[Preparation and application of polyclonal antibody against amelotin polypeptide].

OBJECTIVE: To prepare and identify the rabbit anti-mouse polyclonal antibody against amelotin polypeptide. METHODS: The polypeptide was synthesized based on the bioinformatics analysis of amelotin, and coupled with keyhole limpet hemocyanin (KLH) for immunization. The amelotin polypeptide-KLH was injected into New Zealand rabbits to prepare the polyclonal antibody. ELISA technology was used to detect the titer of the antibody. The specificity of the polyclonal antibody was identified by Western blotting. The expression of amelotin in the submandibular tissue of mice was observed by immunohistochemistry. RESULTS: ELISA showed that the titer of amelotin antibody was 1:1,000,000. Western blotting verified that the antibody had a high specificity. Immunohistochemistry indicated that amelotin was highly expressed in odontoprisis full-thick enamel of 3-day-old and 7-day-old mice, and also expressed in duct epithelial cytoplasm of submandibular glands of 7-day-old mice. CONCLUSION: The polyclonal antibody against amelotin polypeptide has been prepared successfully with high titer and high specificity.

Localization and quantitative co-localization of enamelin with amelogenin.

Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 µm height, 33 µm width) and small (7 µm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.

Immunohistochemical and biochemical evidence of ameloblastic origin of amyloid-producing odontogenic tumors in cats.

Amyloid-producing odontogenic tumors (APOT) are rare, and in cats, the histogenesis of the amyloid remains undetermined. In the present study, APOTs in 3 cats were characterized by immunohistochemistry, and the amyloid components analyzed using tandem mass spectrometry. Antiameloblastin antibodies labeled both neoplastic epithelial cells and amyloid in all cases. Neoplastic epithelial cells had strong, diffuse immunoreactivity to antibodies against cytokeratin AE1/AE3, cytokeratin 14, and cytokeratin 19 in all cases and focal immunoreactivity to nerve growth factor receptor antibodies in 2 of 3 cases. Amyloid and some tumor stromal cells were weakly positive for laminin. Calretinin, amelogenin, S100, and glial fibrillary acidic protein antibodies did not label neoplastic epithelial cells or amyloid. Extracted amyloid peptide sequences were compared to the porcine database because the cat genome is not yet complete. Based on this comparison, 1 identical ameloblastin peptide was detected in each tumor. These results suggest that feline APOTs and the amyloid they produce are of ameloblastic lineage.

Enamel defects associated with coeliac disease: putative role of antibodies against gliadin in pathogenesis.

Enamel defects in the permanent teeth of patients with coeliac disease (CD) are often reported as an atypical manifestation, sometimes being suggestive of an undiagnosed atypical disease. We proposed to explore the pathogenesis of these oral defects, which are poorly studied. Sequence analyses of proteins from gluten (gliadins) and of proline-rich enamel proteins (amelogenin and ameloblastin) suggested the presence of common antigenic motifs. Therefore, we analyzed, by ELISA and western blotting, the reactivity of sera from patients with CD against gliadin and enamel-derived peptides. Correlation analyses between the levels of specific antibodies against gliadin and enamel derived peptides and inhibition experiments confirmed the presence of cross-reactive antibodies. Immunoblot analysis revealed that the most prominent component in enamel matrix derivative (of approximately 18.6 kDa), identified by an amelogenin-specific antibody, is recognized by sera from patients with CD; in addition, several fractions of pure gliadin were recognized by amelogenin-specific antibody. In agreement, sera from mice immunized with enamel matrix-derived proteins generated antibodies that recognized a peptide (of approximately 21.2 kDa) derived from gliadin. In conclusion, antibodies against gliadin generated in patients with CD can react in vitro with a major enamel protein. The involvement of anti-gliadin serum in the pathogenesis of enamel defects in children with untreated CD can be hypothesized on the basis of these novel results.

[Preparation and characterization of anti-Amelotin polyclonal antibody].

AIM: To construct a mouse Amelotin recombinant plasmid, express mouse Amelotin protein and prepare its polyclonal antibody. METHODS: The cDNA sequence of mouse Amelotin gene was downloaded from GenBank. The coding region, which did not include signal peptide, was amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a and transformed into E.coli BL21(DE3). After induced by IPTG, the recombinant protein was expressed and purified using affinity purification. The polyclonal antibody was obtained from New Zealand rabbit immunized with the recombinant protein and the titer was identified by ELISA. The polyclonal antibody was identified by Western blot and immunohistochemistry assays. RESULTS: The recombinant prokaryotic expression vector pET32a-Amelotin was constructed and the recombinant prorein was purified successfully. ELISA analysis showed the titer of the generated antiserum was 1:12,800. Western blot and immunohistochemistry analysis demonstrated this antibody bound specifically with Amelotin. CONCLUSION: The anti-Amelotin antibody from the rabbit with high titer and specificity had been prepared with the purified recombinant Amelotin as immunogen, which is helpful for further research into the detection and function of Amelotin.

