Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and m... more Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and mesenchymal tissues. Many molecules, including growth factors, transcription factors, extracellular matrices, cell surface receptors, and matrix degrading enzymes, have been found to be involved in this process. To investigate the molecular mechanism responsible for morphogenesis of the circumvallate papilla/von Ebners ’ gland complex, we examined the expres-sion patterns of selected cell adhesion molecules, extracellular matrix molecules, innervation and cell division in the circumvallate papilla of mouse embryos from embryonic day 11.5 (E11.5) to E14. At E11.5-E13.5, the lingual epithelium, the site where the circumvallate papilla will develop, is negative for BrdU labeling. At E14-E15, we detected cell division in the papillary area, especially in the epithelial invagination where von Ebners ’ minor salivary gland will form. The basement membrane component, laminin, is expressed as a c...
Introduction-Recombinant DNA produced amelogenin protein was compared to calcium hydroxide in a s... more Introduction-Recombinant DNA produced amelogenin protein was compared to calcium hydroxide in a study of immature apex closure conducted in 24 young mongrel dogs. -Root canals of maxillary and mandibular right premolars (n = 240) were instrumented and left open for 14 days. Canals were cleansed, irrigated and split equally for treatment with recombinant mouse amelogenin (n = 120) or calcium hydroxide (n = 120). Results-After 1, 3, and 6 months, the animals were sacrificed and the treated teeth recovered for histological assessment and immunodetection of protein markers associated with odontogenic cells. After 1 month, amelogenin-treated canals revealed calcified tissue formed at the apical foramen and a pulp chamber containing soft connective tissue and hard tissue; amelogenin-treated canals assessed after 3 and 6 month intervals further included apical tissue functionally attached to bone by a periodontal ligament. In contrast, calcified apical tissue was poorly formed in the calcium hydroxide group and soft connective tissue within the pulp chamber was not observed. Conclusions-The findings from this experimental strategy suggest recombinant amelogenin protein can signal cells to enhance apex formation in non-vital immature teeth and promote soft connective tissue regeneration.
We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during t... more We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2010
Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel bi... more Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the (1)H-(15)N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the (1)H-(15)N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12 and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of (1)H-(15)N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of (1)H-(15)N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein.
Positional information on tooth morphogenesis is investigated by the identification of when and w... more Positional information on tooth morphogenesis is investigated by the identification of when and where phenotypic markers are expressed during odontogenesis. This temporal and positional information is correlated with the instructive and permissive signaling required for both dentinogenesis and amelogenesis. Of particular interest is the establishment of a map for the cranial neural crest-derived dental papilla ectomesenchyme and the odontoblast cell lineages. The expression of ectomesenchymederived cytotactin, dentin phosphoprotein, and epithelial-derived enamel proteins was studied in mice using embryonic, fetal, and postnatal mandibular first molar tooth organ development. This review summarizes the observations in the context of instructive epithelial-mesenchymal interactions and suggests that amelogenesis imperfecta and dentinogenesis imperfecta may in part be explained by alterations in these differentiation markers. Recombinant DNA methods should facilitate future investigations of these inherited dental disorders.
Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein ... more Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein indispensable for enamel formation. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) induces osteogenesis in various cell types. Previously, we demonstrated that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists. There is a reciprocal relationship between osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). Wnt10b-mediated activation of canonical Wnt signaling has been shown to regulate mesenchymal stem cell fate. Using the bipotential bone marrow stromal cell line ST2, we have demonstrated that LRAP activates the canonical Wnt/βcatenin signaling pathway. A specific Wnt inhibitor sFRP-1 abolishes the effect of LRAP on the stimulation of osteogenesis and the inhibition of adipogenesis of ST2 cells. LRAP treatment elevates the Wnt10b expression level whereas Wnt10b knockdown by siRNA abrogates the effect of LRAP. We show here that LRAP promotes osteogenesis of mesenchymal stem cells at the expense of adipogenesis through upregulating Wnt10b expression to activate Wnt signaling.
