Biocoating of Electropolished and Ultra‐Hydrophilic Titanium and Cobalt Chromium Molybdenum Alloy Surfaces with Proteins

Bone morphogenetic proteins (BMPs) play a decisive role in bone development and osteogenesis. In the past they have been the subject of widespread research and clinical trials as stimulants of bone growth. Although recently recombinant human BMP‐2 (rhBMP‐2) has been chemically immobilized on implant surfaces leading to enhanced bone growth and accelerated integration in vivo, the non‐covalent immobilization of proteins on metal surfaces is still poorly understood, since the oxide layers on metals like titanium, stainless steel or cobalt chromium alloys are poor adsorbents of proteins. Protein binding surfaces could either be generated by linking ionic groups (ion‐exchange surface) or by coupling hydrophobic residues (hydrophobic interacting surface, HIS) to the surface. In this paper the preparation of protein adsorbing surfaces on titanium and cobalt chromium molybdenum alloy for the adsorption of rhBMP‐2 and ubiquitin will be described. rhBMP‐2 and ubiquitin are bound extremely tight to surfaces containing propyl or hexyl groups of a certain surface concentration and are slowly released over a range of at least 24–100 days making such surfaces applicable as long‐term drug delivery devices for enhancing bone growth or implant integration.     

Oberflächenmodifizierung von Titan zur Verbesserung der Grenzflächenverträglichkeit

Surface Modification of Titanium for Improvement of the Interfacial Biocompatibility We report the CVD‐polymerisation of amino‐functionalized [2,2]‐paracyclophane for polymer coating and functionalization of titanium surfaces. Additionally, the functionalization was carried out by silanization with 3‐aminopropyl‐triethoxysilane. The generated amino‐groups were used for covalent immobilization of bioactive substances to stimulate the adhesion and growth of osteoblasts. As bioactive substances the pentapeptide GRGDS and the growth factor BMP‐2 were chosen. The covalent bonding was achieved by activation with hexamethylene diisocyanate. Each modification step was characterized by X‐ray‐photoelectron‐spectroscopy (XPS), atomic force microscopy (AFM) and contact angle measurements. The covalent bonding of the bioactive substances was proven by radiolabelling and surface‐MALDI‐ToF‐MS. In vitro‐biocompatibility tests with primary, human osteoblasts demonstrated the improved cell adhesion and spreading on the bioactive modified titanium surfaces.     

Aspects of BMP‐2 binding to receptors and collagen: Influence of cell senescence on receptor binding and absence of high‐affinity stoichiometric binding to collagen.

On the basis of their osteoinductive properties bone morphogenetic proteins (BMPs) are used today in clinical therapy such as the treatment of spinal disorders or healing defects in tibia fractures. A major biochemical function of these molecules is their binding to a large number of other proteins to fulfil their biological role. Two of these BMP‐binding proteins are the cellular BMP‐receptor and the collagen molecule which has been implicated as a specific BMP binding protein from the beginning of BMP‐2 research. These two molecules are the subject of this paper. In receptor binding one must distinguish between the binding function (θ) and the state function (r). The binding function describes the equilibrium between effector (e.g. BMP‐2) and the receptor, whereas the state function describes the change in receptor activation elicted by the effector resulting in downstream effects such as the induction of alkaline phosphatase. In studies on the state function of rhBMP‐2 binding to MC3T3‐E1 cells it will be shown that the binding affinity of the receptor decreases fivefold in a highly significant manner (p <0.0002, n = 10, duplicate assays) with the serial passage number from 1.7 ± 0.3 nM as measured with passage 5 cells to 8.9 ± 1.5 nM as measured with passage 10 cells. Since the serial passage model of MC3T3‐E1 cells is a model of senescence it is concluded that the affinity of the rhBMP‐2 receptor interaction decreases with senescence i.e. age. This may have important consequences for the age dependent dosage of rhBMP‐2 in the clinic. In a second series of experiments it will be shown that contrary to widespread belief, BMP‐2 does not show a high affinity stoichiometric binding to collagen as measured by affinity chromatography on collagen Sepharose and in solution by gel filtration. A review of the literature and evaluation of binding stoichiometry, interestingly supported our finding since the calculated stoichiomerties indicate a binding of 10^‐3 to 10^‐4 mol BMP/mol tropocollagen. It is suggested that collagen is substituted by porous calcium phosphates and metals as carriers for BMP applications.     

