Amelogenin-inspired peptide, calcium phosphate solution, fluoride and their synergistic effect on enamel biomimetic remineralization: an in vitro pH-cycling model.

BACKGROUND: Several methods were introduced for enamel biomimetic remineralization that utilize a biomimetic analogue to interact and absorb bioavailable calcium and phosphate ions and induce crystal nucleation on demineralized enamel. Amelogenin is the most predominant enamel matrix protein that is involved in enamel biomineralization. It plays a major role in developing the enamel's hierarchical microstructure. Therefore, this study was conducted to evaluate the ability of an amelogenin-inspired peptide to promote the remineralization potential of fluoride and a supersaturated calcium phosphate solution in treating artificially induced enamel carious lesions under pH-cycling regimen. METHODS: Fifty enamel slices were prepared with a window (4*4 mm2 ) on the surface. Five samples were set as control healthy enamel and 45 samples were subjected to demineralization for 3 days. Another 5 samples were set as control demineralized enamel and 40 enamel samples were assigned into 8 experimental groups (n=5) (P/I, P/II, P/III, P/AS, NP/I, NP/II, NP/III and NP/AS) according to peptide treatment (peptide P or non-peptide NP) and remineralizing solution used (I; calcium phosphate solution, II; calcium phosphate fluoride solution, III; fluoride solution and AS; artificial saliva). Samples were then subjected to demineralization/remineralization cycles for 9 days. Samples in all experimental groups were evaluated using Raman spectroscopy for mineral content recovery percentage, microhardness and nanoindentation as healthy, demineralized enamel and after pH-cycling. Data were statistically analysed using two-way repeated measures Anova followed by Bonferroni-corrected post hoc test for pairwise multiple comparisons between groups. Statistical significance was set at p= 0.05. Additionally, XRD, FESEM and EDXS were used for crystal orientation, surface morphology and elemental analysis after pH-cycling. RESULTS: Nanocrystals clumped in a directional manner were detected in peptide-treated groups. P/II showed the highest significant mean values in mineral content recovery (63.31%), microhardness (268.81±6.52 VHN), elastic modulus (88.74±2.71 GPa), nanohardness (3.08±0.59 GPa) and the best crystal orientation with I002/I300 (1.87±0.08). CONCLUSION: Despite pH changes, the tested peptide was capable of remineralizing enamel with ordered crystals. Moreover, the supplementary use of calcium phosphate fluoride solution with peptide granted an enhancement in enamel mechanical properties after remineralization.

Regeneration of grade 3 ankle sprain, using the recombinant human amelogenin protein (rHAM+ ) in a rat model.

Lateral ligament tears, also known as high-grade ankle sprains, are common, debilitating, and usually heal slowly. Ten to thirty percent of patients continue to suffer from chronic pain and ankle instability even after 3 to 9 months. Previously, we showed that the recombinant human amelogenin (rHAM+ ) induced regeneration of fully transected rat medial collateral ligament, a common proof-of-concept model. Our aim was to evaluate whether rHAM+ can regenerate torn ankle calcaneofibular ligament (CFL), an important component of the lateral ankle stabilizers. Right CFLs of Sabra rats were transected and treated with 0, 0.5, or 1 µg/µL rHAM+ dissolved in propylene glycol alginate (PGA). Results were compared with the normal group, without surgery. Healing was evaluated 12 weeks after treatment by mechanical testing (ratio between the right and left, untransected ligaments of the same rat), and histology including immunohistochemical staining of collagen I and S100. The mechanical properties, structure, and composition of transected ligaments treated with 0.5 µg/µL rHAM+ (experimental) were similar to untransected ligaments. PGA (control) treated ligaments were much weaker, lax, and unorganized compared with untransected ligaments. Treatment with 1 µg/µL rHAM+ was not as efficient as 0.5 µg/µL rHAM+ . Normal arrangement of collagen I fibers and of proprioceptive nerve endings, parallel to the direction of the force, was detected in ligaments treated with 0.5 µg/µL rHAM+ , and scattered arrangement, resembling scar tissue, in control ligaments. In conclusion, we showed that rHAM+ induced significant mechanical and structural regeneration of torn rat CFLs, which might be translated into treatment for grades 2 and 3 ankle sprain injuries.

Analysis of the Preserved Amino Acid Bias in Peptide Profiles of Iron Age Teeth from a Tropical Environment Enable Sexing of Individuals Using Amelogenin MRM.

