Sylvia Francis Zalzal

Chemical modification of titanium surfaces for covalent attachment of biological molecules

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. Surface analyses following coating procedures consisted of X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Enzymatic activity of coupled alkaline phosphatase was assayed colorimetrically, and surface coverage by bound albumin was evaluated by SEM visualization of colloidal gold immunolabeling. Our results indicate that the linkage of the aminoalkylsilane to the oxidized surface is stable and that bound proteins such alkaline phosphatase and albumin retain their enzymatic activity and antigenicity, respectively. The density of immunolabeling for albumin suggests that the binding and surface coverage obtained is in excess of what would be expected for inducing biological activity. In conclusion, this method offers the possibility of covalently linking selected molecules with known biological activity to oxidized titanium surfaces in order to guide and promote the tissue healing that occurs during implant integration in bone and soft tissues.

Improving biocompatibility of implantable metals by nanoscale modification of surfaces: an overview of strategies, fabrication methods, and challenges.

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.

Comparative Immunochemical Analyses of the Developmental Expression and Distribution of Ameloblastin and Amelogenin in Rat Incisors

SUMMARY Mineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and mid-to late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 μm to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation.

Extracellular matrix in tooth cementum and mantle dentin: Localization of osteopontin and other noncollagenous proteins, plasma proteins, and glycoconjugates by electron microscopy

Noncollagenous proteins (NCPs) are considered to have multiple functions related to the formation, turnover, and repair of the collagen‐based mineralized tissues. Collectively, they comprise a class of generally acidic, mineral‐binding proteins showing extensive posttranslational modifications, including glycosylation, phosphorylation, and sulfation.

Nanoscale Oxidative Patterning of Metallic Surfaces to Modulate Cell Activity and Fate

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.

Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures

As part of ongoing studies aimed at clarifying the early events of bone matrix deposition and mineralization, we have characterized primary osteoblast cultures using ultrastructural and immunocytochemical methods. Osteogenic cells were isolated by sequential enzymatic digestion of newborn rat (2–4‐day‐old) calvariae and grown for periods of 7 to 28 days on polystyrene, Thermanox plastic, or sputtered titanium. Bone‐like nodules, showing a stratified organization of cells and collagen, were examined by scanning and transmission electron microscopy, and further characterized for mineral by backscattered electron imaging and X‐ray microanalysis. Colloidal gold immunocytochemistry was used to examine the distribution of osteopontin in these nodules. Cells at the surface of the nodules were rounded, while those within the nodules generally appeared more flattened. Both cell types, particularly at early culture intervals, exhibited well‐developed protein synthetic organelles. Collagen fibrils were present between the cell layers and some individual fibrils appeared mineralized. Aggregates of needle‐shaped crystallites were sometimes apposed to the cell surface, frequently within invaginated regions of the cell membrane, while other mineralized masses of various sizes were present within the collagenous scaffolding. The periphery of the mineralized masses was often delimited by an electron‐dense, lamina limitans‐like layer. Focal accumulations and/or a more complete layer of afibrillar, mineralized organic matrix were sometimes observed at the interface between the cells and the surface of the culture dish. Osteopontin was immunodetected over the afibrillar and collagenous mineralized matrix throughout the cultures and, in some cases, labeling was concentrated over the peripheral, electron‐dense material delimiting the mineralized masses. In conclusion, these data indicate that calvaria‐derived osteoblasts produce an extracellular matrix with structural and compositional similarities to bone. Although not a regular observation, the accumulation of osteopontin on the surface of the culture substrate and at the periphery of masses of mineralized matrix may be analogous to what takes place in vivo at naturally occurring bone interfaces.

Expression profile of the embryonic markers nanog, OCT-4, SSEA-1, SSEA-4, and frizzled-9 receptor in human periodontal ligament mesenchymal stem cells

Mesenchymal stem cells (MSCs) are self‐renewing cells with the ability to differentiate into various mesodermal‐derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL‐MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL‐MSCs contain a subpopulation of frizzled‐9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA‐1, and SSEA‐4. They are additionally positive for nanog and Oct‐4; two critical transcription factors directing self‐renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP‐10. The presence of nanog, Oct‐4, SSEA‐1, and SSEA‐4 suggests that PDL‐MSCs are less differentiated than bone marrow‐derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled‐9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells. J. Cell. Physiol. 225: 123–131, 2010.

A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects.

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.

The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1−/− animals. The enamel of NBCe1−/− animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1−/− animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.

Early Expression of Bone Matrix Proteins in Osteogenic Cell Cultures

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.