Chemical Functionalization of a Hydroxyapatite Based Bone Replacement Material for the Immobilization of Proteins

Hydroxyapatite (HAP) has a great potential as a bone replacement material because of its similarity to the crystal structure of inorganic matrix of bone. Several years ago we showed that porous hydroxyapatite ceramics (Endobon, Merck) can be covalently modified for the immobilization of proteins [Jennissen, H.P. (1999) PCT Patent WO9926674A2] opening the possibility for the immobilization of growth factors and morphogens. Application of this technique to the immobilization of recombinant human bone morphogenetic protein 2 (rhBMP‐2) on hydroxyapatite would be of high medical interest, because of the decisive role of bone morphogenetic proteins in bone development and osteogenesis. In this paper it will be shown that the model protein ubiquitin and rhBMP‐2 can be immobilized non‐covalently and covalently after chemical modification of a hydroxyapatite ceramic surface (Bio‐Oss® Cancellous Block, Geistlich, = HAP‐wafers). It could be shown that only small amounts of protein are adsorbed to non‐functionalized HAP surfaces (control). In contrast ubiquitin and rhBMP‐2 can be very efficiently immobilized non‐covalently (up to 2.4 mg protein/g HAP) and covalently (up to 9.7 mg protein/g HAP) on porous HAP‐wafers. In desorption experiments it is shown that the bound rhBMP‐2 is slowly released making such surfaces applicable as long term drug delivery devices for enhancing bone growth and osteointegration of implant materials.     

Modell zur Analyse des knöchernen Anwachsverhaltens auf bioaktiv beschichteten Implantatoberflächen

Model to analyse the bone on‐growth on bioactive coated implant surfaces Especially on the field of bone regeneration, transient and permanent implants are an important method of therapy in the Orthopaedic Surgery. In this context, bioactive surfaces on metallic implants provide an improved contact to the surrounding bone. The goal of our study was to establish an in‐vitro test system to evaluate the on‐growth of bone‐derived cells on different surface coatings. Therefore, we invented a special kind of clamps made of commercially‐pure (c‐p) titanium and blasted with hydroxyapatite particles followed by electrochemically coating with calcium phosphate (BONIT®‐HA, BONIT®). Definite pieces of human cancellous bone were attached to these clamps, inserted onto tissue culture plates and cultivated in DMEM for ten days. Finally, the contact area between human cancellous bone and the implant surface was analyzed and the spreading of osteoblast‐like cells evaluated by scanning electron microscopy (SEM). A well‐spread morphology of bone cells was observed on the implant surfaces coated with calcium phosphate (CaP). In comparison the clamps without CaP coatings showed only a marginal growth of bone cells on the clamp surface. The presented newly in‐vitro test setup using titanium clamps coated with bioactive layers attached to human cancellous bone represents a well‐functioning model for qualitative evaluation of bone on‐growth.     

Osteogenic Response of C2C12 Cells on Thermoreversible Polymers

Bone Morphogenetic Proteins (BMPs) in combination with biomaterials are being explored for clinical bone regeneration. The current biomaterials used for BMPs delivery are not specifically designed to support BMP‐induced osteogenesis. Towards this goal, we designed synthetic N‐isopropylacrylamide (NiPAM)‐based thermosensitive polymers and investigated their ability to support osteogenic transformation of pluripotent C2C12 cells. Cell attachment to the polymers was limited as compared to attachment to the plastic surfaces optimized for cell culture. Short‐term (<7 days) studies indicated relatively little cell growth on the polymer surfaces. However, C2C12 cells retained their ability to respond to BMP‐2, as determined by alkaline phosphatase (ALP) induction, when cultured on thermoreversible polymers. Some polymers supported ALP induction that was far superior (～10‐fold) to cells grown on tissue culture surfaces. We conclude that thermosensitive polymers, although limited in their ability to support cell attachment and growth, did support the pluripotent cells' ability to be transformed under the influence of BMP‐2. The ALP induction was dependent on the compositional details of the polymers, suggesting that in vivo osteoinduction was likely to be influenced by the physicochemical properties of the polymers.     

