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Dos Santos Neves J, Wazen RM, Kuroda S, Francis Zalzal S, Moffatt P, Nanci A (2011): Odontogenic ameloblast-associated and amelotin are novel basal lamina components. Histochem Cell Biol, doi: 10.1007/s00418-011-0901-4 Odontogenic ameloblast-associated (ODAM) and amelotin (AMTN) are secreted by maturation stage ameloblasts and accumulate at the interface with enamel where an atypical basal lamina (BL) is present. This study aimed at determining and quantifying the ultrastructural distribution of ODAM and AMTN at the cell–tooth interface. Ultrathin sections of enamel organs from the early to mid- and late maturation stage of amelogenesis were processed for immunogold labeling with antibodies against ODAM, AMTN or with the lectins wheat germ agglutinin, Helix pomatia agglutinin (HPA) and Ricinus communis I agglutinin. Immunolabeling showed that both ODAM and AMTN localized to the BL. Quantitative analyses indicated that at the beginning of maturation there is a concentration of ODAM on the cell side of the BL while AMTN appears more concentrated on the enamel side. In the late maturation stage, such differential distribution is no longer apparent. All three lectins are bound to the BL. Competitive incubation with native lectins did not affect the binding efficiency of ODAM; however, AMTN binding was significantly reduced after incubation with HPA. In conclusion, ODAM and AMTN are bona fide components of the BL associated with maturation stage ameloblasts and they organize into different subdomains during the early maturation stage. The data also suggest that the BL is a dynamic structure that rearranges its organization as enamel maturation advances. Finally, the abrogation of AMTN antibody labeling by HPA supports the presence of O-linked sugars in the molecule and/or its close association with other O-glycosylated molecules.

Bartlett JD, Skobe Z, Nanci A, Smith CE (2011): Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino–enamel junction, and a decussating enamel rod pattern. Eur J Oral Sci, 119 (Suppl. 1) : 199-205.Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino–enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ. Enamel development is stage specific. The two predominant stages are the secretory stage and the maturation stage. During the secretory stage, matrix metalloproteinase-20 (MMP20) is expressed while a protein scaffold is formed and while the mineral precipitates as carbonated hydroxyapatite in long thin ribbons that grow out to form the full thickness of the enamel layer. The secretory stage is when the tall columnar ameloblasts of the enamel organ begin moving in rows to form the rod and inter-rod enamel. During the maturation stage, expression of MMP20 ends and the ameloblasts shorten and cease movement. This is when kallikrein-related peptidase 4 (KLK4) assists in removal of the protein scaffold, which allows the enamel ribbons to grow further in width and thickness into large hexagonal crystals (1, 2). The crystals eventually press against one another and interlock as the enamel matures into its final hardened form (3). Matrix metalloproteinases are a family of proteinases that cleave virtually all extracellular matrix proteins. Matrix metalloproteinases play critical roles in reproduction, development, morphogenesis, wound healing, tissue repair, regeneration, remodeling, and cell migration [reviewed in (4)]. Matrix metalloproteinase-20 is required for the development of healthy dental enamel. People and mice with homozygous MMP20 (human)/Mmp20 (mouse) mutations have soft discolored enamel that may be hypoplastic and abrades easily from the dentin surface (5–11). Although the expression of MMP20 during the secretory stage is necessary to cleave enamel matrix proteins (7, 12–22), MMP20 expression is also required to maintain a normal enamel organ morphology during the maturation stage of development when MMP20 is no longer expressed (6, 7). In the absence of MMP20, ectopic calcified nodules appear on the maturation-stage enamel surface, which disrupts the continuity of the almost linear ameloblast layer. Here we demonstrate, using the Mmp20 null mouse, that: (i) calcified nodules form during the maturation stage of enamel development, (ii) the maturation-stage ameloblasts completely cover these nodules, (iii) the enamel is only loosely connected to the underlying dentin, and (iv) the enamel rod pattern is severely malformed or non-existent. These results are consistent with MMP20 playing a key role in cell movement, formation of a strong dentino–enamel junction (DEJ), and prevention of ectopic mineral formation.

Kuroda S, Wazen RM, Moffatt P, Nanci A (2011): Ameloblastin is not implicated in bone remodeling and repair. Eur Cells Mater, 22: 56-67.Ameloblastin (AMBN) is an enamel matrix protein produced by ameloblasts. It has been suggested that AMBN might also be implicated in craniofacial bone formation. Our objective was to determine whether AMBN has an effect on osteogenic mineralisation and infl uences bone remodelling and repair. MC3T3-E1 cells were screened for endogenous expression of enamel proteins using real time PCR. Various osteogenic cells were infected with lentivirus encoding for AMBN and protein expression was verifi ed using immunochemistry. Cultures were stained with alizarin red and mineralisation was quantifi ed. Healing bone was probed for expression of AMBN by DNA microarray analysis. Tooth extraction, experimental tooth movement (ETM), and creation of a non-critical size bone defect in the tibia (BDT) were carried out in wild type and AMBNΔ5-6 mutant mice. Tissues were processed for immunolabelling of AMBN and Bril, an osteoblast specifi c protein associated with active bone formation. MC3T3-E1 cells and healing bone showed no significant expression of AMBN. Overexpression of AMBN in osteogenic cultures induced no noticeable changes in mineralisation. In wild type mice, AMBN was immunodetected in ameloblasts and enamel, but not in normal bone, and at sites where bone remodelling and repair were induced. Bone remodelling during ETM and BDT repair in AMBNΔ5-6 mice were not signifi cantly different from that in wild type animals. Our results suggest that AMBN does not infl uence osteogenic activity in vitro under the conditions used, and does not participate in craniofacial bone remodelling under mechanical stress and in repair of non-critical size bone defects...

Lacruz RS, Smith CE, Moffatt P, Chang EH, Bromage TG, Bringas Jr P, Nanci A, Baniwal SK, Zabner J, Welsh MJ, Kurtz I, Paine ML (2011): Requirements for ion and solute transport, and pH regulation, during enamel maturation. J Cell Physiol, doi: 10.1002/jcp.22911 Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Other proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr and carbonic anhydrases in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR-􀇻F508) and the NBCe1-null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr and Car2, Car3, Car6 and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and carbonic anhydrase gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR-􀇻F508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth...

Duong A, Dubois M-A, Maris T, Métivaud V, Yi J-H, Nanci A, Rocherfort A, Wuest JD (2011): Engineering Homologous Molecular Organization in 2D and 3D. Cocrystallization of Pyridyl-Substituted Diaminotriazines with Alkanecarboxylic Acids. J Phys Chem C, 115: 12908–12919.Isomeric pyridyl-substituted diaminotriazines 2ac and elongated analogue 3 are designed to adopt flattened structures with features that favor adsorption on surfaces and participation in multiple intermolecular interactions. In particular, pyridyl and diaminotriazinyl groups have strong affinities for graphite, and both form coplanar hydrogen-bonded adducts with alkanecarboxylic acids according to reliable motifs. Together, these properties predispose compounds 2ac and 3 to be coadsorbed with alkanecarboxylic acids on graphite and to cocrystallize as structures built from hydrogen-bonded sheets. Comparison of the 2D structures of the ordered adlayers (as determined by scanning tunneling microscopy) with the 3D structures of the cocrystals (as determined by X-ray diffraction) showed striking homology, typically with quantitatively similar structural parameters. Together, these results illustrate how a series of related compounds can be engineered to form ordered adlayers and crystalline solids with closely analogous 2D and 3D structures. Specifically, the molecular components should have an affinity for the underlying surface and should engage in coplanar interactions that are strong relative to the energy of adsorption, thereby ensuring that the components are positioned reliably in sheets despite the effect of the surface. In general, compounds with these features should favor similar organization in different states, including monolayers, thin films, and bulk materials, and they promise to be useful in applications requiring behavior that depends predictably on dimensions, such as in thin-film molecular devices.....

Bueno RBL, Adachi P, Castro-Raucci LMS, Rosa AL, Nanci A, Tambasco de Oliveira P (2011): Oxidative nanopatterning of titanium surfaces promotes production and extracellular accumulation of osteopontin. Braz Dent J, 22: 179-184.The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30’) Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30’) Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity....

Variola F, Brunski JB, Orsini G, Tambasco de Oliveira P, Wazen RM, Nanci A (2011): Nanoscale surface modifications of medically relevant metals: state-of-the art and perspectives. Nanoscale, 3:335-353 Porous metals (sintered beads and meshes) have been used for many years for different orthopedic applications. Metal foams have been recently developed. These foams have the advantage of being more porous than the traditional coatings. Their high porosity provides more space for bone ingrowth and mechanical interlocking and presents more surface for implant-bone contact. The objective of this study was to evaluate in vivo bone ingrowth into Ti implants covered with a novel Ti foam coating. This foam contains 50% in volume of interconnected pores and a higher surface area compared to dense Ti. Both coated implants and dense Ti controls were placed transcortically in the rat tibia....