The first dental proteomic profile of Iron Age individuals (ca. 2000-1000 years B.P.), collected from the site of Long Long Rak rock shelter in northwest Thailand is described. A bias toward the preservation of the positively charged aromatic, and polar amino acids is observed. It is evident that the 212 proteins identified (2 peptide, FDR <1%) comprise a palimpsest of alterations that occurred both ante-mortem and post-mortem. Conservation of amino acids within the taphonomically resistant crystalline matrix enabled the identification of both X and Y chromosome linked amelogenin peptides. A novel multiple reaction monitoring method using the sex specific amelogenin protein isoforms is described and indicate the teeth are of male origin. Functional analysis shows an enrichment of pathways associated with metabolic disorders and shows a capacity for harboring these conditions prior to death. Stable isotope analysis using carbon isotopes highlights the strongly C3 based (≈80%) diet of the Long Long Rak cemetery people, which probably comprised rice combined with protein from freshwater fish among other food items. The combination of proteomics and stable isotope analysis provides a complementary strategy for assessing the demography, diet, lifestyle, and possible diseases experienced by ancient populations.

Diversidad del ADN mitocondrial en restos óseos prehispánicos / Mitochondrial DNA diversity in prehispanic bone remains on the eastern Colombian Andes

Resumen Introducción. El ADN antiguo que se extrae de los restos óseos humanos permite analizar la composición genética de las poblaciones precolombinas y determinar las dinámicas poblacionales que dieron origen a la diversidad de las poblaciones contemporáneas. Objetivo. Determinar la diversidad genética y la relación con otras comunidades contemporáneas y antiguas de América, de los restos óseos asociados al Templo del Sol en Sogamoso, Colombia. Materiales y métodos. Se analizaron 13 individuos pertenecientes al periodo precolombino muisca (siglos IX-XVI d. C.), provenientes de los alrededores del Templo del Sol en Sogamoso, Boyacá, Andes orientales colombianos. Se amplificó el ADN mitocondrial (ADNmt) y se determinaron los polimorfismos de la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP) para los cuatro haplogrupos amerindios (A, B, C y D). Además, se amplificaron y analizaron los marcadores autosómicos, incluida la amelogenina, y los marcadores de los polimorfismos de repeticiones cortas en tándem (Short Tandem Repeat, STR) del cromosoma Y. Resultados. El haplogrupo A fue el linaje mitocondrial más frecuente en esta población, seguido de los haplogrupos B y C; no se detectó el haplogrupo D. Los análisis de variación genética indicaron una diversidad semejante a la de las poblaciones pertenecientes a la familia lingüística chibcha, contemporánea en Colombia y Centroamérica. Se logró hacer la determinación molecular del sexo de los individuos estudiados y compararla con los datos osteológicos. Con una sola excepción, los datos bioantropológicos y moleculares concordaron. Conclusiones. Estos resultados aportan nuevos elementos a la hipótesis del origen centroamericano de los grupos chibchas del altiplano cundiboyacense con base en marcadores genéticos, y permitieron establecer el sexo y las relaciones de parentesco.

Abstract Introduction: DNA extracted from ancient human bones allows to analyze the genetic makeup of preColumbian populations and to determine the dynamics that gave rise to the diversity of contemporary populations. Objective: To determine the genetic diversity of skeletal remains associated with the Templo del Sol (Sun Temple) and their relationship with other contemporary and ancient communities of America. Materials and methods: We analyzed 13 individuals belonging to the pre-Columbian Muisca Period (IX-XVI centuries AD) from the vicinities of the Templo del Sol (Sun Temple) (Sogamoso, Boyacá) in the eastern Colombian Andes. Mitochondrial DNA was amplified and RFLPs were performed in order to type the four traditional Amerindian haplogroups (A, B, C and D). In addition, autosomal markers including amelogenin and Y-chromosome STRs were amplified. Results: Among the observed mitochondrial lineages, haplogroup A was the most frequent, followed by haplogroups B and C; no evidence of haplogroup D was found. The genetic variation analysis indicated a similar diversity of pre-Columbian Muiscas to that of contemporary populations belonging to the Chibcha linguistic family from Colombia and Central America. Molecular sexing was accomplished and it was compared to osteological data. With only one exception, anthropological and molecular data were consistent. Conclusions: Our results contribute new genetic elements supporting the hypothesis of Central American origin of the Chibcha groups of the Cundiboyacense plateau, and allowed sex typing and kinship evaluations.

Cloning and Expression Analysis of Human Amelogenin in Nicotiana benthamiana Plants by Means of a Transient Expression System.