Erforschung Ti–Co‐basierter,bioaktiver Auftraglötschichten auf oxidischen Hochleistungskeramiken in der Medizintechnik

The demand of prostheses and implants made from biomaterials grows as a result of the rising age of patients. For biomaterials, such as those found in joint‐ or hip‐prostheses, that are in direct contact with the organism, not only mechanical stability is required, but also biocompatibility as well as their ability to support bone regeneration. Taking this into account, a thin‐walled bioactive titanium cobalt‐based brazing coating on high‐performance oxide ceramics (Al₂O₃) has been developed. Here, the coating process offers an economical and at the same time technologically simple way for the coating ceramic materials. The biocompatible coating has been enhanced by addition of bioactive particles made of bioglass and calcium phosphates in order to improve bone formation. The reactions between the bioactive particles and the brazing alloys, as well as the particular melting behavior, were determined through thermo analytical methods. The structures of the brazing alloys enriched with bioactive particles were investigated through metallographical methods. The combination of three bioactive additives and two brazing alloys were analyzed in terms of their melting behavior and the resulting porosity, the parameters of the brazing process have been gradually optimized. The results show, that the combination of calcium phosphate particles and Ti–Co alloys effectively meet the requirements for a defined porous, biocompatible brazing coating.     

Stability of Surface‐Enhanced Ultrahydrophilic Metals as a Basis for Bioactive rhBMP‐2 Surfaces

For several years the treatment of metals like cp titanium and 316L stainless steel with concentrated chromosulfuric acid at high temperatures (230‐240 °C) has formed the basis for preparing ultra‐hydrophilic priming coats on these metals (Jennissen et al. Materialwiss. Werkstofftech. 30, 838‐845, 1999). Metals treated in this way have been called surface‐enhanced, displaying a characteristic ultrastructure, and can be easily modified to carry a biocoat of recombinant human bone morphogenetic protein 2 (rhBMP‐2). The major oxide on surface enhanced titanium is TiO₂. Thus this TiO₂‐layer could be responsible for the ultra‐hydrophilic properties of the priming coat. Irradiation of TiO₂ layers by ultraviolet light (Wang et al., Nature 388, 431‐432, 1997) has been shown to endow these layers with ultra‐hydrophilic properties (i.e. contact angles of ～ 0°). However the ultra‐hydrophilic TiO₂‐layers produced by irradiation are unstable and revert to the original high contact angles of ～ 70° within several days. The question of whether the ultra‐hydrophilic surfaces prepared by the chromosulfuric acid method show long‐term stability was therefore important to answer. In addition the question if rhBMP‐2 immobilized on such a surface will retain its biological activity was of great interest. In this paper it will be shown that ultrahydrophilic titanium mini‐plates retain their ultra‐hydrophilicity with contact angles of 0‐8° unchanged for at least 50 days and support the immobilization of rhBMP‐2 in a biologically active form.     

Preparation and properties of gold nanoparticle-electrodeposited titanium substrates with Arg-Gly-Asp-Cys peptides

Titanium metal has good biocompatibility, superior mechanical properties and excellent corrosion resistance. Like most metals, however, it exhibits poor bioactive properties and fails to bond to bone tissue. To improve its bioactivity, bioactive molecules, such as peptides, can be grafted onto titanium surfaces. In order to do this, the first step may be to establish a stable and compatible linking layer on the titanium surface. In this study, we used electrochemical methods to deposit gold (Au) nanoparticles onto titanium substrates, to which we then grafted arginine-glycine-asparagine-cysteine (RGDC) peptides by thiolate covalent coupling. Properties of electrodeposited Au nanoparticles were evaluated using a variety of techniques, including microstructural, chemical and electrochemical measurements. The biological responses of the RGDC-grafted Ti substrates were evaluated using MG3 human osteoblast-like cells. The results of thin-film X-ray diffraction (TFXRD) and scanning electron microscopy (SEM) indicated the polycrystalline orientation of Au nanoparticles deposited on the titanium surfaces with high density and controllable particle size. The RGDC peptide could be covalently bonded to Au-deposited Ti substrates via Au-thiolate species, as expected. Cell morphology showed that, on RGDC-immobilized titanium with Au particles, MG63 cells attached and spread more rapidly than on Ti substrates either without peptide or with direct loading of the peptide. Immunostaining for focal adhesion kinase (FAK) demonstrated that RGDC enhanced cell attachment. The present method for the formation of Au nanoparticles may serve as an alternative route for bioactive molecule immobilization on Ti implants.     

