Biomedical subjects

A Nanci

Publications and source records attributed to A Nanci.

At least 19 recordsLinked to original sources

PubMed2006

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector.

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. The transduction efficiency of the LVs was evaluated using the MC3T3 cell line and primary calvaria-derived osteogenic cells. For in vivo delivery, the LVs were infused using osmotic minipumps through holes created in the bone of the rat hemimandible and tibia. The production of GFP and AMBN in vitro and in vivo was monitored using fluorescence microscopy. Both transgenes were expressed in MC3T3 and primary osteogenic cells. In vivo, GFP was detected at the infusion site and fibroblast-like cells, osteoblasts, osteocytes and osteoclasts expressed AMBN. Our data demonstrate, for the first time, that primary osteogenic cells are efficiently transduced with LVs and that their infusion is advantageous for locally delivering DNA to bone cells.

Animals↗

PubMed2003

Quantitative immunogold labeling of bone sialoprotein and osteopontin in methylmethacrylate-embedded rat bone.

Methylmethacrylate (MMA) embedding of undecalcified bone is routinely employed for histomorphometric analyses. Although MMA-embedded bone has been used for immunolabeling at the light microscopic level after removal of the resin, there are no such reports for electron microscopy. The aim of the present study was to determine whether MMA embedding can be used for ultrastructural immunolabeling and how it compares to LR White (LRW), an acrylic resin frequently used for immunocytochemistry of bone. Rat tibiae were fixed by vascular perfusion with aldehyde and embedded either in MMA or LRW resin. Thin sections were processed for postembedding protein A-gold immunolabeling with antibodies to rat bone sialoprotein (BSP) and osteopontin (OPN). The density of gold particles over bone was quantified. The density and distribution of immunolabeling for BSP and OPN respectively, were comparable between MMA and LRW. These results indicate that MMA performs as well as LRW for the ultrastructural immunolabeling of noncollagenous bone matrix proteins.

Acrylic Resins↗

PubMed2001

Immunodetection of noncollagenous matrix proteins during periodontal tissue regeneration.

The interface between denuded dentin and regenerative periodontal tissue was investigated in a rat alveolar bone defect model using morphological and immunocytochemical approaches. The dentin surface was surgically exposed along the palatal roots of maxillary first molars. At 3 weeks post treatment, animals were perfused and treated regions from decalcified 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 fibronectin) and plasma (alpha2HS-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. This zone sometimes showed an electron-dense, cement line-like, planar accumulation of organic material immunoreactive for osteopontin and bone sialoprotein. Immunolabeling for osteocalcin and alpha2HS-glycoprotein was moderate and diffuse throughout the interfacial zone, whereas labeling with antibodies to albumin and fibronectin resulted in a weak reaction. It is concluded that accumulation of bone sialoprotein and osteopontin is a primary event during the formation of regenerative cementum onto denuded root surfaces.

Animals↗

PubMed2001

The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study.

In a previous report we showed that young rats fed a calcium-free diet for 28 days developed severe hypocalcaemia and showed a significant increase in serum alkaline phosphatase activity. The main histological and cytochemical changes exhibited by these animals in bone of the metaphyseal primary spongiosa were: (1) hyperplasia of osteoblasts, (2) an increase in the frequency of tartrate-resistant acid phosphatase (TRAP)-positive osteoblasts apposed to osteoid, and (3) an excessive amount of osteoid tissue. In addition to typical osteoblasts, there was a subpopulation of osteoblast-like cells with coated pits, lysosome-like bodies and large cytoplasmic processes. In the present study, we investigated how the above parameters change when calcium-depleted rats are placed on a normal diet for 7 days. Such a regimen normalized calcium concentration and alkaline phosphatase activity in the serum. The osteoid thickness returned to normal and, in some areas, was fully calcified. Most osteoblasts no longer showed TRAP activity and their ultrastructure was similar to that found in controls. Despite an intense alkaline phosphatase activity, some of them still exhibited a number of macrophagic characteristics. They were TRAP-positive, and showed electron-dense bodies in the cytoplasm facing bone, an abundance of coated pits, calcified spicules impinging on the cell membrane and large processes extending into the mineralized matrix. We concluded that calcium deficiency causes hyperplasia of osteoblasts in primary spongiosa and an increase in expression of TRAP. It also induces changes in their phenotype characterized by the acquisition of macrophagic cellular features. While TRAP activity is normalized by calcium repletion, macrophagic characteristics persist. These results suggest that the osteoblast can modulate its phenotype according to its physiological status.

