reported that ChE is involved in tooth development. However, further experiments are needed to understand the precise action of ChE in tooth development. This study aimed to localise types of ChE in human tooth germs, and identify their distribution pattern. ChE were localised in frozen sections of jaws which were prepared from dead fetuses, neonates and stillborns who were free from visible abnormalities by Karnovsky and Root method. AChE was identified in the inner and outer enamel epithelia including the cervical loop region, stratum intermedium and preameloblasts of tooth germs at bell stage. Secretory ameloblasts were free from staining. The bud and cap stages of permanent tooth germs showed AChE activity on the lingual aspect and top surface of the epithelial ingrowths, respectively. BuChE activity was localised in the degenerating dental lamina. Our study reported the first evidence of localisation of ChE in human tooth development and identified the possible molecular form of ChE in tooth germs as AChE. Also, our results have provided strong evidence to speculate the action of AChE is on the cells of enamel organ during tooth development.

A survey of carbonic anhydrase mRNA expression in enamel cells.

Enamel formation requires rigid control of pH homeostasis during all stages of development to prevent disruptions to crystal growth. The acceleration of the generation of bicarbonate by carbonic anhydrases (CA) has been suggested as one of the pathways used by ameloblasts cells to regulate extracellular pH yet only two isozymes (CA II and CA VI) have been reported to date during enamel formation. The mammalian CA family contains 16 different isoforms of which 13 are enzymatically active. We have conducted a systematic screening by RT-PCR on the expression of all known CA isoforms in mouse enamel organ epithelium (EOE) cells dissected from new born, in secretory ameloblasts derived from 7-day-old animals, and in the LS8 ameloblast cell line. Results show that all CA isoforms are expressed by EOE/ameloblast cells in vivo. The most highly expressed are the catalytic isozymes CA II, VI, IX, and XIII, and the acatalytic CA XI isoform. Only minor differences were found in CA expression levels between 1-day EOE cells and 7-day-old secretory-stage ameloblasts, whereas LS8 cells expressed fewer CA isoforms than both of these. The broad expression of CAs by ameloblasts reported here contributes to our understanding of pH homeostasis during enamel development and demonstrates its complexity. Our results also highlight the critical role that regulation of pH plays during the development of enamel.

Expression of typical calpains in mouse molar.

OBJECTIVE: Calpain is a calcium ion-dependent cysteine protease, consisting of two primary isoforms (calpain1/calpain2) which mediate crucial cellular functions. The activity of the calpains is tightly regulated by the endogenous inhibitor calpastatin. Calpains have been detected in several studies during the embryonic and foetal stages. The aim of this study is to investigate the temporal transition of typical calpains and their inhibitor calpastatin during odontogenesis. DESIGN: We used the first molar of foetal ICR mice from embryonic day (E) 14 to postnatal day (PN) 7. Using laser microdissection and semi-quantitative real-time PCR, we investigated calpain1, calpain2 and calpastatin expressions in each enamel epithelium, inner enamel epithelium, stellate reticulum and outer enamel epithelium. RESULTS: We found calpain1 and calpain2 mRNA increased in the all enamel epithelia between E18 and PN1. In addition calpastatin mRNA expression increased in the ameloblasts from PN1 to PN7. The immunohistochemistry results demonstrated that calpain1/calpain2 was present in the distal side of ameloblasts from PN1 to PN7, and calpastatin was present in the extracellular enamel matrix from E16 to PN1. Furthermore calpain1/calpain2 was present in the dentin, and calpastatin was detected in dentin producing odontoblasts and predentin at PN7. CONCLUSIONS: In this study the temporal transition of calpain1, calpain2 and calpastatin mRNA and the immunolocalization are identified during tooth development. Our results indicate that the calcium-dependent proteases may play an important role in mouse molar development and extracellular calpain and calpastatin may be involved in molar mineralization.

Lysosomal protease expression in mature enamel.