Surface modification of a novel porous titanium dioxide/glass composite

A novel technique is used to produce an open porous titanium dioxide/glass composite, named Ecopore, with promising structural and biological properties for the development as a bone graft. This study aims at a fast and lasting integration of the new material by means of biochemical surface modification. Surface etching of Ecopore, aminosilanization and covalent coupling of the cellular attachment mediator fibronectin was employed as modification strategy. In a comparison of different etching procedures, alkaline etching led to the highest density of amino functions after subsequent aminosilanization. Fibronectin was immobilized using a bifunctional aminoreactive PEG‐linker. This protein coating improved the attachment of human osteoblast‐like cells (HOB) on non‐porous Ecopore as displayed by vital staining. XTT metabolism assays indicated an enhanced HOB growth in the initial phase of cultivation on fibronectin‐coated versus non‐coated specimens. In a first feasibility study, cultivation of HOB on coated porous Ecopore cylinders with a median pore size diameter of 130 μm showed that cellular growth was uniform and dense on the external surface of the specimen, but was sparse in the interior pore system. Ecopore batches with larger pores will be modified and investigated in vitro and in vivo in the next step of the study.     

Functionalized Multi‐Wall Carbon Nanotubes for Lipase Immobilization

We examine the immobilization of lipase B from Candida antarctica on functionalized multi‐wall carbon nanotubes (MWCNTs) through physical adsorption. MWCNTs functionalized with carboxyl‐, amine‐ and ester‐ terminal groups on their surface are used as immobilization carriers. Dispersion of the nanotubes and the immobilization procedure take place in aqueous and low‐water media. High enzyme loadings are attained, up to 25% of the weight of the carbon nanotubes. These novel biomaterials are characterized though FT‐IR and Raman spectroscopy. The MWCNT–lipase bioconjugates exhibit high catalytic activity and increased storage and operational stability. The biomaterials retain more than 55% of their initial activity after 6 months at 4 °C, while they retain approximately 25% of their initial activity after 30 d of incubation in hexane at 60 °C. The catalytic behaviour of the immobilized enzyme depends on the terminal group of the carbon nanotubes, the concentration of the enzyme and the immobilization method employed.     

Two Dimensional Nanoarrays of Individual Protein Molecules

Protein molecules on solid surfaces are essential to a number of applications, such as biosensors, biomaterials, and drug delivery. In most approaches for protein immobilization, inter‐molecular distances on the solid surface are not controlled and this may lead to aggregation and crowding. Here, a simple approach to immobilize individual protein molecules in a well‐ordered 2D array is shown, using nanopatterns obtained from a polystyrene‐block‐poly(2‐hydroxyethyl methacrylate) (PS‐b‐PHEMA) diblock copolymer thin film. This water‐stable and protein‐resistant polymer film contains hexagonally ordered PS cylindrical domains in a PHEMA matrix. The PS domains are activated by incorporating alkyne‐functionalized PS and immobilizing azide‐tagged proteins specifically onto each PS domain using “Click” chemistry. The nanometer size of the PS domain dictates that each domain can accommodate no more than one protein molecule, as verified by atomic force microscopy imaging. Immunoassay shows that the amount of specifically bound antibody scales with the number density of individual protein molecules on the 2D nanoarrays.     

Simple Multipurpose Surface Functionalization by Phase Transited Protein Adhesion

Engineered surface modification is of prime importance in modern material science. This work reports how to manipulate a surface property towards ultra‐performance, multi‐functionalities, and smartness/responsiveness by utilizing newly‐discovered lysozyme phase transition. The phase‐transited lysozyme product consisting of amyloid‐contained microfiber network could stably attach onto metals, oxides, semiconductors, and polymers. Such priming processes imparts moderate hydrophilicity and enhanced corrosion resistance to surfaces. The priming also affords mild positive charges and enriched C‐H bonds on surfaces, which consequently supports the growth of a series of functional building blocks including polymer brushes, colloids, small molecules, and bio‐macromolecules based on chemically specific interactions. The bio‐related applications based on this strategy are further emphasized. First, a bioactive surface is conveniently obtained by this modification to specifically and sensitively detect biomarkers from buffer and undiluted serum. Second, a one‐pot protein immobilization method is developed, which features a high integration of all the protein immobilization steps in single incubation process. Third, a smart stimuli‐responsive surface that is capable of performing 100% reversible transition between bioactive and bioinert surface is constructed. This work lays a foundation for the use of phase‐transited proteins as a universal surface modification tool.     