Acid Phosphatase↗

PubMed2001

Localized infusion of tunicamycin in rat hemimandibles: alteration of the basal lamina associated with maturation stage ameloblasts.

At the beginning of the maturation stage of amelogenesis, ameloblasts deposit a basal lamina (BL) at the interface between their apical surface and maturing enamel. This structure is rich in glycoconjugates and is proposed to exhibit adhesive and/or filtering functions. To clarify its role, we have applied a recently developed surgical window model to locally administer tunicamycin (TM), an antibiotic that interferes with N-glycosylation, in the rat hemimandible using an osmotic minipump. Male Wistar rats were infused with either TM or saline as a control. Lectin-gold cytochemistry was performed to reveal glycoconjugates in the BL. Immunogold labeling of enamel proteins and albumin was carried out to verify whether depletion of N-linked sugars in the BL affects the content and distribution of endogenous and exogenous proteins in the enamel layer. Under the influence of the drug, the BL became irregular and exhibited alterations in structural organization and composition. The number of Helix pomatia agglutinin binding sites was not significantly affected but their distribution was altered. The labeling density of wheat germ agglutinin over the BL was slightly reduced. Immunoreactivity for enamel proteins showed only a small decrease, but that of albumin, both between ameloblasts and within the enamel layer, increased significantly. No structural alterations were observed in the contralateral incisor and in other sampled tissues and organs. These results demonstrate that it is possible to achieve a localized administration of TM without systemic side effects and lend support to the proposal that the BL represents a specialized structure with filtering functions.(J Histochem Cytochem 49:165-176, 2001)

Acetylgalactosamine↗

PubMed2001

Immunochemical characterization of a chicken egg yolk antibody to secretory forms of rat incisor amelogenin.

Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentino-enamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.

Amelogenin↗

PubMed2001

High-resolution immunocytochemistry of noncollagenous matrix proteins in rat mandibles processed with microwave irradiation.

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. Sections of incisor, molars, and alveolar bone were processed for postembedding colloidal gold immunolabeling using antibodies against ameloblastin, amelogenin, bone sialoprotein, or osteopontin. Ultrastructural preservation of tissues was in most cases comparable to that obtained by perfusion-fixation, and there was no difference in distribution of labeling with those previously reported for the antibodies used. However, the immunoreactivities obtained were generally more intense, particularly at early stages of tooth formation. Amelogenin was abundant between differentiating ameloblasts and labeling for osteopontin appeared over the Golgi apparatus of odontoblasts after initiation of dentine mineralization. We conclude that MW irradiation represents a simple method that can accelerate the processing of calcified tissues while yielding good structural preservation and antigen retention. (J Histochem Cytochem 49:1099-1109, 2001)

Amelogenin↗

PubMed2001

Alkaline phosphatase and tartrate-resistant acid phosphatase in osteoblasts of normal and pathologic bone.

A review of histochemical and immunohistochemical studies on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) in osteoblasts leads to the following conclusions: 1) AP is a typical (non-specific) marker of osteoblasts, and TRAP is a typical (non-specific) marker of osteoclasts; 2) both enzymes may be used to identify the precursors of these cells and are found in young osteocytes; 3) both are released into the extracellular space; 4) both are expressed by metaphyseal osteoblast-like cells whose ultrastructural characteristics are similar to those of post-osteoclastic cells of the basic multicellular unit (BMU) and are also exhibited by osteoblasts and macrophages; 5) the increased numbers of these cells in hypocalcemic animals suggests that the local calcium ion concentration may trigger the transition of the reversal into the formation phase of the BMU.