2009-2010

Richert L, Variola F, Rosei F, Wuest JD, Nanci A (2010): Adsorption of proteins on nanoporous Ti surfaces. Surf Sci, 604:1445-1451.The cascade of events that regulate cell-substrate interactions is not yet fully understood. However, it is now generally recognized that proteins adsorbed on a substrate prior to its colonization have a major influence on initiating and directing cellular activities. Protein adsorption and the characteristics of the adsorbed layer are determined in part by the physical/chemical properties of the underlying surface. Chemical oxidation can be used to generate nanoscale textures on various metals used as implants in medicine. In this study, we exploit a mixture of H2SO4/H2O2 to etch sputtered titanium, and we evaluate the adsorption of a broad range of proteins on the resulting nanoporous surface. Untreated and nanoporous surfaces were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and Fourier-transform infrared spectroscopy (FTIR)...

Lacruz RS, Nanci A, White SN, Wen X, Wang HJ, Francis Zalzal S, Luong VQ, Schuetter VL, Conti PS, Kurtz I, Paine ML (2010): The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition. J. Biol Chem, 285: 24432-24438.Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonateresulting in metabolic acidosis. Patients with mutations to theSLC4A4 gene (coding for the sodium bicarbonate cotransporterNBCe1), have pRTA, growth delay, ocular defects, and enamelabnormalities. In an earlier report, we provided the first evi-dence that enamel cells, the ameloblasts, express NBCe1 in apolarized fashion, thereby contributing to trans-cellular bicar-bonate transport. To determine whether NBCe1 plays a criticalrole in enamel development, we studied the expression ofNBCe1 at various stages of enamel formation in wild-type miceand characterized the biophysical properties of enamel inNBCe1 / animals. The enamel of NBCe1 / animals was extremely hypomineralized and weak with an abnormal pris-matic architecture. The expression profile of amelogenin, aknown enamel-specific gene, was not altered in NBCe1 / ani-mals. Our results show for the first time that NBCe1 expressionis required for the development of normal enamel. This studyprovides a mechanistic model to account for enamel abnormalities...

Nishio C, Wazen RM, Kuroda S, Moffatt P, Nanci A (2010): Expression pattern of odontogenic ameloblast-associated and amelotin during formation and regeneration of the junctional epithelium. Eur Cell Mater, 20:393-402.Porous metals (sintered beads and meshes) have been used for many years for different orthopedic applications. Metal foams have been recently developed. These foams have the advantage of being more porous than the traditional coatings. Their high porosity provides more space for bone ingrowth and mechanical interlocking and presents more surface for implant-bone contact. The objective of this study was to evaluate in vivo bone ingrowth into Ti implants covered with a novel Ti foam coating. This foam contains 50% in volume of interconnected pores and a higher surface area compared to dense Ti. Both coated implants and dense Ti controls were placed transcortically in the rat tibia....

Nishio C, Wazen RM, Kuroda S, Moffatt P, Nanci A (2010): Disruption of periodontal integrity induces expression of APIN by epithelial cell rests of Malassez. J Periodontal Res, 45(6):709-13.Porous metals (sintered beads and meshes) have been used for many years for different orthopedic applications. Metal foams have been recently developed. These foams have the advantage of being more porous than the traditional coatings. Their high porosity provides more space for bone ingrowth and mechanical interlocking and presents more surface for implant-bone contact. The objective of this study was to evaluate in vivo bone ingrowth into Ti implants covered with a novel Ti foam coating. This foam contains 50% in volume of interconnected pores and a higher surface area compared to dense Ti. Both coated implants and dense Ti controls were placed transcortically in the rat tibia....

Trubiani O, Francis Zalzal S, Paganelli R, Marchisio M, Giancola R, Pizzicannella J, Bühring HJ, Piattelli M, Caputi S, Nanci A (2010): Expression profile of NANOG, OCT-4, SSEA-1, SSEA-4, embryonic markers and frizzled-9 receptor in human periodontal ligament mesenchymal stem cells. J Cell Physiol, 225:123-31 Porous metals (sintered beads and meshes) have been used for many years for different orthopedic applications. Metal foams have been recently developed. These foams have the advantage of being more porous than the traditional coatings. Their high porosity provides more space for bone ingrowth and mechanical interlocking and presents more surface for implant-bone contact. The objective of this study was to evaluate in vivo bone ingrowth into Ti implants covered with a novel Ti foam coating. This foam contains 50% in volume of interconnected pores and a higher surface area compared to dense Ti. Both coated implants and dense Ti controls were placed transcortically in the rat tibia....

Wazen RM, Lefebvre LP, Baril E, Nanci A (2010): Initial evaluation of bone ingrowth into novel porous titanium coating. J Biomed Mater Res, Part B: Appl Biomater, 94B: 64-71. Porous metals (sintered beads and meshes) have been used for many years for different orthopedic applications. Metal foams have been recently developed. These foams have the advantage of being more porous than the traditional coatings. Their high porosity provides more space for bone ingrowth and mechanical interlocking and presents more surface for implant-bone contact. The objective of this study was to evaluate in vivo bone ingrowth into Ti implants covered with a novel Ti foam coating. This foam contains 50% in volume of interconnected pores and a higher surface area compared to dense Ti. Both coated implants and dense Ti controls were placed transcortically in the rat tibia....

Lacruz RS, Nanci A, Kurtz I, Wright JT, Paine ML (2010): Regulation of pH during amelogenesis. Calcif Tissue Int, 86: 91-103. During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation...

Variola F, Lauria A, Nanci A, Rosei F (2009): Influence of Treatment Conditions on the Chemical Oxidative Activity of H2SO4/H2O2 Mixtures for Modulating the Topography of Titanium. Adv Eng Mat, 11: B227-B234. Host-tissue integration of medical implants is governed by their surface properties. The capacity to rationally design the surface physico-chemical cues of implantable materials is thus a fundamental prerequisite to confer enhanced biocompatibility. Our previous work demonstrated that different cellular processes are elicited by the nanotexture generated on titanium (cpTi) and Ti6Al4V alloy by chemical oxidation with a H2SO4/H2O2 mixture. Here, we illustrate that by varying the etching parameters such as temperature, concentration, and treatment time, we can create a variety of surface features on titanium which are expected to impact its biological response. The modified submicron and nanotextured surfaces were characterized by scanning electron (SEM) and atomic force (AFM) microscopies. Contact angle measurements revealed the higher hydrophilicity of the modified surfaces compared to untreated samples and Fourier transform infrared spectroscopy (FT-IR) established that the etching generated a TiO2 layer with a thickness in the 40–60nm range....

Trivellato AE, Campos Ribeiro M, Edvard Sverzut C, Bonucci E, Nanci A, Tambasco de Oliveira P (2009): Osteopetrosis Complicated by Osteomyelitis of the Maxilla and Mandible: Light and Electron Microscopic Findings. Head and Neck Pathol, 3:320–326. This report presents a case of osteopetrosis in a 25-year-old male, which was complicated by the development of osteomyelitis in the maxilla and mandible following traumatic injury and tooth extractions. The osteomyelitis in the mandible was refractory to marginal resection and antibiotic therapy. Partial resection with mandible reconstruction was then carried out. Light and backscattered electron scanning microscopy revealed sclerosis of spongy bone and variations in mineral density of the bone matrix. There was also a prominent periosteal bone formation in regions affected by osteomyelitis. An 18-month follow-up showed absence of active infections in the face and oral structures, with a focal area of bone exposure in the right parasymphysis. However, development of anemia and bone marrow deficiency will likely affect prognosis. The importance of preventive oral health care and dental/periodontal managements in osteopetrosis is emphasized.

Variola F, Nanci A, Rosei F (2009): Assessment of the titanium dioxide absorption coefficient by grazing angle FT-IR and ellipsometric measurements. Appl Spectrosc, 63: 1187-1190. Thin films, either deposited or native (i.e., oxide layers), have been widely exploited in different technological fields (e.g., optics, electronics, and biomedicine) to improve the efficiency of devices and extend their range of applications. Among the different physical/chemical characteristics of thin layers that determine their overall properties and therefore their potential for new applications, thickness has been demonstrated to play a pivotal role in affecting different phenomena; this is particularly significant at nanoscale dimensions, since nanostructured materials often behave very differently from their bulk counterparts.

Vetrone F, Variola F, Tambasco de Oliveira P, Francis Zalzal S, Yi JH, Sam J, Bombonato-Prado KF, Sarkissian A, Perepichka DF, Wuest JD, Rosei F, Nanci A (2009): Nanoscale oxidative patterning of metallic surfaces to control cell activity and fate. Nano Letters, 11: 659-665. In the field of regenerative medicine, nanoscale physical cuing is clearly becoming a compelling determinant of cell behavior. Developing effective methods for making nanostructured surfaces with well-defined physicochemical properties is thus mandatory for the rational design of functional biomaterials. Here, we demonstrate the versatility of simple chemical oxidative patterning to create unique nanotopographical surfaces that influence the behavior of various cell types, modulate the expression of key determinants of cell activity, and offer the potential of harnessing the power of stem cells. These findings promise to lead to a new generation of improved metal implants with intelligent surfaces that can control biological response at the site of healing.