Modelling of Protein‐Protein Interactions of Bone Morphogenetic Protein‐2 (BMP‐2) by 3D‐Rapid Prototyping

Recently we were able to apply the technique of 3D‐Rapid Prototyping (3D‐RP) to the construction of highly accurate three‐dimensional plastic models of biomolecules [Laub, M. et al. (2001), Materialwiss. Werkstofftech. 32, 926]. These models are derived from x‐ray crystallographic data and therefore represent exact replicas of the depicted molecules. Due to their accuracy these models should be suitable for the modelling of protein‐protein‐interactions. In a first study using 3D‐Rapid Prototyping models of bone morphogenetic protein‐2 (BMP‐2) we were able to identify a novel structural motive on the concave side of this protein which we termed anthelix since a left‐handed helix (radius ca. 0.8–1 nm, pitch 8–9 nm) can be fitted into this groove. Based on these structural findings we identified a 15mer polypeptide (KNMTPYRSPPPYVPP) from the Brookhaven database as a potential physiological ligand. Molecular docking studies using a geometric recognition approach confirmed the anthelix as a possible binding site for this peptide. However in affinity chromatography experiments no binding between BMP‐2 and the immobilized peptide was observed. As the question arose whether 3D‐Rapid Prototyping is in general suitable for modelling protein‐protein interaction we used dimeric BMP‐2 to study exemplary monomer‐dimer interaction. Molecular docking studies using the monomeric BMP‐2 subunits predicted a structure which is nearly identical to that found in dimeric BMP‐2 (root mean square deviation < 1 Å) proving the suitability of geometric docking. 3D‐RP‐BMP‐2‐monomers (size 140 mm × 75 mm × 65; magnification ca. 22 × 10⁶ fold) constructed from dimeric BMP‐2 could be assembled by hand yielding a structure highly homologous to dimeric BMP‐2. Differences between the 3D‐Rapid Prototyping model of dimeric BMP‐2 and the assembled monomers arose in several gaps at the interface between the two monomers which are not visible in the dimeric structure. These gaps can be explained by the way the solvent‐accessible molecular surface is generated. During this process an exterior probe sphere is rolled over the spherical atoms of the molecule. Distances between the monomers smaller than the diameter of this sphere are bridged thus resulting in a coherent surface. We conclude that 3D‐Rapid Prototyping is in general eligible for the modelling of protein‐protein‐interaction though there are further efforts needed to increase our understanding of this process.     

A Versatile Toolbox for Multiplexed Protein Micropatterning by Laser Lithography

Photocleavable oligohistidine peptides (POHP) allow in situ spatial organization of multiple His‐tagged proteins onto surfaces functionalized with tris(nitrilotriacetic acid) (tris‐NTA). Here, a second generation of POHPs is presented with improved photoresponse and site‐specific covalent coupling is introduced for generating stable protein assemblies. POHPs with different numbers of histidine residues and a photocleavable linker based on the 4,5‐dimethoxy‐o‐nitrophenyl ethyl chromophore are prepared. These peptides show better photosensitivity than the previously used o‐nitrophenyl ethyl derivative. Efficient and stable caging of tris‐NTA‐functionalized surfaces by POHPs comprising 12 histidine residues is demonstrated by multiparameter solid‐phase detection techniques. Laser lithographic uncaging by photofragmentation of the POHPs is possible with substantially reduced photodamage as compared to previous approaches. Thus, in situ micropatterning of His‐tagged proteins under physiological conditions is demonstrated for the first time. In combination with a short peptide tag for a site‐specific enzymatic coupling reaction, covalent immobilization of multiple proteins into target micropatterns is possible under physiological conditions.     

