Vascular endothelial growth factor and nitric oxide synthase expression in human tooth germ development.

Vascular Endothelia Growth Factor (VEGF) and Nitric Oxide Synthase (NOS) expression, were evaluated in human tooth germs at two different stages of embryogenesis, to clarify the role of angiogenesis during tooth tissue differentiation and growth. Seventy-two third molar germ specimens were selected during oral surgery. Thirty-six were in the early stage and 36 in the later stage of tooth development. The samples were evaluated with Semi-quantitative Reverse Transcription-Polymerase chain Reaction analyses (RT-PcR), Western blot analysis (WB) and immunohistochemical analysis. Western blot and immunohistochemical analysis showed a VEGF and NOS 1-2-3 positive reaction in all samples analysed. VEGF high positive decrease reaction was observed in stellate reticulum cells, ameloblast and odontoblast clusters in early stage compared to later stage of tooth germ development. Comparable VEGF expression was observed in endothelial cells of early and advanced stage growth. NOS1 and NOS3 expressions showed a high increased value in stellate reticulum cells, and ameloblast and odontoblast clusters in advanced stage compared to early stage of development. The absence or only moderate positive reaction of NOS2 was detected in all the different tissues. Positive NOS2 expression showed in advanced stage of tissue development compared to early stage. The action of VEGF and NOS molecules are important mediators of angiogenesis during dental tissue development. VEGF high positive expression in stellate reticulum cells in the early stage of tooth development compared to the later stage and the other cell types, suggests a critical role of the stellate reticulum during dental embryo-morphogenesis.

Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs.

Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.

Expression, localization and synthesis of small leucine-rich proteoglycans in developing mouse molar tooth germ.

The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.

Detection of Notch signaling molecules in ameloblastomas.

BACKGROUND: To evaluate the roles of Notch signaling in the oncogenesis and cytodifferentiation of odontogenic tumors, expression of Notch receptors and ligands was analyzed in ameloblastomas as well as in tooth germs. METHODS: Tissue specimens of nine tooth germs and 32 ameloblastomas were examined by reverse transcriptase polymerase chain reaction and by in situ hybridization to determine the expression of Notch1, Notch2, Notch3, Delta1, and Jagged1. RESULTS: mRNA expression of Notch1, Notch2, Notch3, Delta1, and Jagged1 was detected in all samples of normal and neoplastic odontogenic tissues. In tooth germs, Notch receptors were expressed in odontogenic epithelium (except for inner enamel epithelium), and expression of Notch ligands was lower in inner enamel epithelium than in other epithelial components. Odontogenic mesenchymal components were weakly reactive with these Notch signaling molecules. Ameloblastomas showed expression of Notch receptors and ligands in central polyhedral neoplastic cells. Notch2, Delta1, and Jagged1 were expressed in some neoplastic cells neighboring the basement membrane. Expression of Notch receptors and ligands was not found in keratinizing cells or granular cells in ameloblastoma variants. Stromal cells were weakly reactive with these Notch signaling molecules. CONCLUSION: Expression of Notch receptors and ligands in tooth germs and ameloblastomas suggests that Notch signaling might control cell differentiation and proliferation of normal and neoplastic odontogenic epithelium.

Prion protein (PrP) in human teeth: an unprecedented pointer to PrP's function.

Although prion protein's (PrP) involvement in transmission of degenerative neurological diseases has been subjected to considerable scrutiny, its physiological role is still obscure. The distribution of PrP in dental tissues was investigated using three different methods: immunohistochemistry, cell culture, and scanning electron microscopy. PrP knockout mice were found to have marked anomalies in dentin structure. In human teeth, cementoblasts and odontoblasts showed prominent staining for PrP at levels comparable to those of nerve fibers. Epithelial rests of Malassez, which are remnants of a cell type formerly forming enamel, were also positive. Thus, all PrP-positive cells in human dentition are in some way involved in calcified tissue formation. This suggests a previously undetected function of prion protein in healthy vertebrates as evidenced by an obvious phenotype in PrP knockout mice. Periodontal and pulpal tissue exposed by disease or trauma might represent a clinically relevant entry point for prions incorporated orally and thus a possible mode of infection.

Immunohistochemical localization of alpha-Smooth muscle actin during rat molar tooth development.

