Comparison of the capacity of enamel matrix derivative gel and enamel matrix derivative in liquid formulation to adsorb to bone grafting materials.

BACKGROUND: The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS: Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS: The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS: The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation.

Titanium implants are most commonly used for bone augmentation and replacement due to their favorable osseointegration properties. Here, hyperhydrophilic sand-blasted and acid-etched (SBA) titanium surfaces were produced by alkali treatment and their responses to partially heparinized whole human blood were analyzed. Blood clot formation, platelet activation and activation of the complement system was analyzed revealing that exposure time between blood and the material surface is crucial as increasing exposure time results in higher amount of activated platelets, more blood clots formed and stronger complement activation. In contrast, the number of macrophages/monocytes found on alkali-treated surfaces was significantly reduced as compared to untreated SBA Ti surfaces. Interestingly, when comparing untreated to modified SBA Ti surfaces very different blood clots formed on their surfaces. On untreated Ti surfaces blood clots remain thin (below 15 mm), patchy and non-structured lacking large fibrin fiber networks whereas blood clots on differentiated surfaces assemble in an organized and layered architecture of more than 30 mm thickness. Close to the material surface most nucleated cells adhere, above large amounts of non-nucleated platelets remain entrapped within a dense fibrin fiber network providing a continuous cover of the entire surface. These findings might indicate that, combined with findings of previous in vivo studies demonstrating that alkali-treated SBA Ti surfaces perform better in terms of osseointegration, a continuous and structured layer of blood components on the blood-facing surface supports later tissue integration of an endosseous implant.

Preparation of superhydrophilic microrough titanium implant surfaces by alkali treatment.

A new strategy to render intrinsically hydrophobic microrough titanium implant surfaces superhydrophilic is reported, which is based on a rapid treatment with diluted aqueous sodium hydroxide solutions. The physicochemical characterization and protein interaction of the resulting superhydrophilic implant surfaces are presented. The superhydrophilicity of alkali treated microrough titanium substrates was mainly attributed to deprotonation and ion exchange processes in combination with a strong enhancement of wettability due to the roughness of the used substrates. Albeit these minor and mostly reversible chemical changes qualitative and quantitative differences between the protein adsorption on untreated and alkali treated microrough titanium substrates were detected. These differences in protein adsorption might account for the enhanced osseointegrative potential of superhydrophilic alkali treated microrough implant surfaces. The presented alkali treatment protocol represents a new clinically applicable route to superhydrophilic microrough titanium substrates by rendering the implant surface superhydrophilic "in situ of implantation".

Stability and nonfouling properties of poly(poly(ethylene glycol) methacrylate) brushes under cell culture conditions.

This paper investigates the stability and nonfouling properties of poly(poly(ethylene glycol) methacrylate) (PPEGMA) brushes prepared by surface-initiated atom transfer radical polymerization from SiO(x) substrates modified with a trimethoxysilane-based ATRP initiator. At high chain densities, PPEGMA brushes were found to detach rapidly from glass or silicon substrates. Detachment of the PPEGMA brushes could be monitored with contact angle measurements, which indicated a decrease in the receding water contact angle upon detachment. Detachment of the PPEGMA brushes also resulted in an increase in nonspecific protein adsorption. The stability, and as a consequence the long-term nonfouling properties, of the PPEGMA brushes could be improved by tailoring the brush density and, to a lesser extent, the molecular weight of the polymer chains. By appropriate decrease of the grafting density, the stability of the brushes in cell culture medium could be improved from less than 1 to more than 7 days, without compromising the nonfouling properties.

RGD-Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells.

This report demonstrates the feasibility of surface-initiated atom transfer radical polymerization to prepare thin polymer layers ("brushes") that can be functionalized with short peptide ligands and which may be of use as coatings to promote endothelialization of blood-contacting biomaterials. The brushes are composed of poly(2-hydroxyethyl methacrylate) (PHEMA) or poly(poly(ethylene glycol) methacrylate) (PPEGMA), which do not only suppress non-specific adhesion of proteins and cells but also contain hydroxyl groups that can be used to introduce small peptide ligands. A protocol has been developed that allows functionalization of the brushes with RGD containing peptide ligands resulting in surface concentrations ranging from approximately 0.5-12 pmol/cm(2). At peptide surface concentrations >1-5.3 pmol/cm(2), human umbilical vascular endothelial cells (HUVECs) were found to adhere and spread rapidly. A difference in size and morphology of focal adhesions between HUVECs immobilized on PHEMA and PPEGMA brushes was observed. It is proposed that this is due to the increased ethylene glycol spacer length and hydrophilicity of the PPEGMA brushes, which may lead to increased ligand mobility and reduced ligand-integrin affinity. HUVECs immobilized on the polymer brushes were also found to be able to retain homeostasis when exposed to shear stresses that simulated arterial blood flow.

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins.

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.

A novel star PEG-derived surface coating for specific cell adhesion.

In this study a novel approach for the coating and functionalization of substrates for cell culture and tissue engineering is presented. Glass, silicon, and titanium panes were coated with an ultrathin film (30 +/- 5 nm) of reactive star-shaped poly(ethylene glycol) prepolymers (Star PEG). Homogeneity of the films was checked by optical microscopy and scanning force microscopy. These coatings prevent unspecific protein adsorption as monitored by fluorescence microscopy and ellipsometry. In order to allow specific cell adhesion the films were modified with linear RGD peptides (gRGDsc) in different concentrations. After sterilization, fibroblast, SaOS, and human mesenchymal stem cells (hMSC) were seeded on these substrates. Cell adhesion, spreading, and survival was observed for up to 30 days on linear RGD peptide (gRGDsc)-modified coatings, whereas no cell adhesion could be detected on unmodified Star PEG layers. By variation of the RGD concentration within the film the amount of cells that became adhesive could be controlled. When differentiation conditions are used for cultivation of hMSCs the cells show the expression of osteogenic marker genes after 14 days which is comparable to cultivation on cell culture plastic. Thus, the Star PEG/RGD film did not negatively influence the differentiation process. The high flexibility of the system considering the incorporation of biologically active compounds opens a broad field of future experiments.

Protein-functionalized polymer brushes.

A new strategy for the preparation of protein-functionalized polymer brushes is reported, which is based on a combination of surface-initiated atom transfer radical polymerization (ATRP), p-nitrophenyl chloroformate activation of the surface hydroxyl groups, and subsequent O(6)-benzylguanine (BG) functionalization. The BG-functionalized brushes are used to chemoselectively immobilize O(6)-alkylguanine-DNA-alkyltransferase (AGT) fusion proteins with a defined orientation and surface density. These protein-modified polymer brushes are attractive candidates for the development of protein microarrays.