The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

Evidence by signal peptide trap technology for the expression of carbonic anhydrase 6 in rat incisor enamel organs.

During screening of a rat incisor enamel organ cDNA library by signal peptide trap technology, we identified a DNA fragment matching a predicted translation sequence for rat carbonic anhydrase 6 (CA6). This result was unexpected because CA6, to date, has been associated primarily with secretions from glandular tissues. To further characterize this observation, reverse transcription-polymerase chain reaction (RT-PCR) amplifications were carried out on total RNA extracted from freeze-dried secretory and maturation-stage rat incisor enamel organs. A cDNA fragment of the expected size was detected in control samples from rat salivary glands as well as within maturation-stage enamel organ samples. This CA6 RT-PCR fragment was further cloned and sequenced and found to match the nucleotide sequence 770-1079 from clone XM_216584 of GenBank. Northern blot analyses with the rat CA6 cDNA fragment confirmed its expression relative to maturation-stage enamel organ samples. It is at present unclear whether the CA6 expressed by enamel organ cells is secreted into the enamel layer or into the intercellular spaces of the enamel organ itself to assist in neutralizing excess protons arising from the growth of apatite crystals during the maturation stage of amelogenesis.

Characteristic distribution of immunoreaction for estrogen receptor alpha in rat ameloblasts.

Estrogen has a diverse function, including cell proliferation and differentiation via estrogen receptors (ER), which have been reported to be the case in various tissues in addition to female reproductive organs. A recent immunocytochemical study has reported the expression of ERalpha, a subtype of ER, in rat odontoblasts, suggesting an involvement of estrogen in the differentiation of tooth-forming cells. However, there is no information on the ERalpha immunoexpression in ameloblasts. The present study was therefore undertaken to examine the localization of ERalpha immunoreaction in rat ameloblasts during amelogenesis. A computer-assisted quantitative analysis under a confocal laser scanning microscope was employed to demonstrate the stage-specific localization pattern of ERalpha immunoreaction. Immunohistochemistry of the rat enamel organ revealed ERalpha expression as nuclear localization in ameloblasts, stratum intermedium, stellate reticulum, and papillary layer, in addition to mature and immature odontoblasts. The ratio of immunopositive nuclei to total nuclei (immunopositive ratio) in ameloblasts was high at the apical loop region and gradually declined at the presecretory stage to zero at the secretory stage with statistically significant difference. The ERalpha immunolabeling pattern exhibited a periodic change at the maturation stage proper with constant higher labeling in ruffle-ended ameloblasts than in smooth-ended ameloblasts. The positive ratio was then followed by a statistically significant increase in immunolabeling thereafter. This stage-specific immunolabeling pattern during amelogenesis suggests a possible role of ERalpha in ameloblast proliferation and differentiation.

Fine structural and cytochemical mapping of enamel organ during the enameloid formation stages in gars, Lepisosteus oculatus, Actinopterygii.