Smith CE, Wazen RM, Hu Y, Francis Zalzal S, Nanci A, Simmer JP, Hu JCC (2009): Consequences to enamel development and mineralization resulting from loss of function of ameloblastin or enamelin. Eur J Oral Sci, 117: 485-497. Although the nonamelogenin proteins, ameloblastin and enamelin, are both lowabundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function (Ambn+/+, +/)5,6, )5,6/)5,6, Enam+/+, +/) ,)/)). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn)5,6/)5,6 and Enam)/) homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.

Potvin-Lapointe S, Nishio C, Wazen RM, Macedo Crivelini M, Moffatt P, Nanci A (2009): L’étude du génome de la dent permet la découverte de deux protéines - Impact sur la santé parodontale. J Dent Que, 46: 13-15. u cours des dernières années, diverses initiatives de décodage ont généré une vaste quantité d’informations sur le génome de certains animaux et de l’homme. Le génome est l’ensemble du matériel génétique d’un individu ou d’une espèce contenant les séquences d’ADN codantes pour les protéines produites par les cellules. Collectivement, l’ensemble des protéines représente le protéome d’une cellule. Celui-ci est subdivisé en diverses sous-catégories, dont le sécrétome qui regroupe les protéines destinées à la membrane cellulaire ou à être relâchées à l’extérieur de la cellule. Ces dernières, dites « protéines sécrétées », sont capitales puisqu’elles régulent d’importantes activités cellulaires telles la différenciation et la communication intercellulaire, et qu’elles établissent aussi l’environnement dans lequel les cellules vivent.

Wazen RM, Moffatt P, Francis Zalzal S, Yamada Y, Nanci A (2009): A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects. Mat Biol, 28: 292-303. Ameloblastin (AMBN) is the second most abundant extracellular matrix protein produced by the epithelial cells called ameloblasts and is found mainly in forming dental enamel. Inactivation of its expression by gene knockout results in absence of the enamel layer and its replacement by a thin layer of dysplastic mineralized matrix. The objective of this study was to further characterize the enamel organ and mineralized matrix produced in the AMBN knockout mouse. However, in the course of our study, we unexpectedly found that this mouse is in fact a mutant that does not express the full-length protein but that produces a truncated form of AMBN. Mandibles from wild type and mutant mice were processed for morphological analyses and immunolabeling. Microdissected enamel organs and associated matrix were also prepared for molecular and biochemical analyses. In incisors from mutants, ameloblasts lost their polarized organization and the enamel organ detached from the tooth surface and became disorganized. A thin layer of dysplastic mineralized material was deposited onto dentin, and mineralized masses were present within the enamel organ. These mineralized materials generated lower backscattered electron contrast than normal enamel, and immunocytochemistry with colloidal gold revealed the presence of amelogenin, bone sialoprotein and osteopontin. In addition, the height of the alveolar bone was reduced, and the junctional epithelium lost its integrity. Immunochemical and RT–PCR results revealed that the altered enamel organ in the mutant mice produced a shorter AMBN protein that is translated from truncated RNA missing exons 5 and 6. These results indicate that absence of full-length protein and/or expression of an incomplete protein have direct/indirect effects beyond structuring of mineral during enamel formation, and highlight potential functional regions on the AMBN molecule.

Andrade de Oliva MA, Amarú Maximiano WM, Spínola de Castro LM, da Silva PE Jr, Fernandes RR, Ciancaglini P, Beloti MM, Nanci A, Rosa AL, Tambasco de Oliveira P (2009): Treatment with a growth factor-protein mixture inhibits formation of mineralized nodules in osteogenic cell cultures grown on titanium. J Histochem Cytochem, 57: 265-76. Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-b1, TGF-b2, albumin, fibronectin, and thrombospondin [growth factors (GFs) 1 proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bonederived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs 1 proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose– response experiments were carried out using rat primary calvarial cells exposed to GFs 1 proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red–stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation...

Variola F, Vetrone F, Richert L, Jedrzejowski P, Yi JH, Francis Zalzal S, Clair S, Sarkissian A, Perepichka DF, Wuest JD, Rosei F, Nanci A (2009): Improving biocompatibility of materials by nanoscale modification of surfaces: an overview of strategies, fabrication methods and challenges. Small, 5: 996-1006. The human body is an intricate biochemical–mechanical system, with an exceedingly precise hierarchical organization in which all components work together in harmony across a wide range of dimensions. Many fundamental biological processes take place at surfaces and interfaces (e.g., cell–matrix interactions), and these occur on the nanoscale. For this reason, current health-related research is actively following a biomimetic approach in learning how to create new biocompatible materials with nanostructured features. The ultimate aim is to reproduce and enhance the natural nanoscale elements present in the human body and to thereby develop new materials with improved biological activities. Progress in this area requires a multidisciplinary effort at the interface of biology, physics, and chemistry. In this Review, the major techniques that have been adopted to yield novel nanostructured versions of familiar biomaterials, focusing particularly on metals, are presented and the way in which nanometric surface cues can beneficially guide biological processes, exerting influence on cellular behavior, is illustrated.

2008-2007

Tambasco de Oliveira P, Andrade de Oliva M, Amarú Maximiano WM, Sebastião KE, Crippa GE, Ciancaglini P, Beloti MM, Nanci A, Rosa AL (2008): Effects of a Mixture of Growth Factors and Proteins on the Development of the Osteogenic Phenotype in Human Alveolar Bone Cell Cultures. J Histochem Cytochem, 56: 629–638. Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs 1 proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-b1, TGF-b2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red–stained nodular areas were detected in cultures treated with GFs 1 proteins...

Clair S, Variola F, Kondratenko M, Jedrzejowski P, Nanci A, Rosei F, and Perepichka DF (2008): Self-assembled monolayer of alkanephosphoric acid on nanotextured Ti. J Chem Phys, 128:144705.1-144705.6. Surface modification of titanium and its alloys is of great importance for their practical application as biomedical implants. We have studied and compared assembly of dodecylphosphoric acid on commercial polished and on nanostructured titanium disks. The latter were produced by chemical etching that created nanoscale pits of typical size of about 20 nm. Enhanced hydrophobicity and high molecular density were obtained after functionalization of the nanotextured substrate. Aging tests showed a lifetime of the organic films of about one month in phosphate buffer. The samples were characterized by means of infrared spectroscopy, contact angle measurements, ellipsometry, and atomic force and scanning tunneling microscopies.

Hartikka J, Geall A, Bozoukova V, Kurniadi D, Rusalov D, Enas J, Yi JH, Nanci A, Rolland A (2008): Physical characterization and in vivo evaluation of poloxamer-based DNA vaccine formulations. J Gene Med, 10:770-782. Background. Plasmid DNA (pDNA) vaccines have generated significant interest for the prevention or treatment of infectious diseases. Broader applications may benefit from the identification of safe and potent vaccine adjuvants. This report describes the development of a novel polymer-based formulation to enhance the immunogenicity of pDNA-based vaccines. Methods. Plasmid DNA was formulated with a nonionic block copolymer, poloxamer CRL1005, and the cationic surfactant benzalkonium chloride (BAK) to produce a thermodynamically stable, self-assembling system. The influence of parameters such as polymer concentration and BAK composition on the immune responses was evaluated in mice vaccinated with pDNA encoding influenza nucleoprotein....

Moffatt P, Gaumond MH, Salois P, Sellin K, Bessette MC, Godin E, Tambasco de Oliveira P, Atkins GJ, Nanci A, Thomas G (2008): Bril: a novel bone-specific modulator of mineralization. J Bone Miner Res, 23:1497-1508. In the course of attempting to define the bone “secretome” using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 134 amino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues....

Orsini G, Ruggeri A, Mazzoni A, Nato F, Falconi M, Putignano A, Di Lenarda R, Nanci A, Breschi L (2008): Immunohistochemical localization of dentin matrix protein 1 in human dentin. Eur J Histochem, 52:215-220. Dentin matrix protein 1 (DMP1) is a non-collagenous matrix protein with a recognized role in the formation of mineralized tissues such as dentin. The aim of this study was to analyze the distribution of DMP1 in human dentin by means of immunofluorescence and high-resolution immunogold labeling. Fully developed, sound human dentin specimens were submitted to fluorescence labeling and post-embedding immunolabeling techniques with a rabbit polyclonal antihuman DMP1 antibody followed by corresponding fluorochrome- conjugated or gold-conjugated secondary antibodies. Both immunofluorescence and immunogold labeling showed an intense labeling associated with the peritubular dentin. In addition, at the ultrastructural level, there was also a moderate and diffuse immunoreaction over intertubular dentin, and a weak labeling within predentin which increased in density towards the mineralization front...

Nanci A, Wazen RM, Nishio C, Francis Zalzal S (2008): Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section. Eur. J. Histochem. 52: 201-14. The organic matrix of calcified tissues comprises collagenous and/or noncollagenous matrix proteins (NCPs). Identification and precise mapping of these matrix components is essential for determining their function, formulating coherent hypotheses on their mechanism(s) of action, and developing novel therapeutic approaches based on biologics. Fibrillar collagen can be readily identified by its conspicuous structure, however, NCPs, in general, do not individually exhibit characteristic structural features that permit to identify them and morphologically determine their localization. To address this limitation, we have used immunocytochemistry, a form of “biochemistry on section”, because it allows correlating composition with structure. For cytochemical characterizations, including immunolabeling, the group has opted for colloidal gold labelings and pioneered their application to calcified tissues because this approach has a high spatial resolution and is quantitative....