The dental follicle contains mesenchymal cells that differentiate into osteoblasts, cementoblasts, and fibroblasts. However, the characteristics of these mesenchymal cells are still unknown. alpha-Smooth muscle actin (alpha-SMA) is known to localize in stem cells and precursor cells of various tissues. In the present study, to characterize the undifferentiated cells in the dental follicle, immunohistochemical localization of alpha-SMA was examined during rat molar tooth development. Rat mandibles were collected at embryonic days (E) 15-20 and postnatal days (P) 7-28. Immunohistochemical stainings for alpha-SMA, periostin, Runt-related transcription factor-2 (Runx2), tissue nonspecific alkaline phosphatase (TNAP), and bone sialoprotein (BSP) were carried out using paraffin-embedded sections. alpha-SMA localization was hardly detected in the bud and cap stages. At the early bell stage, alpha-SMA-positive cells were visible in the dental follicle around the cervical loop. At the late bell to early root formation stage (P14), these cells were detected throughout the dental follicle, but they were confined to the apical root area at P28. Double immunostaining for alpha-SMA and periostin demonstrated that alpha-SMA-positive cells localized to the outer side of periostin-positive area. Runx2-positive cells were visible in the alpha-SMA-positive region. TNAP-positive cells in the dental follicle localized nearer to alveolar bone than Runx2-positive cells. BSP was detected in osteoblasts as well as in alveolar bone matrix. These results demonstrate that alpha-SMA-positive cells localize on the alveolar bone side of the dental follicle and may play a role in alveolar bone formation.

The dentin sialoprotein (DSP) expression in rat tooth germs following fluoride treatment: an immunohistochemical study.

UNLABELLED: Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications. OBJECTIVE: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride. DESIGN AND METHODS: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry. RESULTS: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (F(L)) but not in the group supplemented with the high fluoride concentration (F(H)). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong. CONCLUSIONS: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs.

Collagen analysis in human tooth germ papillae.

The extracellular matrix (ECM) performs a very important role in growth regulation and tissue differentiation and organization. In view of this, the purpose of this study was to analyze the collagen, the major organic component of dental pulp ECM, in papillae of human tooth germs in different developmental phases. The maxillas and mandibles of 9 human fetuses ranging from 10 to 22 weeks of intrauterine life were removed and 16 tooth germs (1 in the cap stage, 8 in the early bell stage and 7 in the late bell stage) were obtained. The pieces were processed for histological analysis and stained with hematoxylin-eosin, Masson's Trichrome and picrosirius staining technique. Both types of collagen in the dental papilla were only detected by the picrosirius staining technique under polarized light microscopy. Type III collagen was detected in all specimens. Type I collagen was present in focal areas of the dental papilla only in some specimens. In conclusion, the findings of this study showed that type III collagen is a regular component of the papillae of human tooth germs whereas type I collagen is present in a significantly lesser amount.

Vascular endothelial growth factor (VEGF) in human tooth germ center.

Many oncogenis and tumour suppressor genes found inside normal and pathological cells are fundamental for the processes of development, proliferation and tissue differentiation. The purpose of our study is to show the presence and a possible relationship of the VEGF protein during different phases of the development of human dental germ centers. After cephalometric investigation in 8 orthodontic patients with a mean age of 13 years, (4 females and 4 males), hyperdivergence of the third molars were extracted. The 40 surgical samples were tested with monoclonal human anti-VEGFs antibodies carrying out a semi-quantitative analysis to look for a positive reaction. Reaction for anti-VEGF antibodies was detected in normal embryological tissues and in microvessels near odontogenic cells. During different phases of embryologic development of the dental bud our search showed intracytoplasmatic positive immunoreactions both in the ameloblastic and odontoblastic cells. Additionally, a positive reaction was observed for the VEGF protein in the cells of the stellate reticulum and in those endothelial tissue surrounding the microvessels in all the samples examined.

The expression pattern of hyaluronan synthase during human tooth development.