The induction and possible subsequent effect of human antibodies against porcine enamel matrix derivative.

BACKGROUND: The amino acid sequence of porcine amelogenin, the major component of enamel matrix derivative (EMD), is approximately 91% identical to that of its human counterpart. Whether porcine EMD (pEMD) can elicit neutralizing antibodies after the first surgery, thereby reducing the clinical effect of secondary surgery, has not been established. METHODS: The sera of patients receiving periodontal surgery with or without pEMD were collected before and after surgery. The pEMD product was subjected to electrophoresis and transferred for a Western blot using the purified antibodies from patients as the primary antibodies. To clarify whether the antibodies in patients could inhibit the production of transforming growth factor-beta1 (TGF-beta1), we added different amounts of purified antibodies from pEMD patients (before versus after surgery) into the cell cultures of periodontal ligament fibroblasts. The conditioned media were then collected for an immunoassay of TGF-beta1. RESULTS: The result of the Western blot demonstrated that human antibodies against pEMD were elicited 10 days after surgery. Using mass spectrometry, a non-specific band on the Western blot appeared to be porcine immunoglobulin G (IgG). The results of the immunoassay showed the antibodies from pEMD-treated patients did not hinder the subsequent production of TGF-beta1. CONCLUSIONS: The pEMD product was contaminated with porcine IgG. The application of the product could induce antibodies against different isoforms of porcine amelogenin in humans. However, the increased antibodies did not hinder the production of TGF-beta1, one of the established in vitro functions of pEMD on periodontal ligament fibroblasts.

Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana.

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.

A multicenter study evaluating the sensitization potential of enamel matrix derivative after treatment of two infrabony defects.

BACKGROUND: Several studies reported some success toward regeneration in infrabony defects using enamel matrix derivative (EMD). Clinically and statistically significant improvements in probing depth reduction, clinical attachment levels, and bone fill have been demonstrated. This multi-center study evaluated the potential for sensitization to EMD in a subgroup of periodontal patients treated at least twice with at least 2 months between treatments. METHODS: Three hundred seventy-six (376) patients in 11 university-based postgraduate periodontics programs and five private practices were selected. Surgeries were performed on infrabony defects. Following reflection of mucoperiosteal flaps and debridement of the root surface and defect, root conditioning (either citric acid pH = 1 or 24% EDTA) was performed and the site was irrigated with sterile saline. Enamel matrix derivative was reconstituted and applied to the exposed root surface and the bony defect. Flaps were sutured and pressure applied for 5 minutes. The second test defect was treated in a similar manner at least 8 weeks after the first surgery. The patient was given a diary card where any subjective adverse events (erythema, swelling, itching, headache, root hypersensitivity, or pain) were recorded at weeks 1 and 2 post-surgery. In addition, objective adverse events (gingival inflammation, ulcers, abscess, cratering, and lesions) were recorded by the investigator on an adverse event form. RESULTS: No clinical adverse reactions to multiple applications of EMD were noted. Of 376 patients, two were referred to a dermatologist for evaluation, but neither had signs indicating any adverse events due to EMD treatment. Instead their reactions were classified as a small local abscess and tinea cruris. The single immunoassay performed (on the patient with a small local abscess) did not demonstrate any EMD-reactive antibodies, neither IgE nor IgG. Other subjective/objective reactions that occurred during this study were of the type that are commonly experienced by patients immediately following periodontal surgery, but were not related to EMD. They included headache, swelling, itching, pain, and root hypersensitivity. CONCLUSIONS: This study demonstrated a lack of clinical adverse reactions following two separate applications of EMD. Any subjective/objective adverse reactions experienced by the patient were typical complications following routine periodontal surgery and were not directly related to the use of enamel matrix derivative.

[Extraction and purification of porcine amelogenin and preparation for the polyclonal amelogenin antibody].

OBJECTIVE: To prepare the polyclonal antibody to amelogenin. METHODS: The fetal porcine dental enamel was collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7.4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund's adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test. RESULTS: The Gel filtration showed that amelogenin components is at molecular weights of 15 kD and 13 kD apparently, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1:1000. CONCLUSION: The antibody prepared in this study can be used for the detection of amelogenin.

Conservation and variation in enamel protein distribution during vertebrate tooth development.

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.

Immunologic effects of emdogain in humans: one-year results.