Richert L, Vetrone F, Yi J H, Francis Zalzal S, Wuest JD, Rosei F, Nanci A (2008): Surface nanopatterning to control cell growth. Adv. Mater. 15:1-5. A significant challenge in implantology is the design of biomaterials that actively promote functional regeneration of the host tissue while avoiding undesirable tissue responses. This requires selective control of interactions at the tissue/implant interface, the site of a series of complex events that depend on synergistic parameters including surface chemistry, elasticity, topography, and energy. To date, efforts have focused largely on defining how microtexture influences the molecular and cellular events of tissue repair. However, it is now widely recognized that biological substrates on which cells thrive consist of nanostructured molecular networks, and the sensing apparatus of cells operates on the nanometer scale. Reports have emerged showing that nanometer-scale surface features can influence cellular attachment, differentiation, and alignment...

Francis Zalzal S, Smith CE, Nanci A (2008): Ameloblastin and amelogenin share a common secretory pathway and are co-secreted during enamel formation. Matrix Biology, 27:352-359. The epithelially-derived ameloblasts secrete two main categories of extracellular matrix proteins, amelogenins (AMEL) and nonamelogenins. These proteins assume differential distributions in the forming enamel layer and thereby regulate deposition and structuring of the mineral phase. The objective of this study was to elucidate whether their distribution results from distinctive physicochemical behaviors or differences in intracellular routing. Dual-immunogold labeling was used to visualize the presence of AMEL and ameloblastin (AMBN), the major nonamelogenin, and quantify the proportion of secretory granules containing one or both of these proteins in ameloblasts during the phase of appositional growth of the enamel layer in continuously-erupting rat incisors. Some rats were treated with brefeldin A (BFA) to generate a synchronized cohort of newlyformed secretory granules....

Trubiani O, Isgro A, Zini N, Antonucci I, Aiuti F, Di Primio R, Nanci A, Caputi S, Paganelli R (2008): Functional interleukin-7/interleukin-7Ralpha, and SDF-1alpha/CXCR4 are expressed by human periodontal ligament derived mesenchymal stem cells. J Cell Physiol, 214:706-713. Hematopoiesis in the bone marrow (BM) is maintained by specific interactions between both hematopoietic and non-hematopoietic stromal cells, which are mesenchymal stem cells (MSCs) capable of giving rise to several cell types. The human periodontal ligament (PDL), a tissue of ectomesenchymal origin, has been shown to also be a source of MSCs.Wehave investigated whether MSCs expanded from the PDL of healthy volunteers express characteristics similar to BM-derived stem cells using structural, immunocytochemical and molecular approaches. Their ability to support the growth of hematopoietic progenitors was also analyzed. The PDL-MSCs exhibited a fibroblast-like morphology and their chromatin was dispersed, indicating active gene transcription. The mesenchymal-related antigens CD90, CD29, CD166, CD105, and CD44 were homogeneously detected by cytofluorimetric analysis, whereas membrane CXCR4 was expressed only by a minority of cells. The PDL-MSCs differentiated in vitro into osteogenic and adipogenic cells. Immunolocalization of IL-7, IL-7Ra, SDF-1a, and CXCR4 resulted in a diffuse but specific labeling. RT-PCR analysis confirmed the expression of the above-mentioned transcripts...

Variola F, Yi JH, Richert, L, Wuest, J. D, Rosei F, Nanci A (2008): Tailoring the surface properties of Ti6Al4V by controlled chemical oxidation. Biomaterials, 29:1285-1298. Many efforts have been made to promote cell activity at the surface of implants, mainly by modifying their topography and physicochemical properties. Here we demonstrate the feasibility of creating Ti6Al4V surfaces having both a microtexture and a nanotexture, and show that their properties can be tailored by controlling the length of exposure to a mixture of H2SO4 and H2O2. Scanning electron microscopy (SEM), combined with energy-dispersive X-ray spectroscopy (EDX), indicated that b-phase grains, which surround larger a-phase grains, are etched more rapidly, resulting in a surface composed of microscale cavities with a-grain boundaries. Furthermore, high-resolution SEM and atomic force microscopy (AFM) revealed the presence on the surfaces of both a- and b-phase grains of a network of nanopits with mean diameters ranging between 13 and 21 nm. The grain surface roughness increases from about 4 nm on untreated samples to about 12 nm after 4 h of treatment...

Moffatt P, Smith CE, St-Arnaud, R. Nanci A (2008): Characterization of Apin, a secreted protein highly expressed in tooth associated epithelia. J Cell Biochem, 103: 941-956. We previously reported expression of a protein by enamel organ (EO) cells in rat incisors, originally isolated from the amyloid of Pindborg odontogenic tumors called Apin. The aim of the present study was to further characterize the Apin gene and its protein in various species, assess tissue specificity, and clarify its localization within the EO. Northern blotting and RT-PCR revealed that expression of Apin was highest in the EO and gingiva, moderate in nasal and salivary glands, and lowest in the epididymis. The protein sequences deduced from the cloned cDNA for rat, mouse, pig, and human were aligned together with those obtained from four other mammal genomes. Apin is highly conserved in mammals but is absent in fish, birds, and amphibians. Comparative SDS–PAGE analyses of the protein obtained from bacteria, transfected cells, and extracted from EOs all indicated that Apin is post-translationally modified, a finding consistent with the presence of predicted sites for phosphorylation and O-linked glycosylation. In rodent incisors, Apin was detected only in the ameloblast layer of the EO, starting at post-secretory transition and extending throughout the maturation stage...

Tambasco De Oliveira P, Francis Zalzal S, Beloti MM, Rosa AL, Nanci A (2007): Enhancement of in vitro osteogenesis on titanium by chemically produced nanotopography. J. Biomed. Mater. Res. A, 80A:554-564. The surface characteristics of biomaterials can influence protein adsorption, cellular functions, and ultimately tissue formation. Controlled chemical oxidation of titanium-based surfaces with a mixture of H2SO4/H2O2 creates a nanopatterned surface that has been shown to affect early osteogenic events. The objective of this study was to evaluate the effect over time of this nanopattern on various key parameters of osteogenesis, and determine whether these effects ultimately translate into more mineralized matrix production. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvaria and grown on treated and untreated titanium discs for periods of up to 14 days. Alkaline phosphatase activity peaked earlier and cell number was higher as of day 7 on the nanopatterned discs.

Leucht P, Kim J, Wazen, RM, Currey J, Nanci A, Brunski JB, Helms JA (2007): Effect of mechanical stimuli on skeletal regeneration around implants. Bone, 40: 919-930. Due to the aging population and the increasing need for total joint replacements, osseointegration is of a great interest for various clinical disciplines. Our objective was to investigate the molecular and cellular foundation that underlies this process. Here, we used an in vivo mouse model to study the cellular and molecular response in three distinct areas of unloaded implants: the periosteum, the gap between implant and cortical bone, and the marrow space. Our analyses began with the early phases of healing, and continued until the implants were completely osseointegrated...

Wazen RM, Tye CE, Goldberg HA, Hunter GK, Smith CE, Nanci A (2007): In Vivo Functional Analysis of Polyglutamic Acid Domains in Recombinant Bone Sialoprotein. J Histochem Cytochem, 55(1): 35-42. Bone sialoprotein (BSP) is an anionic phosphoprotein expressed in mineralizing connective tissues that binds to hydroxyapatite and nucleates its formation in vitro. Two polyglutamic acid regions (poly [E]) are believed to participate in these activities. The aim of this study was to evaluate the contribution of these acidic regions to the binding of prokaryote recombinant BSP (prBSPE) within an actual in vivo environment. Full-length prBSPE and prBSPE in which the poly [E] domains were replaced by polyalanine (prBSPA) were tagged with dinitrophenol (DNP). The tagged preparations comprised intact molecules and some fragmented forms...

Colnot C, Romero D, Huang S, Rahman J, Currey J, Nanci A, Brunski JB, Helms JA (2007): Molecular analysis of healing at a bone-implant interface. J Dent, 86: 862-7. While bone healing occurs around implants, the extent to which this differs from healing at sites without implants remains unknown. We tested the hypothesis that an implant surface may affect the early stages of healing. In a new mouse model, we made cellular and molecular evaluations of healing at bone-implant interfaces vs. empty cortical defects. We assessed healing around Ti- 6Al-4V, poly(L-lactide-co-D,L,-lactide), and 303 stainless steel implants with surface characteristics comparable with those of commercial implants. Our qualitative cellular and molecular evaluations showed that osteoblast differentiation and new bone deposition began sooner around the implants, suggesting that the implant surface and microenvironment around implants favored osteogenesis...