In previous studies, hyaluronan (HA) and its major cell surface receptor CD44 have been suggested to play an important role during tooth development. HA synthases (HASs) are the enzymes that polymerize hyaluronan. Data on the expression pattern of HASs during tooth development is lacking and the aim of the present study was to investigate the localisation of HAS by immunohistochemistry in human tooth germs from different developmental stages. The distribution pattern of HAS in the various tissues of the "bell stage" tooth primordia corresponded to that of hyaluronan in most locations: positive HAS immunoreactivity was observed in the dental lamina cells, inner- and outer-enamel epithelium. On the stellate reticulum cells, moderate HAS signal was observed, similar to the layers of the oral epithelium, where faint HAS immunoreactivity was detected. At the early phase of dental hard tissues mineralization, strong HAS immunoreactivity was detected in the odontoblasts and their processes, as well as in the secretory ameloblasts and their apical processes and also, the pulpal mesenchymal cells. The HAS signals observed in odontoblasts and ameloblasts gradually decreased with age. Our results demonstrate that hyaluronan synthesised locally by different dental cells and these results provide additional indirect support to the suggestion that HA may contribute both to the regulation of tooth morphogenesis and dental hard tissue formation.

Integrin regulatory switching in development: oscillation of beta 5 integrin mRNA expression during epithelial-mesenchymal interactions in tooth development.

Integrin receptors for extracellular matrix molecules are thought to play important roles in morphogenesis since they mediate aspects of embryonic cell adhesion and migration. Using in situ hybridization, the mRNA expression pattern of the beta 5 integrin receptor subunit was examined during murine tooth development, a classical system for studying morphogenesis. In developing tooth, high-level expression of beta 5 integrin mRNA alternates from epithelium to mesenchyme and back to epithelium. Each switch in localization occurs within one day. These results demonstrate that an integrin mRNA can be precisely and rapidly up- and down-regulated over unexpectedly short time spans, and that expression can oscillate between adjacent, interacting epithelial and mesenchymal tissues during morphogenesis. This rapid modulation of mRNA expression suggests a potential regulatory role for the beta 5 integrin receptor in morphogenesis.

Ameloblastin expression in rat incisors and human tooth germs.

We recently identified ameloblastin as an ameloblast-specific gene product from a rat incisor cDNA library (Krebsbach et al., J. Biol. Chem. 271: 4431-4435, 1996). Here we report the developmental pattern of expression of ameloblastin in rat incisors and human tooth germs as visualized by in situ hybridization and immunochemistry. Compared to the expression of amelogenin, the major ameloblast product, ameloblastin mRNA was more widely expressed in ameloblasts from the presecretory to the late maturation stage of development. Ameloblastin mRNA was first observed in the juxtanuclear cytoplasm or presecretory stage ameloblasts, gradually increased in the distal cytoplasm of secretory stage ameloblasts and was found throughout the cytoplasm of early to late maturation stage ameloblasts. The immunostaining of ameloblastin, using a monospecific antibody raised against a recombinant protein, showed intense reactivity in Tomes' processes of secretory stage ameloblasts and surrounding enamel. The immunoreaction was concentrated in the juxtanuclear cytoplasm of late maturation stage ameloblasts. High-resolution colloidal gold immunocytochemistry established the presence of ameloblastin antigenicity in the Golgi apparatus, secretory granules in Tomes' process and enamel. Human tooth germs in early to late bell stage also expressed ameloblastin mRNA and ameloblastin antigenicity in the ameloblasts. Western blot analysis of protein extracts from rat incisor tissues indicated that ameloblastin can be found in the enamel epithelial tissue and in mineralized enamel, as well as in the EDTA decalcification solution. These data indicate that ameloblastin is an ameloblast secretory product which is sequentially expressed from the presecretory to the late maturation stage in rat and human teeth. This unique developmental pattern suggests that ameloblastin may have a broader role in amelogenesis than amelogenin and tuftelin.

Neurotrophins in odontogenesis.

Neurotrophins (NTFs) are a family of structurally related proteins with specific effects on the developing nervous system and a wide range of non-neuronal differentiating cells. To date, four NTFs have been characterized: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). To perform their biological effects, the NTFs must bind to appropriate receptors on the surface of responsive cells. High- and low-affinity receptors for NTFs have been identified. The high-affinity receptors are members of the trk protein tyrosine kinase receptor family. The low-affinity neurotrophin receptor gp75NTFR is a common receptor for all NTFs. Here we summarize some of our previous findings on the expression patterns of NGF, gp75NTFR, TrkB, and TrkC in the developing molar tooth of the rat. Both NGF and gp75NTFR are localized in dental epithelium and mesenchyme but often their expression patterns differ. Concomitant expression of NGF and gp75NTFR in mesenchyme is correlated with odontoblast differentiation. The trkB and trkC receptors show distinct cell-specific expression patterns in developing tooth, suggesting that other NTFs, apart from NGF, may be involved in odontogenesis. These data demonstrate that NTFs participate in the cascade of molecular events that direct tooth development, and support the notion that NTFs may have multiple and distinct roles in dental tissues.

