Immobilizing a single growth factor such as bone morphogenetic protein 2 (rhBMP-2) on Implant materials can significantly increase the integration of implants into bone in animals [1,2]. For as yet unknown reasons the effective osteoinductive dose of rhBMP-2 in humans is ca. 100-1000-fold higher than in animals [3,4]. These unphysiologically high doses in humans have led to side effects and serious complications in spine fusion surgery [5]. In physiological secondary bone healing at least 10 different growth and differentiation factors are involved in the first two healing phases leading to a callus and/or woven bone. It is therefore hypothesized that the mandatory 10-100-fold dose reduction for a rhBMP-2 application in humans can be achieved by a combination of rhBMP-2 with a second essential mediator such as vascular endothelial growth factor (rhVEGF165). To this end a new generation of multimodal bioactive hybrid carriers with specific spatio-temporal release kinetics for each factor is being synthesized. Such a carrier is engineered to release rhVEGF with a shorter half-life than rhBMP-2 based on the physiological bone healing time-scale. A versatile material for developing such a carrier is poly-(D,L)-lactide (PDLLA), where a growth factor can either be immobilized on the surface or inside the bulk material e.g. by foaming technologies [6,7]. rhBMP-2 containing foamed tablets show a sustained release of rhBMP-2 with half-lives ranging between 85 and 350 days depending on the temperature [7] and are bioactive in vitro and in vivo. Experiments with rhVEGF are underway. Another technology which is in a successful collaborative development consists of electrospinning PDLLA to a bioactive nanofiber fleece with multimodal mediator release properties.

The objective of the present work is to fabricate and develop growth factor-PDLLA nano-composites by electrospinning into 2D fleece or 3D tubular scaffolds that can mimic the nature and function of the native extracellular matrix of bone tissue or artificial blood vessels. The bioactive growth factors I-125-rhVEGF(165), I-125-rhBMP-2 and the fibrous protein collagen were incorporated directly into the nascent PDLLA fiber during the electrospinning process. The morphology and distribution of the bioactive molecules in the electrospun fibers were investigated using TEM. The micro mechanical deformation was studied in tension tests using ESEM. The electrospun PDLLA nanofibers loaded with rhVEGF(165) and collagen showed a ductile behavior with necking and cold drawing deformation on stretching. The incorporation of rhVEGF(165), rhBMP-2 and collagen molecules into the scaffolds of PDLLA nanofibers could enhance cell attachment, reduce the scaffolds lifetime and promote angiogenesis and osteogenesis process, and thereby induce bone formation.

For the successful regeneration of lost bone, the use of biomaterial-based substitutes in combination with factors that promote the regenerative process, such as vascular endothelial growth factor (VEGF) or bone morphogenetic protein 2 (BMP-2), might be promising. In this study, a model was used which generated capillary-like vessels by co-cultivating human outgrowth endothelial cells (OEC) with human primary osteoblasts (pOB). Using this model the effect of VEGF-or BMP-2-loaded polylactide scaffolds on the development of microvessel-like structures was analyzed ultra-structurally using transmission electron microscopy (TEM). The co-cultivation of OEC with pOB on VEGF-loaded polylactide scaffolds resulted in the formation of capillary-like structures after 7 days of cultivation. After 14 days of co-cultivation on BMP-2-loaded polylactide constructs, OEC showed an initial angiogenic induction. TEM analysis proved to be an excellent method to analyze morphological changes of cells when cultivated on differently treated bone biomaterials.

Two methods of contact angle measurement are applied on screw type dental implants composed of titanium with different surfaces: The standard Wilhelmy method (dynamic measurement) and an extended sessile drop method (static measurement), where droplets with a volume in the picoliter range are generated. Both methods lead to comparable but in detail different results.

Electro spun PDLLA-nanofibers were functionalized with I-125-rhBMP-2 and I-125-rhVEGF(165), either by protein-adsorption to, or by incorporation into electrospun PDLLA-nanofibers. Both, adsorbed and incorporated growth factors could be visualized via autoradiography. By fitting the release data according the two phase exponential decay, release rates as well as release-half-lives could be calculated mathematically. Moreover different release rates could be observed, suitable to design multimodal hybrid materials. Loading the PDLLA-nanofibers with rhBMP-2 and/or rhVEGF(165) would be of benefit in bone regeneration.

The assembly and stability of polyelectrolyte multilayers (PEMs) was studied with the tracer I-125-Poly-L-lysine together with chondroitin sulphate on microstructured titanium surfaces (mu SLA). PEM assembly followed an exponential growth curve, which is mimicked by rhBMP-2 binding on glass slides during assembly. PEM disassembly occurred with a half life of 695 +/- 205 days over 21 days.

Sandblasted titanium screw type implants were acid-etched by two different methods using a semiautomatic wet chemical processing platform. Both etching methods led to homogenous, micro-structured and hyperhydrophilic surfaces making them especially suitable for dental applications. However processing time and procedural complexity differ greatly between these methods. In addition a discussion of contact angle measurement of dental implants by the method of Wilhelmy is given.