Dang H, Maris T, Yi JH, Rosei F, Nanci A, Wuest JD (2007): Ensuring homology between 2D and 3D molecular crystals. Langmuir, 23: 11980-85. Integrated studies using scanning tunneling microscopy and X-ray crystallography have established that 4,5,9,- 10-tetrahydropyrene-2,7-dicarboxylic acid and pyrene-2,7-dicarboxylic acid crystallize in 2D and 3D with striking homology. Different behavior is shown by related biphenyls that lack the planarizing conformational constraints of the pyrenyl core and the directing effects of intermolecular hydrogen bonding. The results of these studies show that molecules specifically designed to engage in multiple strong directional interadsorbate interactions are promising tools for imposing particular nanopatterns on surfaces and for revealing subtle aspects of crystallization...

Nath KG, Ivasenko O, MacLeod JM, Miwa JA, Wuest JD, Nanci A, Perepichka DF, Rosei F (2007): Crystal engineering in two dimensions: An approach to molecular nanopatterning. J Phys Chem C, 111:16996-17007. We describe a surprising cooperative adsorption process observed by scanning tunneling microscopy (STM) at the liquid-solid interface. The process involves the association of a threefold hydrogen-bonding unit, trimesic acid (TMA), with straight-chain aliphatic alcohols of varying length (from C7 to C30), which coadsorb on highly oriented pyrolytic graphite (HOPG) to form linear patterns. In certain cases, the known TMA “flower pattern” can coexist temporarily with the linear TMA-alcohol patterns, but it eventually disappears. Time-lapsed STM imaging shows that the evolution of the flower pattern is a classical ripening phenomenon. The periodicity of the linear TMA-alcohol patterns can be modulated by choosing alcohols with appropriate chain lengths, and the precise structure of the patterns depends on the parity of the carbon count in the alkyl chain. Interactions that lead to this odd-even effect are analyzed in detail. The molecular components of the patterns are achiral, yet their association by hydrogen bonding leads to the formation of enantiomeric domains on the surface....

Zhou H, Dang H, Nanci A, Rochefort A and Wuest JD. Frustrated 2D molecular crystallization (2007): J Am Chem Soc; 129:13774-13775. We describe a surprising cooperative adsorption process observed by scanning tunneling microscopy (STM) at the liquid-solid interface. The process involves the association of a threefold hydrogen-bonding unit, trimesic acid (TMA), with straight-chain aliphatic alcohols of varying length (from C7 to C30), which coadsorb on highly oriented pyrolytic graphite (HOPG) to form linear patterns. In certain cases, the known TMA “flower pattern” can coexist temporarily with the linear TMA-alcohol patterns, but it eventually disappears. Time-lapsed STM imaging shows that the evolution of the flower pattern is a classical ripening phenomenon. The periodicity of the linear TMA-alcohol patterns can be modulated by choosing alcohols with appropriate chain lengths, and the precise structure of the patterns depends on the parity of the carbon count in the alkyl chain. Interactions that lead to this odd-even effect are analyzed in detail. The molecular components of the patterns are achiral, yet their association by hydrogen bonding leads to the formation of enantiomeric domains on the surface....

2006-2005

Yi JH, Bernard C, Variola F, Fracis Zalzal S, Wuest JD, Rosei, F. Nanci A (2006): Characterization of a bioactive nanotextured surface created by controlled chemical oxidation of titanium. Surf. Sci, 600: 4613-4621. Events at bone–implant interfaces are influenced by implant surface properties. Our previous work has revealed that osteogenic activity is enhanced by a nanotextured Ti surface, obtained by controlled chemical oxidation using a H2SO4/H2O2 mixture. To better understand the origin of this biological effect, we have carried out a characterization of the modified surface at the nanoscale. In particular, the morphology, structure, and chemical composition of the Ti surface were examined thoroughly. X-ray photoelectron spectroscopy (XPS), combined with grazing-angle Fourier-transform infrared (FTIR) spectroscopy, revealed that the oxidized Ti surface consists of almost pure TiO2 with Ti:O ratio ranging between 1:2.02 and 1:2.08. Raman spectroscopy and X-ray diffraction (XRD) indicated that the chemically treated Ti surface is mainly composed of amorphous titania....

Lebel O, Perron ME, Maris T, Francis Zalzal S, Nanci A, Wuest JD (2006): A new class of selective low-molecular-weight gelators based on salts of diaminotriazinecarboxylic acids. Chem. Mater, 18:3616-3626. New low-molecular-weight gelators can be discovered by an approach that integrates classical methods for identifying potential gelators with strategies recently developed by crystal engineers to build porous molecular networks. This hybrid approach has yielded a potent new class of selective gelators based on salts of 4,6-bis(arylamino)-1,3,5-triazine-2-carboxylic acids. These compounds lack the high degree of conformational flexibility and long alkyl chains typical of classical gelators, and Na+ and DMSO play specific roles in the mechanism of gelation. Scanning electron microscopy and atomic force microscopy showed that the resulting gels consist of elemental nanofibers that are approximately 30-100 nm in width, and X-ray diffraction yielded the structure of needle-shaped crystals of a gelator obtained directly from its gel...

Moffatt P, Smith CE, St-Arnaud R, Simmons C, Wright JT, Nanci A (2006): Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium. Biochem. J, 399: 37-46. Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently describedmouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao,Wendel and Ganss (2005) J. Dent. Res. 84, 1127–1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences...

Wazen RM, Moffatt P, Fracis Zalzal S, Daniel NG, Westerman KA, Nanci A (2006): Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector. Gene Ther,13: 1595-1602. Gene transfer using viral vectors offers the potential for the sustained expression of proteins in specific target tissues. However, in the case of calcified tissues, in vivo delivery remains problematic because of limited accessibility. The aim of this study was to test the efficiency of lentiviral vectors (LVs) on osteogenic cells in vitro, and determine the feasibility of directly transducing resident bone cells in vivo. LVs encoding for green fluorescent protein (GFP) and ameloblastin (AMBN), a protein associated with mineralization not reported in bone, were generated...

Smith CE, Nanci A, Moffatt, P (2006): Evidence by signal peptide trap technology for the expression of carbonic anhydrase 6 in rat incisor enamel organs. Eur. J. Oral Sci, 114 (Suppl.1): 147-153. During screening of a rat incisor enamel organ cDNA library by signal peptide trap technology, we identified a DNA fragment matching a predicted translation sequence for rat carbonic anhydrase 6 (CA6). This result was unexpected because CA6, to date, has been associated primarily with secretions from glandular tissues. To further characterize this observation, reverse transcription–polymerase chain reaction (RT– PCR) amplifications were carried out on total RNA extracted from freeze-dried secretory and maturation-stage rat incisor enamel organs. A cDNA fragment of the expected size was detected in control samples from rat salivary glands as well as within maturation-stage enamel organ samples. This CA6 RT–PCR fragment was further cloned and sequenced and found to match the nucleotide sequence 770–1079 from clone XM_216584 of GenBank...

Moffatt P, Smith CE, Sooknanan, R, St-Arnaud, R. Nanci A (2006): Identification of secreted and membrane proteins in the rat incisor enamel organ using a signal-trap screening approach. Eur J Oral Sci, 114 (Suppl.1): 139-146. The secretome represents the subset of proteins that are targeted by signal peptides to the endoplasmic reticulum. Among those, secreted proteins play a pivotal role because they regulate determinant cell activities such as differentiation and intercellular communication. In calcified tissues, they also represent key players in extracellular mineralization. This study was carried out to establish a secretome profile of rat enamel organ (EO) cells. A functional genomic technology, based on the signal trap methodology, was applied, starting with a library of 5¢-enriched cDNA fragments prepared from rat incisor EOs. A total of 2,592 clones were analyzed by means of macroarray hybridizations and DNA sequencing. Ninety-four unique clones encoding a signal peptide were retrieved...

Nath KG, Ivasenko O, Miwa JA, Dang H, Wuest JD, Nanci A, Perepichka DF, Rosei F (2006): Rational modulation of the periodicity in linear hydrogen-bonded assemblies of trimesic acid on surfaces. J Am Soc Chem, 128: 4412-4413. Self-assembled structures physisorbed on surfaces are a rich source of information. The nature and transformations of these structures shed new light on the fundamental interactions between complex molecules.2-8 In many self-assembled molecular networks (SAMNs), hydrogen bonding is the primary link among adjacent molecules and governs the resulting structure. One of the most extensively studied models is trimesic acid (TMA), a tricarboxylic acid with three-fold symmetry.

Nanci A, Bosshardt DD (2006): Structure of periodontal tissues in health and disease. Periodontol. 2000, 40: 11-28. The periodontium, defined as those tissues supporting and investing the tooth, comprises root cementum, periodontal ligament, bone lining the tooth socket (alveolar bone), and that part of the gingiva facing the tooth (dentogingival junction). The widespread occurrence of periodontal diseases and the realization that lost tissues can be repaired and, perhaps, regenerated has generated considerable interest in the factors and cells regulating their formation and maintenance...