Acid Phosphatase↗

PubMed2000

Purification and characterization of beta-actin-rich tumor cell pseudopodia: role of glycolysis.

The MSV-MDCK-INV invasive variant of Moloney sarcoma virus (mos) transformed MDCK cells express multiple beta-actin-rich pseudopodia (P. U. Le et al., Cancer Res. 58, 1631-1635, 1998). We show here that the tips of these actively protruding cellular domains are morphologically distinct presenting numerous blebs and selectively pass through 1-microm-pore filters. The pseudopodia were purified from the underside of the filters and a major protein component was identified as the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). By confocal microscopy, GAPDH colocalized with actin in MSV-MDCK-INV pseudopodia localizing this glycolytic enzyme to this site of active actin polymerization. Inhibition of glycolysis with 2-deoxyglucose or oxamate induced a rapid transformation of beta-actin-rich pseudopodia into extended lamellipodia and prevented cell motility. A localized glycolytic supply of energy therefore regulates the formation of beta-actin-rich pseudopodial protrusions and thereby the motility of invasive tumor cells.

Actins↗

PubMed2000

Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff- and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.

Albumins↗

PubMed2000

A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats.

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.

Acid Phosphatase↗

PubMed1999

Content and distribution of noncollagenous matrix proteins in bone and cementum: relationship to speed of formation and collagen packing density.

The organic matrix of collagen-based calcified tissues consists of a supporting collagen meshwork and various noncollagenous matrix proteins (NCPs). Together, they contribute to determining the structure and biomechanical properties of the tissue. Their respective organization and interrelation can advantageously be examined by immunocytochemistry, an approach which allows correlation of composition with structure. The aim of this article is to review postembedding immuno- and lectin-gold-labeling data on the characterization of the noncollagenous compartment in rat and human bone and cementum, and on its relationship to collagen. The two major NCPs, bone sialoprotein and osteopontin, generally codistribute and accumulate in cement lines and in the spaces among the mineralized collagen fibrils. However, there are variations in their distribution and density of labeling throughout the tissue. Indeed, bone and cementum can form in environments that are either poor or enriched in NCPs. The amount of NCPs generally correlates with bone and cementum types and with speed of formation of the tissue and packing density of collagen fibrils. Taken together, the data suggest that production of both collagenous and noncollagenous constituents can be "modulated" during formation of collagen-based calcified tissues. It is concluded that, in addition to structural and compositional parameters, tissue dynamics must be taken into consideration in order to understand the significance of the apparent accumulation of NCPs at some sites and to determine the mechanisms of normal and pathological calcified tissue formation.

Animals↗

PubMed1999

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.

Adsorption↗

PubMed1999

In vivo model for the experimental manipulation of calcified tissues: a surgical approach for accessing the odontogenic organ and associated tissues of the rat incisor.

The tooth organ is extensively used in developmental biology to investigate organogenesis and cell differentiation. It also represents an advantageous system for the study of the various cellular and extracellular matrix events that regulate the formation of both collagenous and noncollagenous calcified tissues. This article describes an in vivo surgical approach to access and experimentally manipulate the tooth organ and supporting tissues of the rat incisor. By use of a dental drill, a "window" was created through the alveolar bone on the buccal aspect of the hemimandible at the apical end of the incisor. It is at this site that epithelial and mesenchymal precursors are situated and undergo cellular differentiation to give rise to cells of the odontogenic organ. Active bone remodeling is also observed in this area to accommodate posterior growth of the tooth. An osmotic minipump connected to the bony window through an outlet catheter was used for controlled and continuous administration of experimental agents over a predetermined period of time. To validate the model, vinblastine sulfate, fetuingold, and dinitrophenylated albumin were thus infused. The animals were then sacrificed and the hemimandibles were processed for histological and immunocytochemical analyses. The effects of the drug and the presence of tracers were restricted to the treated hemimandible and were found in the enamel organ and pulp, as well as in the tooth supporting tissues. Cellular changes typically associated with the administration of vinblastine were obtained, and tracers were localized both in the extracellular milieu and within the endosomal/lysosomal elements of cells. These results suggest that this new surgical approach could serve as an advantageous in vivo model in which various chemical agents, therapeutic drugs, molecular probes are locally administered to study the molecular events that regulate calcified tissue formation.