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF-MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.

Genes Involved in the Enamel Development Are Associated with Calcium and Phosphorus Level in Saliva.

Saliva components play a crucial role in the integrity of the dental enamel and in caries susceptibility. The saliva characteristics are controlled by many factors, including genetic factors. Therefore, this study aimed to evaluate the association between the genetic variations in genes expressed in enamel development with calcium and phosphorus levels in saliva. We collected 276 unrelated 12-year-old children from private and public schools. Saliva was collected for DNA extraction from oral cells and for measurement of calcium and phosphorus. Inductively coupled plasma-mass spectrometry determined calcium and phosphorus levels in whole saliva. Fifteen genetic variations in 9 genes were analyzed. The genotype was determined by real-time polymerase chain reactions. Data were analyzed using Plink with an alpha of 5%. Genetic variations in AMELX, AMNB and ESRRB were associated with the calcium level in saliva (p < 0.05). A borderline association was observed in ENAM allele distribution shown with phosphate level in saliva (p = 0.049). In conclusion, our results are the first to report that genetic variations contribute to calcium and phosphorus levels in saliva.

Promotion of enamel caries remineralization by an amelogenin-derived peptide in a rat model.

OBJECTIVE: An amelogenin-derived peptide has been shown to promote remineralization of demineralized enamel in an in vitro model of initial caries induced by pH cycling. The present study examines whether the peptide exerts similar effects within the complex oral environment in vivo. DESIGN: Specific pathogen-free Sprague-Dawley rats (n=36) were infected with Streptococcus mutans, given ad libitum access to Diet 2000 and drinking water supplemented with sucrose (10%, w/v), and then randomly divided into three groups treated with 25µM peptide solution, 1g/L NaF or deionized water. Molar teeth were swabbed twice daily with the respective solutions for 24days. Then animals were killed, their jaws were removed and caries lesions were analyzed using the quantitative light-induced fluorescence-digital (QLF-D) technique to measure changes in mineral content. To verify QLF-D results, caries were scored for lesion depth and size using the Keyes method, and analyzed using polarized light microscopy (PLM). RESULTS: Mineral gain was significantly higher in teeth treated with peptide or NaF than in teeth treated with water (p<0.05), based on the QLF-D results (ΔF and ΔQ). Incidence of smooth-surface and sulcal caries based on Keyes scores was similar in rats treated with peptide or NaF, and significantly lower in these groups than in rats treated with water (p<0.05). Lesions on teeth treated with peptide or NaF were shallower, based on PLM. No significant differences were observed between molar enamel caries treated with peptide or NaF. CONCLUSIONS: This amelogenin-derived peptide can promote remineralization in a rat caries model, indicating strong potential for clinical use.

[Mitochondrial DNA diversity in prehispanic bone remains on the eastern Colombian Andes].

INTRODUCTION: DNA extracted from ancient human bones allows to analyze the genetic makeup of pre-Columbian populations and to determine the dynamics that gave rise to the diversity of contemporary populations. OBJECTIVE: To determine the genetic diversity of skeletal remains associated with the Templo del Sol (Sun Temple) and their relationship with other contemporary and ancient communities of America. MATERIALS AND METHODS: We analyzed 13 individuals belonging to the pre-Columbian Muisca Period (IX-XVI centuries AD) from the vicinities of the Templo del Sol (Sun Temple) (Sogamoso, Boyacá) in the eastern Colombian Andes. Mitochondrial DNA was amplified and RFLPs were performed in order to type the four traditional Amerindian haplogroups (A, B, C and D). In addition, autosomal markers including amelogenin and Y-chromosome STRs were amplified. RESULTS: Among the observed mitochondrial lineages, haplogroup A was the most frequent, followed by haplogroups B and C; no evidence of haplogroup D was found. The genetic variation analysis indicated a similar diversity of pre-ColumbianMuiscas to that of contemporary populations belonging to the Chibcha linguistic family from Colombia and Central America. Molecular sexing was accomplished and it was compared to osteological data. With only one exception, anthropological and molecular data were consistent. CONCLUSIONS: Our results contribute new genetic elements supporting the hypothesis of Central American origin of the Chibcha groups of the Cundiboyacense plateau, and allowed sex typing and kinship evaluations.

Full-length amelogenin influences the differentiation of human dental pulp stem cells.