During cap enameloid formation in gars (Lepisosteus oculatus), the dental epithelial cells that constitute the enamel organ were observed by means of transmission electron microscopy and enzyme cytochemistry to detect the hydrolytic enzyme activities, alkaline phosphatase (ALPase), acid phosphatase (ACPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase) (sodium, potassium-activated adenoshine triphosphatase (Na-K-ATPase)). The enameloid formation process in gars was divided into three stages: matrix formation, mineralisation and maturation. The enamel organ consisted of the outer dental epithelial (ODE) cells, stellate reticulum (SR), stratum intermedium (SI) and the inner dental epithelial (IDE) cells during the whole of the cap enameloid formation stages. During the matrix formation stage, many cisternae of rough endoplasmic reticulum and widely distributed Golgi apparatus, in which the procollagen granules containing cross-striations were often found, were remarkable elements in the IDE cells. During the stage of mineralisation, the IDE cells were tall columnar, and infoldings of distal plasma membrane of the IDE cells became marked. The most developed Golgi apparatus was visible at this stage, and large secretory granules containing fine granular or tubular materials were found in the distal cytoplasm that was close to the infoldings of the distal end. Many lysosomes that were ACPase positive were seen near the Golgi apparatus and in the distal cytoplasm of the IDE cells. ACPase positive granules often contained the cross-striation structure resembling procollagen, suggesting that the procollagen is degenerated in the IDE cells. During the maturation stage, the distal infoldings became unclear, and there were no large granules containing tubular materials, but many ACPase positive lysosomes were still present in the IDE cells. Non-specific ALPase was detected at the plasma membrane of the IDE cells at the mineralisation and maturation stages. K-NPPase was markedly detected at the plasma membrane of the IDE cells at the maturation stage. These results demonstrate that the IDE cells might be mainly involved in the removal of degenerated organic matrix from enameloid during the later formation stages. Strong Ca-ATPase activity was observed at the entire plasma membrane of the stratum intermedium cells, and there was slightly weak activity at the plasma membrane of the IDE cells during the mineralisation and maturation stages, implying that these cells are related to the active Ca transport to the maturing enameloid. It is likely that although the structure of the enamel organ is different, the function, especially at the mineralisation and maturation stages, is similar to other actinopterygians having well-mineralized cap enameloid.

Expression of protein kinases C betaI, betaII, and VEGF during the differentiation of enamel epithelium in tooth development.

Protein kinase C (PKC) is an important molecule involved in various cell function, and mediates induced secretion of vascular endothelial growth factor (VEGF). It is hypothesized that PKC and VEGF may be associated with tooth development. Using the laser microdissection method and real-time reverse-transcription-polymerase chain-reaction (RT-PCR), we investigated the expression of PKC betaI and betaII, VEGF, and amelogenin (used as a marker of differentiation to ameloblasts) in the inner and outer enamel epithelia, stellate reticulum, and dental papilla in each stage of the dental germ. We found that the expression levels of PKC betaI and betaII were increased in the inner enamel epithelium during the early bell stage. In addition, the increased expression levels of PKC betaI and betaII were accompanied by increased VEGF expression. These results indicate that PKC betaI, betaII, and VEGF are closely associated with the differentiation of the inner enamel epithelium to ameloblasts.

Histochemical localization of cholinesterase activity in the dental epithelium of guinea pig teeth.

Cholinesterase is known for its remarkable diversity in distribution and function. An association of this enzyme with proliferative and morpho-differentiating tissues has been reported in several species. Here we report on the first evidence of the presence of cholinesterase in the enamel organ of continuously erupting incisors and molars of the guinea pig. Frozen sections of the incisors and molars of the guinea pig were incubated for histochemical demonstration of cholinesterase activity by means of the thiocholine method as described by Karnovsky and Root. The cholinesterase activity was observed in several types of cells of the dental epithelium; cells forming the basal portion of the enamel organ, outer enamel epithelium and maturation stage ameloblasts of both the incisors and molars. In the crown analogue side, the outer enamel epithelial cells gained strong reactions for cholinesterase and maintained the reaction throughout the secretory and maturation stages of amelogenesis. In contrast, cholinesterase reactions were lacking in the inner enamel epithelium, pre-ameloblasts, and secretory ameloblasts. In the early stage of enamel maturation, ameloblasts began to show positive reactions for cholinesterase, which was upregulated in the incisal direction. Although both tooth types showed similar reactive patterns for cholinesterase at the growing ends, maturation ameloblasts depicted a different pattern of staining displaying the reactions only sporadically in molars. These data indicate the role of cholinesterase in the enamel organ in tooth morphogenesis and function of guinea pig teeth.

Fine structure and Ca-ATPase activity of the stratum intermedium cells during odontogenesis in gars, Lepisosteus, Actinopterygii.

This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

Enamelysin and kallikrein-4 mRNA expression in developing mouse molars.