Animals↗

PubMed1999

Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin.

Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues. (J Histochem Cytochem 47:1237-1245, 1999)

Ameloblasts↗

PubMed1998

Developmental appearance and distribution of bone sialoprotein and osteopontin in human and rat cementum.

BACKGROUND: 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 (immuno)cytochemistry. RESULTS: Incubations with antibodies against BSP and OPN and with lectins recognizing prominent sugars in these proteins generally revealed similar labeling patterns in both human and rat teeth, with gold particles accumulating mainly in the interfibrillar spaces. The lectin Helix pomatia, specific for N-acetyl-D-galactosamine, was distinctive in that it consistently reacted with human cementum, but only sporadically labeled rat cementum. Regardless of both the species and the stage of root development, mineralization initiated in mantle predentin in association with distinct foci immunoreactive for BSP and OPN. In human teeth, the deposition of cementum collagen began before the start of dentin mineralization and thus prior to any detectable labeling for BSP and OPN. However, at early stages of root formation in the rat, cementum collagen appeared after BSP and OPN accumulated on the root surface, whereas at advanced stages the deposition of cementum collagen, BSP and OPN coincided. CONCLUSIONS: The temporo-spatial differences in the appearance of BSP and OPN relative to cementum collagen correlate well with known differences in the speed of root elongation and explain the variable appearance of the dentino-cemental junction. The data reveal no causal relationship between BSP and OPN and the differentiation of cementoprogenitor cells and indicate that the distribution of collagen fibrils ultimately determines the amount and pattern of accumulation of these NCPs. There also is no consistent planar accumulation of BSP and OPN between dentin and cementum such as the cement lines found between "old" and "new" bone. It is concluded that the interlacement of collagen fibrils at the dentino-cemental junction, across which mineralization spreads, represents the primary attachment mechanism between cementum and dentin.

Adolescent↗

PubMed1998

Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue.

Enzymatic digestion of bone tissue potentially releases a mixture of precursor, differentiating, and mature cells. Conceptually, early fetal osteogenic tissue should provide a more uniform population of cells than late embryonic or newborn bone in which cells have already differentiated. In this context, we have applied sequential enzymatic digestion to obtain and culture cells from 15-16-day fetal rat cranial tissue, a developmental age where deposition of bone matrix has not yet started at this site. These cultures were compared with those of osteogenic cells isolated from newborn rat calvariae and grown under similar conditions. Matrix production and composition were examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The plated cells formed mineralized nodules by day 14. The presence of mineral was determined by von Kossa staining and backscattered electron imaging (BEI), and the accumulation of calcium and phosphorus within the nodules was demonstrated by X-ray microanalysis and elemental mapping. At early time intervals, cells were generally cuboidal in shape and showed a well-developed Golgi apparatus, which occasionally was immunoreactive for OPN. Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery, of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface. Osteocalcin immunoreactivity was also associated with electron-dense portions of the bone-like matrix. These data demonstrate the potential of presumptive fetal rat calvarial cells to form a bone-like matrix in vitro and suggest that the assembly and mineralization pattern show similarities to the process of intramembranous ossification. Such a culture system is of interest not only for studying cellular and matrix events of bone formation, but also factors which influence mesenchymal cells in committing themselves to the osteogenic pathway.

Animals↗

PubMed1998

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.

Biological Factors↗