BACKGROUND: Amelogenin is an extracellular matrix protein well known for its role in the organization and mineralization of enamel. Clinically, it is used for periodontal regeneration and, due to its finding also in predentin and intercellular spaces of dental pulp cells, it has recently been suggested for pulp capping procedures. The aim of this study was to analyse in vitro the effect of the recombinant human full-length amelogenin on the growth and differentiation of human dental pulp stem cells (hDPSCs). METHODS: Human DPSCs were treated with a supplement of amelogenin at a concentration of 10 ng/ml, 100 ng/ml and 1000 ng/ml. The groups were compared to the unstimulated control in terms of cell morphology and proliferation, mineralization and gene expression for ALP (alkaline phosphatase), DMP1 (dentin matrix protein-1) and DSPP (dentin sialophosphoprotein). RESULTS: Amelogenin affects hDPSCs differently than PDL (periodontal ligament) cells and other cell lines. The proliferation rate at two weeks is significantly reduced in presence of the highest concentration of amelogenin as compared to the unstimulated control. hDPSCs treated with low concentrations present a downregulation of DMP1 and DSPP, which is significant for DSPP (p = 0.011), but not for DMP1 (p = 0.395). CONCLUSIONS: These finding suggest that the role of full-length amelogenin is not restricted to participation in tooth structure. It influences the differentiation of hDPSC according to various concentrations and this might impair the clinical results of pulp capping.

[DNA extraction and sex determination for human teeth dated 3000 years ago unearthed in Xi'an].

OBJECTIVE: To extracted DNA from ancient human teeth dated 3000 years ago unearthed in Xi'an and determine the genders for the individuals. METHODS: Thirty five ancient human teeth were studied. A 'Reverse-root-canal' technique and a Chelex-100 solution were used to extract the DNA. Specific primers for Amelogenin gene were designed for PCR amplification. RESULTS: Genomic DNA was successfully extracted from 30 samples, for which 8 were determined to be males and 22 were females. CONCLUSION: The 'Reverse-root-canal' technique may be used for extracting DNA from ancient human teeth. Genetics method can supplement physical anthropology for determination of sex for ancient samples.

DNA extraction and sex determination for human teeth dated 3000 years ago unearthed in Xi'an / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To extracted DNA from ancient human teeth dated 3000 years ago unearthed in Xi'an and determine the genders for the individuals.</p><p><b>METHODS</b>Thirty five ancient human teeth were studied. A 'Reverse-root-canal' technique and a Chelex-100 solution were used to extract the DNA. Specific primers for Amelogenin gene were designed for PCR amplification.</p><p><b>RESULTS</b>Genomic DNA was successfully extracted from 30 samples, for which 8 were determined to be males and 22 were females.</p><p><b>CONCLUSION</b>The 'Reverse-root-canal' technique may be used for extracting DNA from ancient human teeth. Genetics method can supplement physical anthropology for determination of sex for ancient samples.</p>

[Effect of fluoride on the expression of MMP-20/TIMP-2 in ameloblast of rat incisor and the antagonistic effect of melatonin against fluorosis].

PURPOSE: To study the effect of concentration of fluoride on the expression of matrix metalloproteinase-20(MMP-20) and tissue inhibitors of metalloproteinase-2 (TIMP-2) in the ameloblast of rat incisor,and explore the formation mechanism of dental fluorosis. By comparing the different expression of MMP-20,TIMP-2 between fluoride group and the melatonin group,to decide whether melatonin has antagonitic effect on dental fluorosis. METHODS: Forty Wistar rats were randomly divided into 6 groups. The groups were as follows: control group,low-dose group, high-dose group,normal saline group and melatonin group. The animals were sacrificed 10 weeks after treatment. HE and immunohistochemical staining were used to observe the changes of ameloblasts and the expression of MMP-20 and TIMP-2 in rat incisors. MetaMorph microscope images analysis system was used to analyze the images, and SPSS12.0 software package was used for data analysis. RESULTS: The surface of rat incisors fed with fluoride had chalky color change and cross stritations could be seen on the enamel surface.In the fluoride group,the ameloblasts were disarranged, cells arranged in multi-layer,even showing vacuolar change.The changes in the high-dose group was severer than the low-dose group. MMP-20, TIMP-2 were expressed both in the secretory ameloblasts, and in the odontoblasts.The expression of MMP-20 in rat's ameloblasts in the experimental group was significantly lower than that in the control group (P < 0.01); and no significant difference was found between the low-dose and high-dose groups(P > 0.05). The difference of expression of TIMP-2 was not significant among all the groups. The difference of expression of MMP-20 and TIMP-2 was not significant between the melatonin and the fluoride groups. CONCLUSIONS: The excessive fluoride can inhibit the secretion of MMP-20 and disturb the balance between MMP-20 and TIMP-2,which lead to the delay of amelogenin removal and enamel demineralization. Melatonin has no antagonistic effect on the dental fluorosis. Supported by National Natural Science Foundation of China (30600509) and Natural Science Foundation of Liaoning Province (20102278).