2004-2003

Nanci A, Wazen RM, Francis Zalzal S, Fortin M, Goldberg HA, Hunter GK, Ghitescu DL (2004): A Tracer Study with Systemically and Locally Administered Dinitrophenylated Osteopontin. J Histochem Cytochem, 52:1591-1600

Laboux,O, Dion N, Arana-Chavez VE, Ste-Marie L-G, Nanci A (2004): Microwave irradiation of ethanol-fixed bone improves preservation, reduces procesing time and allows both light and electron micorscopy on a same sample. J Histochem Cytochem, 52:1267-1275.

Bosshardt DD, Nanci A (2004): Hertwig's epithelial root sheath, enamel matrix proteins, and initiation of cementogenesis in procine teeth. J Clin Periodontol, 31:184-192.

Tambasco De Oliveira P, Nanci A (2004): Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells. Biomaterials, 25:403-413.

Bosshardt DD, Nanci A (2003): Immunocytochemical characterization of ectopic enamel deposits and cementicles in human teeth. Eur.J.Oral Sci, 111:51-59.

Gascon-Barré M, Demers C, Mirshahi A, Neron S, Francis Zalzal S, Nanci A (2003): The normal liver harbors the vitamin D nuclear receptor in nonparenchymal and biliary epithilial cells. Hepatology, 37:1034-1042.

Laboux O, Ste-Marie L-G, Glorieux FH, Nanci A (2003): Quantitative immunogold labeling of bone sialoprotein and osteopontin in methylmethacrylate-embedded rat bone. J Histochem Cytochem, 51:61-67.

Tambasco De Oliveira P, Francis Zalzal S, Irie K, Nanci A (2003): Early expression of bone matrix proteins in osteogenic cell cultures. J Histochem Cytochem, 51:633-641.

2002-2000

Shimazu Y, Nanci A, Aoba T (2002): Immunodetection of osteopontin at sites of resorption in the pulp of rat molars. J Histochem Cytochem, 50:911-921.

Arana-Chavez VE, Nanci A (2001): High-resolution immunocytochemistry of noncollagenous matrix proteins in rat mandibles processed with microwave irradiation. J Histochem Cytochem, 49:1099-1109. The mineral phase in calcified tissues represents an additional factor to be considered during their preservation for ultrastructural analyses. Microwave (MW) irradiation has been shown to facilitate fixative penetration and to improve structural preservation and immunolabeling in a variety of soft tissues. The aim of the present study was to determine whether MW processing could offer similar advantages for hard tissues. Rat hemimandibles were immersed in 4% formaldehyde + 0.1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2, and exposed to MWs for three periods of 5 min at temperatures not exceeding 37C. They were then decalcified in 4.13% EDTA, pH 7.2, for 15 hr, also under MW irradiation. Osmicated and non-osmicated samples were dehydrated in graded concentrations of ethanol and embedded in LR White resin...

Bonucci E, Mocetti P, Silverstrini G, Ballanti P, Francis Zalzal S, Fortin M, Nanci A (2001): The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study. J Electron Microsc, 50:333-347.

Kawaguchi H, Ogawa T, Kurihara H, Nanci A (2001): Immunodetection of noncollagenous matrix proteins during periodontal tissue regeneration. J Periodont Res, 36:205-213. The interface between denuded dentin and regenerative periodontal tissue was investigated in a rat alveolar bone defect model using morphological and immuno- cytochemical approaches. The dentin surface was surgically exposed along the palatal roots of maxillary ®rst molars. At 3 weeks post treatment, animals were perfused and treated regions from decalci®ed mandibles were embedded in Epon for ultrastructural studies or LR White for post-embedding immunogold labeling. Thin tissue sections were incubated with antibodies against noncollagenous matrix (osteopontin, bone sialoprotein, osteocalcin and ®bronectin) and plasma (a2HS-glycoprotein and albumin) proteins. While in some cases, regenerative events took place directly on the denuded dentin surface, the interface between the denuded dentin and regenerating periodontal tissue was frequently characterized by the presence of an interfacial zone....

Orsini,G, Francis Zalzal S, Nanci A. (2001): Localized infusion of tunicamycin in rat hemimandibles: alteration of the basal lamina associated with maturation stage ameloblasts. J Histochem Cytochem, 49:165-176.

Orsini G, Lavoie P, Smith C.E, Nanci A. (2001): Immunochemical characterization of a chicken egg yolk antibody to secretory forms of rat incisor amelogenin. J Histochem Cytochem, 49:285-292.

Brunski JB, Puleo DA, Nanci A. (2000): Biomaterials and biomechanics of oral and maxillofacial implants: current status and future developments. Int J Oral Maxillofac Implants, 15:15-46. Major advances have occurred over the last 3 decades in the clinical use of oral and maxillofacial implants. Statistics on the use of dental implants bear this out; about 100,000 to 300,000 dental implants are placed per year,1 which approximates the numbers of artificial hip and knee joints placed per year.2 Implants are currently used to replace missing teeth, rebuild the craniofacial skeleton, provide anchorage during orthodontic treatments, and even to help form new bone in the process of distraction osteogenesis. Despite the impressive clinical accomplishments with oral and maxillofacial implants—and the undisputed fact that implants have improved the lives of millions of patients—it is nevertheless disquieting that key information is still missing about fundamental principles underlying their design and clinical use. With some important exceptions, the design and use of oral and maxillofacial implants has often been driven by an aggressive, “copycat” marketing environment, rather than by basic advances in biomaterials, biomechanics, or bone biology.....

Mocetti P, Ballanti P, Francis Zalzal S, Silvestrini G, Bonucci E, Nanci A (2000): A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats. J Histochem Cytochem, 48:1059-1077.

Nanci A, Mocetti,P, Sakamoto,Y, Kunikata,M, Lozupone,E, and Bonucci,E. (2000): Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor. J Histochem Cytochem, 48:1043-1057.

Nguyen TN, Wang HJ, Francis Zalzal S, Nanci A, Nabi IR (2000): Purification and characterization of beta-actin-rich tumor cell pseudopodia: role of glycolysis. Exp Cell Res, 258:171-183.

1999-1995

Josephsen K, Smith CE, Nanci A (1999): Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin. J Histochem Cytochem, 47:1237-1245.

Nanci A (1999): Content and distribution of noncollagenous matrix proteins in bone and cementum: Relationship to speed of formation and collagen packing density. J Struct Biol, 126:256-269.

Puleo DA, Nanci A (1999): Understanding and controlling the bone-implant interface. Biomaterials, 20:2311-2321.

Vu D-D, Daniel NG, Nanci A (1999): In vivo model for the experimental manipulation of calcified tissues: A surgical approach for accessing the odontogenic organ and associated tissues in the rat incisor. J Histochem Cytochem, 47:1-14.

Bosshardt DD, Francis Zalzal S, McKee MD, Nanci A (1998): Developmental appearance and distribution of bone sialoprotein and osteopontin in human and rat cementum. Anat Rec, 250:1-21. Bone sialoprotein (BSP) and osteopontin (OPN), two major noncollagenous proteins (NCPs) in collagen-based mineralized tissues, have been implicated in mineral deposition and cell- and matrix-matrix interactions during root development. However, their role in cementogenesis is still a subject of debate. Since distribution of proteins is indicative of function, we have analyzed their temporo-spatial appearance in relation to that of cementum collagen. Methods: Human premolars and rat molars at various stages of root development characterized by differing rates of formation were fixed in aldehyde and embedded in epoxy and LR White resin. Sections were processed for ultrastructural analysis and postembedding colloidal gold (immune) cytochemistry...

Bosshardt DD, Nanci A (1998): Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells. J Histochem Cytochem, 46:135-142. After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMEN), bone sialoprotein (BSP), and osteopontin (OPN)...

Irie K, Francis Zalzal S, Ozawa H, McKee MD, Nanci A (1998): Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue. Anat Rec, 252:554-567.

Nanci A, Wuest JD, Péru L, Brunet P, Sharma V, Francis Zalzal S, McKee MD. (1998): Chemical modification of titanium surfaces for covalent attachment of biological molecules. J Biomed Mater Res, 40:324-335. The surface of implantable biomaterials is in direct contact with the host tissue and plays a critical role in determining biocompatibility. In order to improve the integration of implants, it is desirable to control interfacial reactions such that nonspecific adsorption of proteins is minimized and tissue-healing phenomena can be controlled. In this regard, our goal has been do develop a method to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive organic molecules. Titanium first was chemically treated with a mixture of sulfuric acid and hydrogen peroxide to eliminate surface contaminants and to produce a consistent and reproducible titanium oxide surface layer. An intermediary aminoalkylsilane spacer molecule was then covalently linked to the oxide layer, followed by the covalent binding of either alkaline phosphatase or albumin to the free terminal NH2 groups using glutaraldehyde as a coupling agent....

Nanci A, Francis Zalzal S, Lavoie P, Kunikata M, Chen W-Y, Krebsbach PH, Yamada Y, Hammarström L, Simmer JP, Fincham AG, Snead ML, and Smith CE (1998): Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors. J Histochem Cytochem, 46:911-934.