Tissue regeneration after therapeutic manipulations is essential in periodontology, oral surgery, and trauma of the periodontal tissues. Local inflammation because of poor oral hygiene also plays a crucial role in the above situations. Local inflammatory reaction, accompanied by the local production of cytokines, profoundly influences bone turnover and regeneration. Several products of low immunogenicity for augmenting tissue regeneration have been recently proposed as boosters of soft and mineralized tissue regeneration. Among them, Emdogain, an amelogenin derivative of porcine origin, has recently been introduced. Clinical results indicate that this product might be a good additive, producing fast tissue regeneration with no apparent clinical side effects. In contrast, very little is known about its in vivo immunologic effects. A previous study showed that Emdogain does not modify the cellular or humoral immune response in vitro. In the present work, performed in 10 patients, only a slight, nonsignificant activation of the immune system occurred during the first year following Emdogain application. Neither cellular immunity nor humoral immune response was significantly modified. In addition, the in vitro response of the patients' lymphocytes to Emdogain was assayed 2 and 12 months postoperative. We did not find any significant specific lymphocyte transformation in the presence of Emdogain, although lymphocytes could be stimulated by nonselective mitogens. These results indicate the immunologic safety of the agent in vivo, at least after 1 year.

Immunochemical characterization of a chicken egg yolk antibody to secretory forms of rat incisor amelogenin.

Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentino-enamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.

Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin.

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelial nests.

Internalization of amelogenin by dendritic cells of the papillary layer during transition and early maturation stages.

The existence of amelogenin in the papillary layer facing transition and early maturation zones of ameloblasts of rat incisors and the role of dendritic cells were examined by light microscopic immunocytochemistry and immunoelectron microscopy using anti-porcine 25 kDa amelogenin antibodies and anti-MHC class II antibodies. At the light microscopic level, no overall relationship was observed between anti-class II-positive dendritic cells and anti-amelogenin-positive materials located intercellularly: anti-amelogenin-positive dots were scattered in papillary layers and ameloblasts. Anti-MHC class II-positive cells were present in the papillary layer at the transition and maturation stages. Under electron microscopy, however, the dendritic cells occasionally endocytosed anti-amelogenin-positive materials and formed small vesicles. The results suggest that the dendritic cells play a role in eliminating amelogenin from the enamel organ.

Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin.

Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues. (J Histochem Cytochem 47:1237-1245, 1999)

Low stimulation of peripheral lymphocytes, following in vitro application of Emdogain.

Fast tissue regeneration after therapeutic manipulations is a central problem of periodontology, oral surgery and trauma of the periodontal tissues, including bone. Several products, which augment tissue regeneration, have been manufactured and assayed in clinical practice with positive results. Emdogain is a recent addition in this field, as a tissue-regenerating product. The substance is a derivative of amelogenin, obtained from porcine embryonic tissues. At the present time, it is not known whether the substance can induce a local (due to the uptake of the substance) or systemic immune response. The aim of the present study was to evaluate, in vitro, the ability of Emdogain to influence, in vitro, the immune system. Peripheral blood lymphocytes, isolated for 10 healthy donors, were cultured in the presence of various concentrations of the substance, in order to determine the rate of cell proliferation, the expression of surface antigens and the production of cytokines and immunoglobulins. Under our experimental conditions, Emdogain produced a slight increase of the proliferation of lymphocytes, restricted to the CD25 (IL-2 receptor) fraction of the CD4 positive T-lymphocytes, and a concomitant decrease of CD19 positive B-lymphocytes. Other cell fractions (CD8 positive T-cells, B-cells and NK-cells) were not affected. Under our conditions too, immunoglobulin and cytokin (IL-2 and IL-6) production was not modified, even after a 3-day application of concentrations much higher than those used in clinical practice. Our data suggest that Emdogain slightly induce an immune response, restricted to the activated fraction of CD4 T-lymphocytes in vitro.

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs.

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.

Derived protein and cDNA sequences of hamster amelogenin.

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3' rapid amplification of cDNA ends (3' RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3' non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5' PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.

Isolation of amelogenin-positive ameloblasts from rat mandibular incisor enamel organs by flow cytometry and fluorescence activated cell sorting.

The purpose of this study was to use amelogenin as a marker to examine the feasibility of isolating ameloblasts from enamel organ cell populations by fluorescence activated cell sorting. After treating dissected rat enamel organs with proteolytic enzymes to loosen cell attachments and labial connective tissues, dissociated cell suspensions were fixed, then immunostained with rabbit anti-rM179 recombinant amelogenin antibody and FITC-conjugated goat anti-rabbit Ig G antibody. Flow cytometry indicated that about 70% of the total cell sample and virtually all the larger cells therein were amelogenin-positive. Fluorescence activated cell sorting yielded a sample of amelogenin-positive cells at 97% purity. Immunofluorescence microscopy indicated that these isolated amelogenin-positive cells varied widely in size and morphology. This was attributed to loss of intercellular support for ameloblasts once they were dissociated from each other, and to some fragmentation caused when the cells were initially physically removed from the teeth. The results demonstrate that viable ameloblast cell fractions, especially representing cells at the secretory stage, can be purified from enzymic digests of rat enamel organ by sorting on the basis of cell size alone. From these fractions, subpopulations of ameloblasts may be identified when differentiation specific cell surface markers become available.