Differentiation of human ameloblast-lineage cells in vitro.

Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19-24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5-15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering.

Transcription factor sumoylation and factor YY1 serve to modulate mouse amelogenin gene expression.

Amelogenin proteins are essential in the control of enamel biomineralization and the amelogenin gene therefore is spatiotemporally regulated to ensure proper amelogenin protein expression. In this study, we examined the role of sumoylation to alter CCAAT/enhancer-binding protein alpha (C/EBPalpha) activity, and performed a search using a protein/DNA array system for other proteins that act co-operatively with C/EBPalpha to alter amelogenin expression. We observed that C/EBPalpha was modified by sumoylation, and that this modification played an indirect inhibitory role on the regulation of C/EBPalpha activity which appeared to act through other transcription factors. The protein/DNA array allowed us to single out the transcription factor, YY1, which acts in the absence of direct DNA binding to repress both the basal amelogenin promoter activity and C/EBPalpha-mediated transactivation. Taken together, these pathways may account for part of the physiological modulation of the amelogenin gene expression in accordance with tooth developmental and enamel biomineralization requirements.

Enamel matrix protein interactions.

UNLABELLED: The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self-assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein-protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins. INTRODUCTION: The purpose of this study was to identify and catalog additional proteins that play a significant role in enamel formation. MATERIALS AND METHODS: We used the yeast two-hybrid assay to identify protein partners for amelogenin, ameloblastin, and enamelin. Once identified, RT-PCR was used to assess gene transcription of these newly identified and potential "enamel" proteins in ameloblast-like LS8 cells. RESULTS: In the context of this yeast assay, we identified a number of secreted proteins and integral membrane proteins that interact with amelogenin, ameloblastin, and enamelin. Additionally, proteins whose functions range from the inhibition of soft tissue mineralization, calcium ion transport, and phosphorylation events have been identified as protein partners to these enamel matrix proteins. For each protein identified using this screening strategy, future studies are planned to confirm this physiological relationship to biomineralization in vivo. CONCLUSION: Identifying integral membrane proteins of the secretory surface of ameloblast cells (Tomes' processes) and additional enamel matrix proteins, based on their abilities to interact with the most abundant enamel matrix proteins, will better define the molecular mechanisms of enamel formation at its most rudimentary level.

Design of a multiplex PCR for genotyping 16 short tandem repeats in degraded DNA samples.

The molecular genotyping of individuals and reconstruction of kinship through short and high polymorphic DNA markers, so-called short tandem repeats (STR), has become an important and efficient method in anthropology and forensic science. The here introduced experimental design describes a multiplex PCR capable of simultaneously amplifying 16 STRs and the sex determinant locus amelogenin in a short fragment lengths range from 84 bp to 275 bp. Thus, the design depends predominantly on the routines for DNA typing of historical samples with highly degraded ancient DNA. It is shown, that the newly designed multiplex PCR is suitable for successful typing of both forensic and historical material.

Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line.

Tooth development was cooperatively regulated by the epithelial ameloblasts and mesenchymal odontoblasts. Ameloblasts secrete enamel matrix, critical for enamel formation. While there are several reports about establishment of immortalized ameloblast-like cells by introducing viral oncogene, we tried to establish a spontaneously immortalized ameloblast-lineage cell line, maintaining the cell type specific character, including the ability to induce in vitro bio-mineralization. The established cell line (ameloblast-lineage cell; ALC) maintained the expression of several ameloblast specific genes (Amelogenin, Tuftelin, and Enamelin) in long-term culture. They formed calcified nodules after the induction by medium switching from SMEM to DMEM, having high-level alkaline-phosphatase activity. The size and number of calcified nodule formation were enhanced by TGF-beta treatment. Six weeks after sub-cutaneous implantation of ALC to athymic nude mice, we ectopically observed enamel epithelium like structure formation, chondrogenesis, and calcification. These data indicate that ALC is a useful experimental tool to analyze ameloblast character.

Polypeptides translated from alternatively spliced transcripts of the amelogenin gene, devoid of the exon 6a, b, c region, have specific effects on tooth germ development in culture.