Rittling SR, Matsumoto HN, McKee MD, Nanci A, An X-R, Novick KE, Kowalski AJ, Noda M, and Denhardt DT (1998): Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro. J Bone Miner Res, 13:1101-1111. We have used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of the osteopontin (Opn, or Spp1, for secreted phosphoprotein 1) gene. Mice homozygous for this disruption fail to express osteopontin (OPN) as assessed at both the mRNA and protein level, although an N-terminal fragment of OPN is detectable at extremely low levels in the bones of 2/2 animals. The Opn2/2 mice are fertile, their litter size is normal, and they develop normally. The bones and teeth of animals not expressing OPN are morphologically normal at the level of light and electron microscopy, and the skeletal structure of young animals is normal as assessed by radiography. Ultrastructurally, proteinaceous structures normally rich in OPN, such as cement lines, persist in the bones of the Opn2/2 animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild-type mice. Spleen cells from Opn2/2 mice cells formed osteoclasts 3- to 13-fold more frequently than did control Opn1/1 cells, while the extent of osteoclast development from Opn2/2 bone marrow cells was about 2- to 4-fold more than from the corresponding wild-type cells.....

Bosshardt DD, Nanci A (1997): Immunodetection of enamel- and cementum-related (bone) proteins at the enamel-free area and cervical portion of the tooth in rat molars. J Bone Miner Res, 12:367-379. Enamel and dentin at the cervical portion of the tooth are frequently covered by a collagen-free matrix referred to as acellular afibrillar cementum (AAC). It is believed that AAC deposition occurs when the enamel organ is displaced or disrupted, and mesenchymal cells from the dental follicle gain access to the tooth surface, differentiate into cementoblasts, and secrete noncollagenous proteins typically found in collagen-based mineralized tissues. A similar thin layer of mineralized matrix is found at the enamel-free area (EFA) of rodent molars, but in this case the matrix is covered by inner enamel epithelium (IEE) throughout development. We have, therefore, used this site as a paradigm to test the hypothesis that typical mesenchymal matrix proteins can also be found in association with epithelial cells. To this end, we have analyzed the presence and distribution of enamel- and cementum-related matrix proteins at the EFA and at the cervical portion of the tooth. Rat mandibular molars were processed for colloidal gold immunolabeling with antibodies to amelogenins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and dentin sialoprotein (DSP), and the plasma proteins a2HS-glycoprotein and albumin. The EFA matrix was immunoreactive for amelogenins as well as for BSP, OPN, OC, and a2HS-glycoprotein, but not for albumin and DSP. The AAC was, similar to the EFA matrix, labeled for BSP, OPN, OC, and a2HSglycoprotein...

Zylberberg L, Sire J-Y, Nanci A (1997): Immunodetection of amelogenin-like proteins in the ganoine of experimentally regenerating scales of Calamoichthys calabaricus, a primitive actinopterygian fish. Anat Rec, 249:86-95.

Berdal A, Hotton D, Saffar JL, Thomasset M, Nanci A (1996): Calbindin-D 9K and calbindin-D 28K expression in rat mineralized tissues in vivo. J Bone Miner Res, 11:768-779.

Hashimoto J, Nanci A (1996): Disassembly and reformation of the Golgi apparatus in rat incisor secretory stage ameloblasts after brefeldin A treatment: A dynamic in vivo analysis. Acta Histochem Cytochem, 29:188-189.

Lee S-K, Krebsbach PH, Matsuki Y, Nanci A, Yamada K Yamada Y (1996): Ameloblastin expression in rat incisors and human tooth germs. Int J Dev Biol, 40:1141-1150.

McKee MD, Nanci A (1996): Osteopontin: An interfacial extracellular matrix protein in mineralized tissues. Connect Tissue Res, 35:197-205.

McKee MD, Nanci A (1996): Secretion of osteopontin by macrophages and its accumulation at tissue surfaces during wound healing in mineralized tissues: A potential requirement for macrophage adhesion and phagocytosis. Anat Rec, 245:394-409.

McKee,M.D. Nanci A. (1996): Osteopontin at mineralized tissue interfaces in bone, teeth and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover and repair. Microsc.Res.Tech, 33:141-164.

McKee MD, Francis Zalzal S, Nanci A (1996): Extracellular matrix in tooth cementum and mantle dentin: Localization of osteopontin and other noncollagenous proteins, plasma proteins and glycoconjugates by electron microscopy. Anat Rec, 245:293-312.

Nanci A, Hashimoto J, Francis Zalzal S, Smith CE (1996): Transient accumulation of proteins at interrod and rod enamel growth sites. Adv.Dent.Res, 10:135-149.

Nanci A, Francis Zalzal S, Gotoh Y, and McKee, MD (1996): Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures. Microsc Res Tech, 33:214-231.

Nanci A, Fortin M, Ghitescu L (1996): Endocytotic functions of ameloblasts and odontoblasts: Immunocytochemical and tracer studies on the uptake of plasma proteins. Anat Rec, 245:219-234.

Smith CE, Nanci A (1996): The protein dynamics of amelogenesis. Anat.Rec, 245:186-207.

Chen W-Y, Nanci A, Smith, CE (1995): Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins. Calcif Tissue Int, 57:145-151.

McKee MD, Nanci A, Khan,SR (1995): Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi. J Bone Miner Res, 10:1913-1929.

McKee MD, Nanci A (1995): Post-embedding colloidal-gold immunocytochemistry of non-collagenous extracellular matrix proteins in mineralized tissues. Microsc Res Tech, 31:44-62.

Sawada T, Nanci A (1995): Spatial distribution of enamel proteins and fibronectin at early stages of rat incisor tooth formation. Archs Oral Biol, 40:1029-1038.

Smith CE, Nanci A (1995): Overview of morphological changes in enamel organ cells associated with major events in amelogenesis. Int J Dev Biol, 39:153-161.

1994-1990

Chen J, McKee MD, Nanci A, Sodek J (1994): Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin. Histochem J, 26:67-78.

Kan FWK, Francis Zalzal S, Roux E, Nanci A (1994): Homogeneity in the distribution of matrix components in the hamster zona pellucida as revealed by backscattered electron imaging fracture-label. Anat Rec, 239:35-46.

Marks SC Jr, McKee MD, Francis Zalzal S, Nanci A (1994): The epithelial attachment and the dental junctional epithelium: Ultrastructural features in porcine molars. Anat.Rec, 238:1-14.

Nakamura,M, Bringas P Jr, Nanci A, Zeichner-David M, Ashdown B, Slavkin HC (1994): Translocation of enamel proteins from inner enamel epithelia to odontoblasts during mouse tooth development. Anat.Rec, 238:383-396.

Nanci A, Kawaguchi H, KogayaY (1994): Ultrastructural studies and immunolocalization of enamel proteins in rodent secretory stage ameloblasts processed by various cryofixation methods. Anat Rec, 238:425-436.

Nanci A, McCarthy GF, Francis Zalzal S, Clokie CML, Warshawsky H, McKee MD (1994): Tissue response to titanium implants in the rat tibia: ultrastructural, immunocytochemical and lectin-cytochemical characterization of the bone-titanium interface. Cells Mater, 4:1-30.

Grenier D, Groleau D, Nanci A (1993): Characterization of the wheat germ agglutinin-binding property of Treponema denticola. J Periodont Res, 28:211-218. Lectins were used to characterize glycoconjugates on the cell surface of Treponema denticola, a suspected periodontopathogen. Bacteria were first screened by light microscopy using fluorescein isothiocyanate-coupled lectins. Wheat germ agglutinin (WGA) showed a high reactivity to T. denticola. While the WGA-binding activity was accentuated following heating or detergent treatments of bacterial cells, the reaction was inhibited by incorporation of competing carbohydrates. Scanning and transmission electron microscope studies were conducted in order to characterize the distribution of the WGA-binding sites on the cell surface of T. denticola. Data from these studies confirmed that heat treatment increases the percentage of labeled profiles and suggest that the WGA-binding sites are concentrated on specific regions on the spirochete surface. Initial biochemical analysis indicated that the high reactivity to WGA resides in a peptidoglycan fraction....

Kawaguchi H, McKee MD, Okamoto H, Nanci A (1993): Immunocytochemical and lectin-gold characterization of the interface between alveolar bone and implanted hydroxyapatite in the rat. Cells Mater, 3:337-350.

McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A (1993): Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and a 2 HS-glycoprotein) proteins in rat bone. J Bone Miner Res, 8:485-496.

McKee MD, Nanci A (1993): Ultrastructural, cytochemical and immunocytochemical studies on bone and its interfaces. Cells Mater, 3:219-243.

Nanci A, Francis Zalzal S, Kan, FWK (1993): High-resolution scanning electron microscopy of rat incisor ameloblasts. Scan Microsc, 7:165-175.

Nanci A, Francis Zalzal S, Kogaya Y (1993): Cytochemical characterization of basement membranes in the enamel organ of the rat incisor. Histochemistry, 99:321-331.

Smith CE, Nanci A, DenBesten PK (1993): Effects of chronic fluoride exposure on morphometric parameters defining the stages of amelogenesis and ameloblast modulation in rat incisors. Anat Rec, 237:243-258.