Proteolytic processing and degradation of enamel matrix proteins appears to be an essential feature of dental enamel formation. The source and character of proteolytic activity in the enamel matrix of developing teeth changes as enamel formation progresses. Two proteinases have been isolated from the extracellular enamel matrix of developing teeth: enamelysin (MMP-20), a matrix metalloproteinase. and kallikrein-4 (KLK4), a serine proteinase. Here, we ask if the expression of MMP-20 and KLK4 correlate with the stage-associated changes in the digestion of enamel proteins. Using in situ hybridization, we localized MMP-20 and KLK4 mRNA in mouse maxillary first molars on postnatal days 1, 2, 3, 5, 6, 7, 9, 11, and 14. Enamelysin signal was first detected in preameloblasts, ameloblasts, and odontoblasts on day 2, but not in ameloblasts covering the enamel-free zone. Enamelysin signal declined in ameloblasts on day 6 but persisted in the dental pulp. In contrast, KLK4 transcripts were first observed on day 3 in pulp and on day 6 in ameloblasts covering the enamel-free zone. the KLK4 signal was present in maturation-stage ameloblasts on days 9, 11, and 14. The expression patterns of MMP-20 and KLK4 by ameloblasts in mouse molars are stage-specific and complementary.

Expression and activity of matrix metalloproteinase-2 (MMP-2) in the development of rat first molar tooth germ.

Tooth germ development is associated with morphological and biochemical changes of the dental papilla and enamel organ. Enzymes with gelatinolytic activities were studied by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography in tooth germ of newborn to 15-day-old rats. Three major bands with gelatinolytic activity were detected at all periods and characterized as the latent and active forms of MMP-2 using their molecular weight and activity dependent on Zn++ and Ca++ ions as criteria. Expression and activity of MMP-2 increased progressively from 0 to 15 days after birth. Mechanical separation of the tooth germ from 10-day-old rats showed that the gelatinolytic activity was localized mainly in the dental papilla and not the dental organ. These data indicate that the expression and activity of MMP-2 varies during the development and maturation of rat first molar tooth germ.

Expression and activity of matrix metalloproteinase-2 (MMP-2) in the development of rat first molar tooth germ

Tooth germ development is associated with morphological and biochemical changes of the dental papilla and enamel organ. Enzymes with gelatinolytic activities were studied by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography in tooth germ of newborn to 15-day-old rats. Three major bands with gelatinolytic activity were detected at all periods and characterized as the latent and active forms of MMP-2 using their molecular weight and activity dependent on Zn++ and Ca++ ions as criteria. Expression and activity of MMP-2 increased progressively from 0 to 15 days after birth. Mechanical separation of the tooth germ from 10-day-old rats showed that the gelatinolytic activity was localized mainly in the dental papilla and not the dental organ. These data indicate that the expression and activity of MMP-2 varies during the development and maturation of rat first molar tooth germ.

Dipeptidyl-peptidase II and cathepsin B activities in amelogenesis of the rat incisor.

A body of published evidence suggests that a significant portion of enamel matrix protein synthesized by ameloblasts localises in the lysosomal-endosomal organelles of these enamel organ cells. Little is known regarding the lysosomal proteolytic activities during amelogenesis. The aims of this study were to detect and measure the activities of lysosomal peptidases cathepsin B (E.C. 3.4.22.1) and dipeptidyl-peptidase II (E.C. 3.4.14.2) in the enamel organ of the rat incisor and to ascertain whether rat enamel matrix proteins are degraded by these peptidases in vitro. Whole enamel organs were dissected from rat mandibular incisors. Enamel protein was also collected from the rat teeth. Analysis indicated that the rat incisor enamel organs contained specific activities of both dipeptidyl-peptidase II and cathepsin B at levels comparable with those of kidney which is rich in both these lysosomal peptidases. Gel electrophoresis and immunoblotting demonstrated that both cathepsin B and dipeptidyl-peptidase II were able to substantially degrade the rat enamel proteins in vitro. Based on these observations, we propose that lysosomal proteases have roles in amelogenesis in the intracellular degradation of amelogenins.