Early dental epithelial transcription factors distinguish ameloblastoma from keratocystic odontogenic tumor.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from 27 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 12 sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into 2 distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX2, MSX2, DLX2, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive growth, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX2 was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription factors. In contrast, gene expression of KCOT indicates differentiation toward keratinocytes.

Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis.

Cementum-derived attachment protein (CAP) is a 56 kDa collagenous protein that promotes attachment of mesenchymal cells. Previous studies have shown that the presence of CAP is restricted to cementum in adult human tissues. In this study, we report generation of a monoclonal antibody against CAP and its use for the investigation of CAP in developing bovine tooth germs. Mice were immunized with CAP purified from bovine cementum, and a monoclonal antibody, 3G9, was produced. Immunohistochemical staining of bovine tooth germ at root forming stage using 3G9 antibody showed that the tissue distribution of CAP expression was limited to cementum matrix and cementoblasts during cementogenesis. Alveolar bone did not stain with the 3G9 antibody, whereas anti-type I collagen stained positively. CAP was purified from bovine tooth germs with immunoaffinity purification using the 3G9 antibody. Examination of the immunoaffinity-purified fraction showed that CAP existed in tooth germ as a 65 kDa protein. The protein was susceptible to bacterial collagenase. To investigate the possible biological function of CAP during cementogenesis, we isolated dental follicle cells from the bovine tooth germ, and showed that they adhered to surfaces containing CAP. These data demonstrate that CAP is expressed by bovine cementoblasts as a 65 kDa protein and that the CAP may have a function in cementogenesis.

Accelerated bone formation and increased osteoblast number contribute to the abnormal tooth germ development in parathyroid hormone-related protein knockout mice.

Our previous study showed that tooth germs at late embryonic stage [later than embryonic day 17.5 (E17.5)] and neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are compressed or penetrated by the surrounding alveolar bone tissue. In vivo and in vitro studies have shown that the development of the tooth germ proper is not disturbed, but insufficient alveolar bone resorption, due to the decreased number and hypofunction of osteoclasts, is the main cause of this abnormality. In addition to the insufficient alveolar bone resorption, progressive bone formation toward tooth germs was observed in homozygous mice, suggesting that accelerated bone formation also contributes to this abnormality. To further investigate this, homozygous mice at E14.0 and E15.5, when alveolar bone is forming, were used for histochemical and bone histomorphometric analyses. In contrast to the late embryonic stage, the alveolar bone did not yet compress developing tooth germs in homozygous mice on E14.0, but a larger amount of bone tissue was seen compared to wild-type littermates. Histomorphometric analysis of bone at E14.0 revealed that the osteoblast numbers and surfaces in the mandibles and in the bone collar of femora of homozygous mice were significantly higher than those of wild-type mice. However, unlike our previous study showing the osteoclast surface on E18.5 in homozygous mice to be significantly lower than that of wild-type mice, this study at E14.0 showed no significant difference between the two genotypes. To evaluate the amount of calcification around tooth germs, 3D images of mandibles were reconstructed from the calcein-labeled sections of the wild-type and mutant mice. Labeling was performed at E14.0, and the mice were sacrificed 1 h after the calcein injection to minimize the effect of bone resorption. Comparison of the 3D images revealed that the labeled surface was larger around developing tooth germs in homozygous mouse than in wild-type mouse. On day E15.5, osteoblasts approached the enamel organ of homozygous mice but this was not observed in wild-type mice. In this study, we report a systemic increase in osteoblast number and accelerated bone formation in homozygous PTHrP-knockout mice, both of which contribute to the abnormal tooth development.

Spectroscopic imaging of mineral maturation in bovine dentin.