Kogaya Y. Nanci A. (1992): Post-embedding staining with high-iron diamine-thiocarbohydrazide-silver proteinate and its application to visualizing sulfated glycoconjugates in cryo-fixed kidney and cartilage. J Histochem Cytochem, 40:1257-1267.

Landis WJ, Hodgens KJ, McKee MD, Nanci A, Song MJ, Kiyonaga,S, Arena J, McEwen B (1992): Extracellular vesicles of calcifying turkey leg tendon characterized by immunocytochemistry and high voltage electron microscopic tomography and 3-D graphic image reconstruction. Bone Miner, 17:237-241.

McKee MD, Glimcher MJ, Nanci A (1992): High-resolution immunolocalization of osteopontin and osteocalcin in bone and cartilage during endochondral ossification in the chicken tibia. Anat Rec, 234:479-492.

Nanci A, McKee MD, Smith CE (1992): Immunolocalization of enamel proteins during amelogenesis in the cat. Anat Rec, 233:335-349.

Nanci A, Mazariegos M, Fortin M. (1992): The use of osmicated tissues for Lowicryl K4M embedding. J Histochem Cytochem, 40:869-874.

Smith CE, Dahan S, Fazel A, Lai W, Nanci A (1992): Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse-chase labeling with L- [ 35S] - and L- [methyl- 3H] -methionine. Anat Rec, 232:1-14.

Berdal A, Nanci A, Smith CE, Ahluwalia JP, Thomasset M, Cuisinier-Gleizes P, MathieuH. (1991): Differential expression of calbindin-D 28 kDa in rat incisor ameloblasts throughout enamel development. Anat Rec, 230:149-163.

McKee MD, Nanci A, Landis WJ, Gotoh Y, Gerstenfeld LC, Glimcher MJ (1991): Effects of fixation and demineralization on the retention of bone phosphoprotein and other matrix components as evaluated by biochemical analyses and quantitative immunocytochemistry. J Bone Miner Res, 6:937-945.

Gerstenfeld LC, Gotoh Y, McKee MD, Nanci A, Landis WJ, Glimcher MJ (1990): Expression and ultrastructural immunolocalization of a major 66 kDa phosphoprotein synthesized by chicken osteoblasts during mineralization in vitro. Anat Rec, 228:93-103.

McKee MD, Nanci A, Landis WJ, Gotoh Y, Gerstenfeld LC, Glimcher MJ (1990): Developmental appearance and ultrastructural immunolocalization of a major 66 kDa phosphoprotein in embryonic and post-natal chicken bone. Anat Rec, 228:77-92.

Nanci A, Francis Zalzal S, Smith CE (1990): Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections. J Histochem Cytochem, 38:403-414.

1989-1980

Gerstenfeld LC, Lian JB, Gotoh Y, Lee DD, Landis WJ, McKee MD, Nanci A, Glimcher MJ. (1989): Use of cultured embryonic chicken osteoblasts as a model of cellular differentiatiom and bone mineralization. Connect.Tissue Res, 21:215-225.

McKee MD, Nanci A, Landis WJ, Gerstenfeld LC, Gotoh Y, Glimcher MJ (1989): Ultrastructural immunolocalization of a major phosphoprotein in embryonic chick bone. Connect Tissue Res, 21:21-29.

McKee MD, Warshawsky H, Nanci A (1989): Cyclical incorporation of 33P into rat incisor enamel in vivo as visualized by whole-mount radioautography. Arch.Oral Biol, 34:989-993.

Nanci A, Ahluwalia JP, Pompura JR, Smith CE (1989): Biosynthesis and secretion of enamel proteins in the rat incisor. Anat.Rec, 224:277-291.

Nanci A, Ahluwalia JP, Francis Zalzal S, Smith, CE (1989): Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor. J Histochem Cytochem, 37:1619-1633.

Smith CE, Pompura JR, Borenstein S, Fazel A, Nanci A (1989): Degradation and loss of matrix proteins from developing enamel. Anat Rec, 224:292-316.

Smith CE, Nanci A (1989): A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection. Anat Rec, 225:257-266.

Smith CE, Nanci A (1989): Secretory activity as a function of the development and maturation of ameloblasts. Connect Tissue Res, 22:147-156.

Kan FWK, Nanci A (1988): Backscattered electron imaging of lectin binding sites in tissues following freeze-fracture cytochemistry. J Electron Microsc Tech, 8:363-370.

Landis WJ, Burke G.Y, Neuringer JR, Paine M.C, Nanci A, Bai P, Warshawsky H (1988): Earliest enamel deposits of the rat incisor examined by electron microscopy, electron diffraction, and electron probe microanalysis. Anat Rec, 220:233-238.

McKee MD, Wedlich L, Pompura JR, Nanci A, Smith CE, Warshawsky H. (1988): Demonstration by staining and autoradiography of cyclical distributions of protein at the enamel surface in rat incisors. Arch.Oral Biol, 33:413-423.

Nanci A, Smith CE (1988): Les protéines de l'émail dentaire. Etude immunocytochimique de l'amélogenèse. M/S , 4:42-45.

Slavkin HC, Bessem C, Bringas P Jr, Zeichner-David M, Nanci A, Snead ML (1988): Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis. Differentiation, 37:26-39.

Bendayan M, Nanci A, Kan FWK (1987): Effect of tissue processing on colloidal gold cytochemistry. J Histochem Cytochem, 35:983-996.

Camarda AJ, Butler WT, Finkelman RD, Nanci A (1987): Immunocytochemical localization of gamma-carboxyglutamic acid-containing proteins (osteocalcin) in rat bone and dentin. Calcif Tissue Int, 40:349-355.

McKee MD, Warshawsky H, Nanci A (1987): Use of backscattered electron imaging on developed radioautographic emulsions: Application to viewing rat incisor enamel maturation pattern following 45calcium injection. J Electron Microsc Tech, 5:357-365.

Nanci A, Francis Zalzal S, and Smith,C.E. (1987): Application of backscattered electron imaging and lectin-gold cytochemistry to visualize the distribution of glycoconjugates in a basal lamina. Scan.Microsc, 1:1963-1970.

Nanci A, Slavkin HC, Smith CE (1987): Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors. Anat Rec, 217:107-123.

Nanci A, Slavkin HC, Smith CE (1987): Application of high-resolution immunocytochemistry to the study of the secretory, resorptive, and degradative functions of ameloblasts. Adv Dent Res, 1:148-161.

Nanci A, Uchida T, Warshawsky H (1987): The effects of vinblastine on the secretory ameloblasts: An ultrastructural, cytochemical, and immunocytochemical study in the rat incisor. Anat Rec, 219:113-126.

Smith CE, McKee MD, Nanci A (1987): Cyclic induction and rapid movement of sequential waves of new smooth-ended ameloblast modulation bands in rat incisors as visualized by polychrome fluorescent labeling and GBHA-staining of maturing enamel. Adv Dent Res, 1:162-175.

Warshawsky,H, Bai,P, Nanci A (1987): Analysis of crystallite shape in rat incisor enamel. Anat.Rec, 218:380-390.

Babaï F, Nanci A, Affoyon F (1986): Caractères ultrastructuraux de la régression de l'hépatome de Novikoff transplanté chez des rats immunisés. Ann Pathol, 6:305-312.

Bendayan M, Nanci A, Herbener GH, Grégoire S, Duhr M-A (1986): A review of the study of protein secretion applying the protein A-gold immunocytochemical approach. Am J Anat, 175:379-400.

Nanci A, Bendayan M Slavkin HC (1985): Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry. J.Histochem.Cytochem, 33:1153-1160.

Nanci A, Warshawsky H (1984): Characterization of putative secretory sites on ameloblasts of the rat incisor. Am.J.Anat, 171:163-189.

Nanci A, Warshawsky H (1984): Relationship between the quality of fixation and the presence of stippled material in newly formed enamel of the rat incisor. Anat Rec, 208:15-31.

Nanci A, Bai P, Warshawsky H (1983): The effect of osmium postfixation and uranyl and lead staining on the ultrastructure of young enamel in the rat incisor. Anat.Rec, 207:1-16.

Nanci A, Bringas P Jr, Samuel N, Slavkin HC (1983): Selachian tooth development: III Ultrastructural features of secretory amelogenesis in squalus acanthias. J Craniofac Genet Dev Biol, 3:53-73.

Samuel N, Bringas P Jr, Santos V, Nanci A, Slavkin, HC (1983): Selachian tooth development: I. Histogenesis, morphogenesis, and anatomical features in squalus acanthias. J Craniofac Genet Dev Biol, 3:29-41.

Slavkin HC, Samuel N, Bringas P Jr, Nanci A, Santos V (1983): Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in squalus acanthias. J Craniofac Genet Dev Biol, 3:43-52.

Warshawsky H, Nanci A (1982): Stereo electron microscopy of enamel crystallites. J Dent Res, 61:1504-1514.

Nanci A, Babaï F, Dumont A (1980): Quantitative and ultrastructural changes of peritoneal macrophages in immunized rats after intraperitoneal injection of Novikoff hepatoma. J Nat Cancer Inst, 65:1273-1283.

 
© 2011 Pedro A. Segura
Faculty of Dentristy