Fincham AG: Hertwig's epithelial root sheath differentiation and initial cementum and hone format... more Fincham AG: Hertwig's epithelial root sheath differentiation and initial cementum and hone formation during long-term organ culture of mouse mandihular first molars using serumless. chemically-defined medium.
Positional information on tooth morphogenesis is investigated by the identification of when and w... more Positional information on tooth morphogenesis is investigated by the identification of when and where phenotypic markers are expressed during odontogenesis. This temporal and positional information is correlated with the instructive and permissive signaling required for both dentinogenesis and amelogenesis. Of particular interest is the establishment of a map for the cranial neural crest-derived dental papilla ectomesenchyme and the odontoblast cell lineages. The expression of ectomesenchymederived cytotactin, dentin phosphoprotein, and epithelial-derived enamel proteins was studied in mice using embryonic, fetal, and postnatal mandibular first molar tooth organ development. This review summarizes the observations in the context of instructive epithelial-mesenchymal interactions and suggests that amelogenesis imperfecta and dentinogenesis imperfecta may in part be explained by alterations in these differentiation markers. Recombinant DNA methods should facilitate future investigations of these inherited dental disorders.
International Journal of Developmental Biology, 2002
Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and m... more Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and mesenchymal tissues. Many molecules, including growth factors, transcription factors, extracellular matrices, cell surface receptors, and matrix degrading enzymes, have been found to be involved in this process. To investigate the molecular mechanism responsible for morphogenesis of the circumvallate papilla/von Ebners' gland complex, we examined the expression patterns of selected cell adhesion molecules, extracellular matrix molecules, innervation and cell division in the circumvallate papilla of mouse embryos from embryonic day 11.5 (E11.5) to E14. At E11.5-E13.5, the lingual epithelium, the site where the circumvallate papilla will develop, is negative for BrdU labeling. At E14-E15, we detected cell division in the papillary area, especially in the epithelial invagination where von Ebners' minor salivary gland will form. The basement membrane component, laminin, is expressed a...
A team of senior scientists was formed in 2006 to create a blueprint for the regeneration of whol... more A team of senior scientists was formed in 2006 to create a blueprint for the regeneration of whole human teeth along with all of the supporting structure of the dentition. The team included experts from diverse fields, each with a reputation for stellar accomplishment. Participants attacked the scientific issues of tooth regeneration but, more importantly, each agreed to work collaboratively with experts from other disciplines to form a learning organization. A commitment to learn from one another produced a unique interdisciplinary and multidisciplinary team. Inspired by the Kennedy space program to send a man to the moon, with its myriad of problems and solutions that no one discipline could solve, this tooth regeneration team devised an ambitious plan that sought to use stem cell biology, engineering, and computational biology to replicate the developmental program for odontogenesis. In this manner, team members envisioned a solution that consisted of known or knowable fundamenta...
Journal of Molecular and Engineering Materials, 2016
Surgical site infection is a common cause of post-operative morbidity, often leading to implant l... more Surgical site infection is a common cause of post-operative morbidity, often leading to implant loosening, ultimately requiring revision surgery, increased costs and worse surgical outcomes. Since implant failure starts at the implant surface, creating and controlling the bio-material interface will play a critical role in reducing infection while improving host cell-to-implant interaction. Here, we engineered a biomimetic interface based upon a chimeric peptide that incorporates a titanium binding peptide (TiBP) with an antimicrobial peptide (AMP) into a single molecule to direct binding to the implant surface and deliver an antimicrobial activity against S. mutans and S. epidermidis, two bacteria which are linked with clinical implant infections. To optimize antimicrobial activity, we investigated the design of the spacer domain separating the two functional domains of the chimeric peptide. Lengthening and changing the amino acid composition of the spacer resulted in an improvemen...
We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during t... more We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.