Dentin is a useful model for the study of mineral maturation. Using Fourier Transform Infrared Imaging (FTIRI), we characterized distinct regions in developing dentin at 7- micro m spatial resolution. Mineral-to-matrix ratio and crystallinity in bovine dentin from cervical and incisal parts of 3rd-trimester fetal compared with one-year-old incisor crowns showed that virtually all maturation stages in dentin could be spectroscopically isolated and analyzed. In the fetal incisors, mantle and circumpulpal dentin presented distinct patterns of mineral maturation. Gradients in both mineral properties examined were observed at the mineralization front and at the dentino-enamel junction.

Amino acid sequence of a major human amelogenin protein employing Edman degradation and cDNA sequencing.

The abundant hydrophobic, proline-glutamine, and histidine-rich (over 90%) amelogenins constitute the major class of proteins in forming extracellular enamel matrix. These are thought to play a major role in the structural organization and mineralization of developing enamel. The present report describes the successful sequencing of the major human amelogenin protein, by use of both Edman degradation and cDNA sequencing. When Edman degradation was used, over 75% of the primary structure of the protein was determined. This sequence was supplemented with cDNA sequencing studies, which revealed the predicted sequence of this protein. Together, they provide the complete sequence of an important human enamel protein. The information complements recent studies on bovine and human amelogenin genes. A comparison between the present results and the protein sequences predicted from the corresponding human amelogenin genomic coding regions and that of cDNA sequences of other species is described.

Stage-specific expression of decapentaplegic-Vg-related genes 2, 4, and 6 (bone morphogenetic proteins 2, 4, and 6) during human tooth morphogenesis.

Members of the decapentaplegic-Vg-related (DVR) gene family are diffusible signaling molecules regulating inductive tissue interactions during vertebrate development. Expression of DVR/bone morphogenetic protein (BMP) 2, 4, and 6 was studied in human fetal teeth. Sequential morphogenetic stage-specific studies of DVR/BMP 2 and 4 mRNA expression by in situ hybridization revealed transcripts for DVR/BMP 4 during compaction of the dental mesenchyme. In contrast, DVR/BMP 2 mRNA appeared later during tooth development and was located in differentiated cells (odontoblasts). These results were confirmed by reverse-transcription polymerase chain reaction (RT-PCR), which detected DVR/BMP 2 and 4 mRNA in human tooth-germ samples. DVR/BMP 6 protein was distributed in the early dental epithelium and, later, in pre-odontoblasts and odontoblasts, where it remained during dentin formation. These results suggest that DVR/BMP 4 is involved in the early tooth morphogenesis. DVR/BMP 6 may, in particular, be implicated in epithelial-mesenchymal interactions controlling cytodifferentiation. DVR/BMP 2 and 6 may also be involved in odontoblast secretory function. The results suggest that members of the DVR gene family may play regulatory roles during human tooth development.

Expression of basement membrane type IV collagen and type IV collagenases (MMP-2 and MMP-9) in human fetal teeth.

Formation and degradation of dental basement membrane (BM) are important for tooth development. Data on the expression of genes for type IV collagen (the major structural component of the BM) and type IV collagenases [MMP-2 (72 kDa) and MMP-9 (92 kDa)], enzymes that degrade type IV collagen during human tooth development, are lacking. We studied expression of type IV collagen and the MMP-2 and MMP-9 in human fetal teeth (from the 13th to the 20th gestational weeks, covering cap stage through early hard tissue formation). During cap and bell stages, in situ hybridization located transcripts for alpha 1 type IV collagen chain in the fibroblasts surrounding the enamel organ. No alpha 1 type IV collagen chain mRNA was detected in tooth germ epithelium or dental papilla. However, type IV collagen immunoreactivity was observed in BM underlying the dental epithelium up to the appositional stage. Transcripts for MMP-2 were located mostly in the cells of the dental papilla and follicle. Transient expression of MMP-2 mRNA was observed in the inner enamel epithelium of late cap/early bell-stage teeth. During early apposition, a high level of MMP-2 was confined to secretory odontoblasts. Transcripts for MMP-9 were detected by the sensitive reverse-transcription polymerase chain reaction (RT-PCR) in developing teeth. Thus, in dental BM, alpha 1 type IV collagen chain may be of mesenchymal cell origin. Further, MMP-2 but not MMP-9 may participate in remodeling and degradation of BM during human tooth morphogenesis.