Antonio Nanci

Understanding and controlling the bone–implant interface

A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, endosseous implants should result in formation of a characteristic interfacial layer and bone matrix with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the interface and of the effects biomaterials have on bone and bone cells is needed. Such knowledge is essential for developing strategies to optimally control osseointegration. This paper reviews current knowledge of the bone-biomaterial interface and methods being investigated for controlling it. Morphological studies have revealed the heterogeneity of the bone-implant interface. One feature often reported, regardless of implant material, is an afibrillar interfacial zone, comparable to cement lines and laminae limitantes at natural bone interfaces. These electron-dense interfacial layers are rich in noncollagenous proteins, such as osteopontin and bone sialoprotein. Several approaches, involving alteration of surface physicochemical, morphological, and/or biochemical properties, are being investigated in an effort to obtain a desirable bone-implant interface. Of particular interest are biochemical methods of surface modification, which immobilize molecules on biomaterials for the purpose of inducing specific cell and tissue responses or, in other words, to control the tissue-implant interface with biomolecules delivered directly to the interface. Although still in its infancy, early studies indicate the value of this methodology for controlling cell and matrix events at the bone-implant interface.

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.

Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro.

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 −/− animals. The Opn−/− 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 Opn−/− animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild‐type mice. Spleen cells from Opn−/− mice cells formed osteoclasts 3‐ to 13‐fold more frequently than did control Opn+/+ cells, while the extent of osteoclast development from Opn−/− bone marrow cells was about 2‐ to 4‐fold more than from the corresponding wild‐type cells. Osteoclast development occurred when Opn−/− spleen cells were differentiated in the presence of Opn−/− osteoblasts, indicating that endogenous OPN is not required for this process. These results suggest that OPN is not essential for normal mouse development and osteogenesis, but can modulate osteoclast differentiation.

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: Ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair

Currently available data describing the gene expression and regulation, secretion, distribution, and protein chemistry of osteopontin (OPN) all are consistent with the notions of this protein functioning as an inhibitor of mineralization and/or as a mediator of cell‐matrix and matrix‐matrix/mineral adhesion (cohesion) during the formation, turnover, and repair of normal and pathological mineralized tissues. The properties and overall integrity of mineralized tissues are in part dictated by the nature of their interfaces—sites where organic and inorganic components of the extracellular matrix interact to provide biomechanical strength, regulate mineral ion homeostasis, and influence cellular events involved in mineralized tissue modeling, remodeling, and repair. High‐resolution, colloidal‐gold immunocytochemistry has been used to characterize the proteinaceous composition of these interfaces and to establish that the phosphorylated sialoprotein, OPN, is a major component found at these sites where it accumulates as a dense, planar “coating” of organic material termed either a cement line or a lamina limitans. Structural/functional features of OPN predict an ability of this protein to regulate calcification in the matrix proper of mineralized tissues and to participate, more specifically, in cell‐matrix and matrix‐matrix/mineral adhesion in laminae limitantes and cement lines, respectively. From the ultrastructural immunocytochemical data presented herein for OPN illustrating the cellular expression and extracellular matrix distribution of this protein, it is demonstrated that the production of OPN is one of the earliest, and latest, secretory activities of the osteoblast lineage and that this activity manifests itself morphologically as a cement line or a lamina limitans, respectively, at bone matrix interfaces. In laminae limitantes at bone surfaces, OPN appears to be involved in osteoclast adhesion and possibly haptotaxis. An OPN‐containing cement line is also present at hard tissue interfaces in rat tooth, against osseointegrated titanium and hydroxyapatite implants and at the margins of surgically created bone defects—and there may influence biological adhesion in a manner similar to that proposed for normal bone. It is suggested, therefore, that in addition to its potential for influencing cell adhesion/dynamics in bones and teeth, OPN in cement lines may act as an interfacial adhesion promotor between apposing substrates, therein maintaining the overall integrity of bone during the bone remodeling sequence and “bonding” dissimilar tissues (or biocompatible materials) together in biological composites such as teeth and osseointegrated implants.

Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells

Bone formation around implants is influenced by surface geometry. Since cell/matrix/substrate interactions associated with cell signaling occur in the nanoscale dimension, we have evaluated the influence of nanotexturing of titanium-based surfaces on the expression of matrix proteins by cultured osteogenic cells at initial time points. Cells were obtained by enzymatic digestion of newborn rat calvaria and grown on titanium and titanium alloy discs with nanotextured or machined surfaces, and on glass coverslips for periods of 6 h, 1 day, and 3 days, under standard culture conditions. Cultures were processed for single or dual immunolabeling with monoclonal and/or polyclonal antibodies against bone sialoprotein (BSP), fibronectin (FN), osteopontin (OPN), type-I pro-collagen, or tubulin, followed by corresponding fluorophore-conjugated secondary antibodies. Some samples were processed for scanning electron microscope analysis of morphology and immunogold labeling. After 6 h, nanotextured surfaces exhibited up to a nine-fold increase in the proportion of cells with peripheral OPN labeling. At day 3, the proportion of OPN and BSP labeled cells was higher, and the intensity of immunoreactivity dramatically increased. No significant differences were observed in the expression pattern and the proportion of cells immunoreactive for FN or type-I pro-collagen. Our results demonstrate that nanotexturing of titanium-based surfaces upregulates the early expression of BSP and OPN in osteogenic cell cultures.

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.

Osteopontin: An Interfacial Extracellular Matrix Protein in Mineralized Tissues

Among the noncollagenous matrix proteins found in mineralized tissues (MTs), colloidal-gold immunocytochemistry has demonstrated that the ultrastructural distribution of osteopontin (OPN) is unique in that this protein preferentially accumulates at MT interfaces. In bone, OPN is present as a major component of cell- and matrix-matrix interfacial structures termed laminae limitantes and cement lines, respectively. Here, we review the implications of this distinct, interfacial tissue distribution as it relates to the properties and functional motifs of OPN (e.g. RGD, polyAsp, phosphorylation) in different MTs, and more specifically, how it pertains to current theory on the cellular and extracellular matrix (ECM) events associated with bone remodeling. The production of OPN as one of the earliest, and latest, secretory activities of the osteoblast lineage is discussed, together with a consideration of the role of OPN in cement lines and laminae limitantes in bone and in other normal, pathological and healing MTs such as teeth, kidney stones, bone wound healing and implant osseointegration. Further to its ability to influence cell dynamics, calcification and possibly tissue cohesion in MTs, it is proposed that OPN in cement lines may also promote adhesion between apposing substrata. With regard to this latter function, it is suggested that the molecular interactions within, and biomechanical properties of, such an OPN-rich interfacial zone may be important in minimizing strain-induced fatigue damage and microcrack propagation in bone and across other MT interfaces.

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.