Enamel, which covers the anatomical crown of the tooth, is the hardest tissue in human body. Supp... more Enamel, which covers the anatomical crown of the tooth, is the hardest tissue in human body. Supported by a soft but tough dentin structure, the tooth is an advanced nanocomposite that can endure mastication stresses throughout a lifetime. A detail understanding the structure of the tooth, and sepecifically detin-enamel junction (DEJ), not only provides a sound basis for a model for synthetic dental restoration, but also provides lessons from nature on biomimetic regeneration with mechanical integrity. Enamel is a non-growing mineralized tissue and is subjected to most mechanical abuse. Dentin-enamel junction plays a critical role of distributing load between two very dissimilar materials - enamel and dentin. Bulk scale mechanical tests have shown that induced cracks on enamel tend to be arrested DEJ. Furthermore, nanoindentation measurements have also shown that there is a gradual decrease in hardness from enamel to dentin in the DEJ zone suggests a strong mechanical coupling in bo...
The aim of the present study was to select the optimal concentration of TiF4 solution to facilita... more The aim of the present study was to select the optimal concentration of TiF4 solution to facilitate the remineralization of early dentine caries lesions. Sixty human dentine specimens were cut and randomly divided into 6 groups (1%, 2%, 3%, 4% TiF4 groups, 2.712% NaF group and distilled deionized water (DDW) control group). Artificial dentine caries-like lesions were created. After being subjected to fluoride treatment and immersed in remineralizing solution for 2weeks, the specimens were observed by microCT, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Data were analysed using linear regression analysis (P<0.05). The lesion depths of the specimens treated by 2% TiF4 solution were statistically less than those of the other groups. Further, the greyscale values of these lesion areas were greater. The 3% and 4% TiF4 solutions caused further lesion demineralization. The 2.712% NaF solution seemed to be detrimental to remineralization during the expe...
Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The... more Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3'-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins....
Silver nanoparticles (AgNP) are promising candidates for fighting drug-resistant infections becau... more Silver nanoparticles (AgNP) are promising candidates for fighting drug-resistant infections because of their intrinsic antimicrobial effect. The design of high-yield antimicrobial molecules may inadvertently cause variation in host cells' biological responses. While many factors affect AgNPs' efficacy, their surface is exposed to the biological environment and thus plays a critical role in both the preservation of antimicrobial efficacy against pathogens and the modulation of host cells cytotoxicity. This work investigated an engineered biomimetic interface approach to controlling AgNP surface properties to provide them a competitive advantage in a biological environment. Here, a fusion protein featuring a silver-binding peptide (AgBP) domain was engineered to enable self-assembly and track assembly by a green fluorescent protein (GFP) reporter. Following AgNP functionalisation with GFP-AgBP, their antimicrobial and cytotoxic properties were evaluated. GFP-AgBP binding affinity to AgNPs was evaluated using localized surface plasmon resonance sensing. The GFP-AgBP biomimetic interface on AgNPs' surfaces provided sustained antibacterial efficacy at low concentrations based on bacterial growth inhibition assays. Viability and cytotoxicity measurements in fibroblast cells exposed to GFP-AgBP proteinfunctionalised AgNPs showed significant improvement compared to controls. Biointerface engineering offers promise towards tailoring AgNP antimicrobial efficacy while addressing safety concerns to maintain optimum cellular interactions.
To investigate the possible biological mechanism of dental fluorosis at a molecular level. Cultur... more To investigate the possible biological mechanism of dental fluorosis at a molecular level. Cultured LS8 were incubated with serum-free medium containing selected concentrations of NaF (0 ∼ 2 mM) for either 24 or 48 h. Subcellular microanatomy was characterized using TEM; meanwhile, selected biomolecules were analysed using various biochemistry techniques. Transient transfection was used to modulate a molecular pathway for apoptosis. Apoptosis of LS8 was induced by NaF treatment that showed both time and concentration dependency. The activity of caspase-3, -8, -9 was found to be increased with NaF in a dose-dependent manner. Western blot revealed that the protein expression of p-ERK and p-JNK were decreased, while the expression of p-P38 was increased. Inhibition of the p-ERK and p-JNK pathways resulted in a similar decrease for caspase-3. During NaF-induced apoptosis of LS8, p-ERK and p-JNK were closely associated with induction of apoptosis, which might be a mechanism of dental flu...
Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and m... more Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and mesenchymal tissues. Many molecules, including growth factors, transcription factors, extracellular matrices, cell surface receptors, and matrix degrading enzymes, have been found to be involved in this process. To investigate the molecular mechanism responsible for morphogenesis of the circumvallate papilla/von Ebners ’ gland complex, we examined the expres-sion patterns of selected cell adhesion molecules, extracellular matrix molecules, innervation and cell division in the circumvallate papilla of mouse embryos from embryonic day 11.5 (E11.5) to E14. At E11.5-E13.5, the lingual epithelium, the site where the circumvallate papilla will develop, is negative for BrdU labeling. At E14-E15, we detected cell division in the papillary area, especially in the epithelial invagination where von Ebners ’ minor salivary gland will form. The basement membrane component, laminin, is expressed as a c...
Introduction-Recombinant DNA produced amelogenin protein was compared to calcium hydroxide in a s... more Introduction-Recombinant DNA produced amelogenin protein was compared to calcium hydroxide in a study of immature apex closure conducted in 24 young mongrel dogs. -Root canals of maxillary and mandibular right premolars (n = 240) were instrumented and left open for 14 days. Canals were cleansed, irrigated and split equally for treatment with recombinant mouse amelogenin (n = 120) or calcium hydroxide (n = 120). Results-After 1, 3, and 6 months, the animals were sacrificed and the treated teeth recovered for histological assessment and immunodetection of protein markers associated with odontogenic cells. After 1 month, amelogenin-treated canals revealed calcified tissue formed at the apical foramen and a pulp chamber containing soft connective tissue and hard tissue; amelogenin-treated canals assessed after 3 and 6 month intervals further included apical tissue functionally attached to bone by a periodontal ligament. In contrast, calcified apical tissue was poorly formed in the calcium hydroxide group and soft connective tissue within the pulp chamber was not observed. Conclusions-The findings from this experimental strategy suggest recombinant amelogenin protein can signal cells to enhance apex formation in non-vital immature teeth and promote soft connective tissue regeneration.
We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during t... more We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2010
Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel bi... more Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the (1)H-(15)N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the (1)H-(15)N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12 and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of (1)H-(15)N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of (1)H-(15)N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein.
Positional information on tooth morphogenesis is investigated by the identification of when and w... more Positional information on tooth morphogenesis is investigated by the identification of when and where phenotypic markers are expressed during odontogenesis. This temporal and positional information is correlated with the instructive and permissive signaling required for both dentinogenesis and amelogenesis. Of particular interest is the establishment of a map for the cranial neural crest-derived dental papilla ectomesenchyme and the odontoblast cell lineages. The expression of ectomesenchymederived cytotactin, dentin phosphoprotein, and epithelial-derived enamel proteins was studied in mice using embryonic, fetal, and postnatal mandibular first molar tooth organ development. This review summarizes the observations in the context of instructive epithelial-mesenchymal interactions and suggests that amelogenesis imperfecta and dentinogenesis imperfecta may in part be explained by alterations in these differentiation markers. Recombinant DNA methods should facilitate future investigations of these inherited dental disorders.
Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein ... more Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein indispensable for enamel formation. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) induces osteogenesis in various cell types. Previously, we demonstrated that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists. There is a reciprocal relationship between osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). Wnt10b-mediated activation of canonical Wnt signaling has been shown to regulate mesenchymal stem cell fate. Using the bipotential bone marrow stromal cell line ST2, we have demonstrated that LRAP activates the canonical Wnt/βcatenin signaling pathway. A specific Wnt inhibitor sFRP-1 abolishes the effect of LRAP on the stimulation of osteogenesis and the inhibition of adipogenesis of ST2 cells. LRAP treatment elevates the Wnt10b expression level whereas Wnt10b knockdown by siRNA abrogates the effect of LRAP. We show here that LRAP promotes osteogenesis of mesenchymal stem cells at the expense of adipogenesis through upregulating Wnt10b expression to activate Wnt signaling.