Biomimetic Titanium Alloy with Sparsely Distributed Nanotubes Could Enhance Osteoblast Functions

Designing the bone implant surface by mimicking the structure of natural tissue is an intriguing means to achieve better osseointegration. In this study, a biomimetic surface with sparsely distributed 80 nm diameter nanotubes (SNT) with a spacing of about 20–80 nm from each other, contrary to the closely distributed nanotubes on pure titanium, is fabricated by anodization of near β titanium alloy Ti‐5Zr‐3Sn‐5Mo‐15Nb (TLM). The structure is more similar to the cross‐section of collagen fibrils than commonly reported nanotubes on pure titanium. The SNT‐textured Ti‐alloy also shows good wettability and low elastic modulus more compatible with bone tissues. The surface bioactivity is evaluated in vitro by primary osteoblast cultures. Compared with the polished non‐textured TLM, the SNT texture exhibits satisfactory bioactivity with protein adsorption, initial cell adhesion, cell differentiation as revealed by ALP activity and osteogenesis‐related gene expression. Although cell proliferation is slightly suppressed, ECM deposition is enhanced. The enhanced cell functions are probably related to the biomimetic structure, biochemical characteristics, and mechanical properties of SNT‐textured Ti‐alloy as well as the potential fluid exchange effect among the nanotubes. The SNT texture which constitutes a bio‐interface with fluid supply from the implant surface can be tailored to enhance biological properties such as cell immigration and differentiation.     

Solid‐Supported Biomolecules on Modified Silica Surfaces—A Tool for Fast Physicochemical Characterization and High‐Throughput Screening

Biofunctionalization for a wide variety of applications can be achieved by coating silica surfaces with biomolecules such as lipids or proteins. However, specific surface optimization of the inorganic SiO₂ is necessary to achieve biocompatible surfaces. Surface shielded porous silica beads can be non‐covalently coated with a single lipid bilayer. The lipids retain their fluidity in this handy solid‐supported system, perfectly mimicking the soft‐surface properties of cellular membranes. A supramolecular architecture can also be used for functional immobilization of membrane proteins: An artificial cytosolic compartment can be created with the aid of polymers; coating by lipid membranes and integration of membrane proteins results in a solid‐supported biofunctional cellular surface. Another surface modification enables a direct immobilization of human serum albumin (HSA) molecules onto silica surfaces. The HSA on this otherwise passivated surface provides a convenient material for the investigation of unspecific protein binding of pharmaceuticals on a high‐throughput scale.     

Diatom‐Inspired Silica Nanostructure Coatings with Controllable Microroughness Using an Engineered Mussel Protein Glue to Accelerate Bone Growth on Titanium‐Based Implants

Silica nanoparticles (SiNPs) have been utilized to construct bioactive nanostructures comprising surface topographic features and bioactivity that enhances the activity of bone cells onto titanium‐based implants. However, there have been no previous attempts to create microrough surfaces based on SiNP nanostructures even though microroughness is established as a characteristic that provides beneficial effects in improving the biomechanical interlocking of titanium implants. Herein, a protein‐based SiNP coating is proposed as an osteopromotive surface functionalization approach to create microroughness on titanium implant surfaces. A bioengineered recombinant mussel adhesive protein fused with a silica‐precipitating R5 peptide (R5‐MAP) enables direct control of the microroughness of the surface through the multilayer assembly of SiNP nanostructures under mild conditions. The assembled SiNP nanostructure significantly enhances the in vitro osteogenic cellular behaviors of preosteoblasts in a roughness‐dependent manner and promotes the in vivo bone tissue formation on a titanium implant within a calvarial defect site. Thus, the R5‐MAP‐based SiNP nanostructure assembly could be practically applied to accelerate bone‐tissue growth to improve the stability and prolong the lifetime of medical implantable devices.     

Absence of genotoxic effects after exposure of mammalian cells to the recombinant human bone morphogenetic protein 2 (BMP‐2) prepared from E. coli

Recombinant human bone morphogenetic protein 2 (rhBMP‐2) has been widely employed for the induction of bone growth in animal models and in clinical trials. Since rhBMP‐2 is commercially available only to a very limited extent, we have prepared our own recombinant material for the current testing and for future applications. As yet, no toxicological data on rhBMP‐2 has been reported to the knowledge of the authors. Therefore, it was considered necessary to apply the in vitro‐micronucleus assay to our preparation to monitor a possible genotoxic effect. A total of approximately 24500 nuclei from V79 Chinese hamster cells and more than 40000 nuclei from human amniotic fluid fibroblast‐like (AFFL) cells were screened for the frequency of micronuclei following incubation with 3‐300 nM rhBMP‐2, which covers the concentration range from initial biological activity to the begin of a maximal response in MC3T3‐E1 cells. It can be concluded from the results that our preparations of recombinant human BMP‐2 prepared in E. coli do not cause DNA damage in the concentration range tested.     

Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns

Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.