Recombinant proteins have been produced from cDNAs corresponding to alternatively spliced transcripts comprised from exons 2,3,4,5,6d,7 and 2,3,5,6d,7 of the rat amelogenin gene. These peptides, designated as [A + 4] and [A - 4], respectively, induce embryonic muscle fibroblasts in culture in vitro to express proteins characteristic of the chondrogenic and osteogenic phenotypes, and in matrix-supported implants into rat muscle, in vivo, induce typical bone matrix proteins. The aim of the present work was to examine the potential role of these proteins on the development of odontogenic tissue. The lower first molars were collected from Charles River CD-1 mice at postnatal days 1 and 2 and were grown on semisolid, serum-free medium supplemented with ascorbic and retinoic acids and transferin. The peptides were added to the serum-free media at 10 ng/ml. As controls, the medium was either 20% fetal bovine serum or the supplemented serum-free medium without either amelogenin peptide. The tooth germs were cultured for 6 days, then fixed and paraffin embedded by standard procedures. The tissue blocks were serially sectioned and stained with hematoxylin-eosin (H&E), or antibodies to collagen 1 (Col1), phosphophoryn (DMP2), or cementum attachment protein (CAP). CAP, DMP2, and Col1 expression was enhanced by the addition of the amelogenin peptides, as compared to the 0% fetal bovine serum (FBS) controls, but the peptides showed different effects. Expression of DMP2, characteristic of dentin matrix, was upregulated by [A + 4], whereas CAP, characteristic of cementum, was upregulated by [A - 4]. Since the recombinant peptides are active, their corresponding tissue forms may be important in the stimulation of mesenchymal tissue differentiation. Thus, these specific amelogenin proteins may be involved in tooth morphogenesis.

Effects of amelogenin on the transforming surface microstructures of Bioglass in a calcifying solution.

Topographies of a bioactive glass (45S5 type Bioglass(R)) during 0-4 h of immersion in a supersaturated calcifying solution (SCS) and the SCS containing recombinant porcine amelogenin rP172 (SCS(rP172)) were observed by atomic force microscopy. Other techniques including X-ray diffraction, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, and transmission electron microscopy were used for some complementary microstructural investigations. The smooth Bioglass surface changed to be very rough after 0.5 h of SCS immersion because of glass network dissolution. Spherical silica-gel particles with diameters of 150-300 nm consisting of substructures of 20-60 nm across had formed on the sample surfaces after 1 h of SCS immersion. The chemisorption of amorphous calcium phosphate and crystallization of nanophase apatite were seen to occur epitaxially on the silica-gel structures during 1-4 h of SCS immersion. During the first 0.5 h of SCS(rP172) immersion, more than 95% of rP172 protein in solution was adsorbed onto the sample surfaces and generated spherical assemblies of 10-60 nm diameters. During 0.5-4 h of SCS(rP172) immersion, the protein assemblies of rP172 remarkably induced the formation of orientated silica-gel plates (approximately 100-nm wide and 50-nm thick) and subsequently of long and thin apatite needle crystals. The recombinant amelogenin rP172-modulated apatite crystals resembled those formed in the early stage of tooth enamel biomineralization, suggesting the functional roles of amelogenins during the oriented growth of enamel crystallites and a great potential for amelogenins in applications designed to fabricate enamel-like calcium phosphate biomaterials.

Reconstruction of kinship by fecal DNA analysis of orangutans.

Genetic analysis is a useful tool for assigning biological relationships. Thus, it will improve genetic management of wild animal populations and breeding colonies. Kinship analysis will give new insights into the behavior, sociobiology and genetic management of orangutans. In this study, chromosomal DNA from orangutan (Pongo pygmaeus ssp.) was extracted from excrements. Feces samples were screened for up to nine microsatellite markers from related zoo populations of orangutans (Pongo pygmaeus ssp.) kept at the Zoological Garden Berlin and the Zoological Garden Heidelberg, Germany. Family structures are documented in the "International Studybook of the Orangutan" (Perkins 1995) and the "Europäisches Erhaltungszucht Programm 1998" (Becker 1998). To examine whether human short tandem repeat loci (STR) are suitable for the reconstruction of kinship in orangutans, nine STRs, commonly used in forensic studies and the amelogenin system, were amplified in a multiplex-PCR approach (AmpFlSTR Profiler Plus). We were able to show that five of the nine human autosomal STRs in question amplified successfully in orangutans. Thus, we could reconstruct kinship structures of the Berlin and Heidelberg populations.