Fincham AG: Hertwig's epithelial root sheath differentiation and initial cementum and hone format... more Fincham AG: Hertwig's epithelial root sheath differentiation and initial cementum and hone formation during long-term organ culture of mouse mandihular first molars using serumless. chemically-defined medium.
Positional information on tooth morphogenesis is investigated by the identification of when and w... more Positional information on tooth morphogenesis is investigated by the identification of when and where phenotypic markers are expressed during odontogenesis. This temporal and positional information is correlated with the instructive and permissive signaling required for both dentinogenesis and amelogenesis. Of particular interest is the establishment of a map for the cranial neural crest-derived dental papilla ectomesenchyme and the odontoblast cell lineages. The expression of ectomesenchymederived cytotactin, dentin phosphoprotein, and epithelial-derived enamel proteins was studied in mice using embryonic, fetal, and postnatal mandibular first molar tooth organ development. This review summarizes the observations in the context of instructive epithelial-mesenchymal interactions and suggests that amelogenesis imperfecta and dentinogenesis imperfecta may in part be explained by alterations in these differentiation markers. Recombinant DNA methods should facilitate future investigations of these inherited dental disorders.
International Journal of Developmental Biology, 2002
Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and m... more Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and mesenchymal tissues. Many molecules, including growth factors, transcription factors, extracellular matrices, cell surface receptors, and matrix degrading enzymes, have been found to be involved in this process. To investigate the molecular mechanism responsible for morphogenesis of the circumvallate papilla/von Ebners' gland complex, we examined the expression patterns of selected cell adhesion molecules, extracellular matrix molecules, innervation and cell division in the circumvallate papilla of mouse embryos from embryonic day 11.5 (E11.5) to E14. At E11.5-E13.5, the lingual epithelium, the site where the circumvallate papilla will develop, is negative for BrdU labeling. At E14-E15, we detected cell division in the papillary area, especially in the epithelial invagination where von Ebners' minor salivary gland will form. The basement membrane component, laminin, is expressed a...
A team of senior scientists was formed in 2006 to create a blueprint for the regeneration of whol... more A team of senior scientists was formed in 2006 to create a blueprint for the regeneration of whole human teeth along with all of the supporting structure of the dentition. The team included experts from diverse fields, each with a reputation for stellar accomplishment. Participants attacked the scientific issues of tooth regeneration but, more importantly, each agreed to work collaboratively with experts from other disciplines to form a learning organization. A commitment to learn from one another produced a unique interdisciplinary and multidisciplinary team. Inspired by the Kennedy space program to send a man to the moon, with its myriad of problems and solutions that no one discipline could solve, this tooth regeneration team devised an ambitious plan that sought to use stem cell biology, engineering, and computational biology to replicate the developmental program for odontogenesis. In this manner, team members envisioned a solution that consisted of known or knowable fundamenta...
Journal of Molecular and Engineering Materials, 2016
Surgical site infection is a common cause of post-operative morbidity, often leading to implant l... more Surgical site infection is a common cause of post-operative morbidity, often leading to implant loosening, ultimately requiring revision surgery, increased costs and worse surgical outcomes. Since implant failure starts at the implant surface, creating and controlling the bio-material interface will play a critical role in reducing infection while improving host cell-to-implant interaction. Here, we engineered a biomimetic interface based upon a chimeric peptide that incorporates a titanium binding peptide (TiBP) with an antimicrobial peptide (AMP) into a single molecule to direct binding to the implant surface and deliver an antimicrobial activity against S. mutans and S. epidermidis, two bacteria which are linked with clinical implant infections. To optimize antimicrobial activity, we investigated the design of the spacer domain separating the two functional domains of the chimeric peptide. Lengthening and changing the amino acid composition of the spacer resulted in an improvemen...
We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during t... more We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.
Enamel, which covers the anatomical crown of the tooth, is the hardest tissue in human body. Supp... more Enamel, which covers the anatomical crown of the tooth, is the hardest tissue in human body. Supported by a soft but tough dentin structure, the tooth is an advanced nanocomposite that can endure mastication stresses throughout a lifetime. A detail understanding the structure of the tooth, and sepecifically detin-enamel junction (DEJ), not only provides a sound basis for a model for synthetic dental restoration, but also provides lessons from nature on biomimetic regeneration with mechanical integrity. Enamel is a non-growing mineralized tissue and is subjected to most mechanical abuse. Dentin-enamel junction plays a critical role of distributing load between two very dissimilar materials - enamel and dentin. Bulk scale mechanical tests have shown that induced cracks on enamel tend to be arrested DEJ. Furthermore, nanoindentation measurements have also shown that there is a gradual decrease in hardness from enamel to dentin in the DEJ zone suggests a strong mechanical coupling in bo...
The aim of the present study was to select the optimal concentration of TiF4 solution to facilita... more The aim of the present study was to select the optimal concentration of TiF4 solution to facilitate the remineralization of early dentine caries lesions. Sixty human dentine specimens were cut and randomly divided into 6 groups (1%, 2%, 3%, 4% TiF4 groups, 2.712% NaF group and distilled deionized water (DDW) control group). Artificial dentine caries-like lesions were created. After being subjected to fluoride treatment and immersed in remineralizing solution for 2weeks, the specimens were observed by microCT, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Data were analysed using linear regression analysis (P<0.05). The lesion depths of the specimens treated by 2% TiF4 solution were statistically less than those of the other groups. Further, the greyscale values of these lesion areas were greater. The 3% and 4% TiF4 solutions caused further lesion demineralization. The 2.712% NaF solution seemed to be detrimental to remineralization during the expe...
Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The... more Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3'-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins....
Silver nanoparticles (AgNP) are promising candidates for fighting drug-resistant infections becau... more Silver nanoparticles (AgNP) are promising candidates for fighting drug-resistant infections because of their intrinsic antimicrobial effect. The design of high-yield antimicrobial molecules may inadvertently cause variation in host cells' biological responses. While many factors affect AgNPs' efficacy, their surface is exposed to the biological environment and thus plays a critical role in both the preservation of antimicrobial efficacy against pathogens and the modulation of host cells cytotoxicity. This work investigated an engineered biomimetic interface approach to controlling AgNP surface properties to provide them a competitive advantage in a biological environment. Here, a fusion protein featuring a silver-binding peptide (AgBP) domain was engineered to enable self-assembly and track assembly by a green fluorescent protein (GFP) reporter. Following AgNP functionalisation with GFP-AgBP, their antimicrobial and cytotoxic properties were evaluated. GFP-AgBP binding affinity to AgNPs was evaluated using localized surface plasmon resonance sensing. The GFP-AgBP biomimetic interface on AgNPs' surfaces provided sustained antibacterial efficacy at low concentrations based on bacterial growth inhibition assays. Viability and cytotoxicity measurements in fibroblast cells exposed to GFP-AgBP proteinfunctionalised AgNPs showed significant improvement compared to controls. Biointerface engineering offers promise towards tailoring AgNP antimicrobial efficacy while addressing safety concerns to maintain optimum cellular interactions.
To investigate the possible biological mechanism of dental fluorosis at a molecular level. Cultur... more To investigate the possible biological mechanism of dental fluorosis at a molecular level. Cultured LS8 were incubated with serum-free medium containing selected concentrations of NaF (0 ∼ 2 mM) for either 24 or 48 h. Subcellular microanatomy was characterized using TEM; meanwhile, selected biomolecules were analysed using various biochemistry techniques. Transient transfection was used to modulate a molecular pathway for apoptosis. Apoptosis of LS8 was induced by NaF treatment that showed both time and concentration dependency. The activity of caspase-3, -8, -9 was found to be increased with NaF in a dose-dependent manner. Western blot revealed that the protein expression of p-ERK and p-JNK were decreased, while the expression of p-P38 was increased. Inhibition of the p-ERK and p-JNK pathways resulted in a similar decrease for caspase-3. During NaF-induced apoptosis of LS8, p-ERK and p-JNK were closely associated with induction of apoptosis, which might be a mechanism of dental flu...
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Papers by Malcolm Snead