Composite tablets of human recombinant bone morphogenetic protein 2 (rhBMP-2) and poly-(D,L)lactide (PDLLA) (1-2 mg rhBMP-2/g PDLLA) were prepared by foaming of PDLLA with supercritical CO2. Release kinetics of rhBMP-2 were measured by a continuous flow and a batch dilution method at 22 °C and 50 °C respectively. In both cases a two-phase first-order release was found. Release half-lives of the sustained release phases (87-90% of total) decreased significantly from 348 days at 22 °C to 86 days at 55 °C i.e. by a factor of 4 indicating a coupling with PDLLA hydrolysis. The released rhBMP-2 was biologically active.

This chapter contains sections titled: Introduction Theoretical Considerations Materials and Methods Results and Discussion Conclusion

With acid hydrophilized rough titanium plasma sprayed (TPS) miniplates (10.4 x 5.2 x 1.7 mm; Ra = 22 mu m; rm = 8) we discovered that ca. 20% of the data in each Wilhelmy balance measurement corresponded to "forbidden" data of the type cos theta > 1.0. It was then found, that the dogma stating "cos theta > 1.0 as undefined" is false. Surprisingly, the equation cos theta > 1.0 is indeed defined, only in imaginary number space. The imaginary dynamic contact angles theta ai of hydrophilized TPS surfaces could then be redetermined to theta ai = 20i degrees-22i degrees (advancing and receding). We have termed this phenomenon "hyperhydrophilicity" or Inverse Lotus Effect which can be defined as the total wetting of rough surfaces with extreme wettability rates generating Wilhelmy force values which can be expressed as imaginary contact angles.

Angiogenesis and osteogenesis are closely related processes sharing some essential mediators like VEGF (Vascular Endothelial Growth Factor) and BMP-2 (Bone Morphogenetic Protein-2). As vascularization is crucial to bone formation biofunctionalization of bone replacement materials with both of these proteins may result in enhanced bone formation. Here we will present immobilization of self prepared biologically active BMP-2 and VEGF on different bone replacement materials and their controlled release. The amount of bound protein can be steered as a function of protein concentration in the incubation solution. Desorption behaviour of proteins differs strongly depending on the type of bone replacement material used and protein ranging from 0.1-2 days for the burst phase and from 17-115 days for the sustained release phase. A combined loading and release of the growth factors is now planned.

Evalauation of safety and efficacey of BMP in revision of failed

The main objectives of the study described below were of two-fold nature: (1) to examine if rhBMP-2-biocoated implants in a pig model could lead to ectopic bone formation and (2) if quantitative and/or qualitative differences could be found between adhesively and covalently bonded BMP II using the scintigraphic method. In order to examine these central questions, 26 Göttingen minipigs were allocated to three groups with a control group (n = 7) and two study groups (n = 9 each) receiving one of three implant types: (a) chromosulfuric acid treated titanium surface as control, (b) non-covalently bonded BMP-2, and (c) covalently bonded and immobilized rhBMP-2. Each animal received four barbell-shaped implants, one in the proximal and distal metaphysis of each femur. The scintigraphic analyses were conducted after four, eight, and 12 weeks postoperatively. The visual (qualitative) analysis failed to show ectopic bone formation in any of the three groups. The statistical analysis of the relative values for bone formation yielded no significant differences between the groups, although the limitation in the applied methods do not enable one to draw conclusions regarding the histomophometric results.

The selective degradation of many proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved protein [1]. Ubiquitylated proteins were degraded by the 26S protea-some in an ATP-depended manner. The degradation of ubiquitylated proteins were controlled by isopeptidase cleavage. A well characterised system of ubiquitylation and deubiquitylation is the calmodulin system in vitro [2]. Detection of ubiquityl-calmodulin conjugtates in vivo have not been shown so far. In this article we discuss the detection of ubiquitin calmodulin conjugates in vivo by incubation with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues. Proteins with a molecular weight of ubiquityl-calmodulin conjugates could be detected in all organs tested. Incubation with ubiquitylprotein-isopeptidase showed clearly a decrease of ubiquitin calmodulin conjugates in vivo with an origination of unbounded ubiquitin. These results suggest that only few ubiquitin calmodulin conjugates exist in rabbit tissues.

Galway, Ireland Synthesis of Osteoinductive Glasses by Immobilization of rhBMP-2 K. Zurlinden, M. Laub, M. Lindner, K. Koczur, A. Kirsten, A. Oliveira, G. Seifert, S. Gemming, C. Müller-Mai, H. Fischer, H.P. Jennissen 1 Institut für Physiologische Chemie, Universität Duisburg-Essen, Universitätsklinikum Essenm Germany Zahnärztliche Werkstoffkunde und Biomaterialforschung, Universitätsklinikum RWTH Aachen, Germany Fachbereich Chemie, Arbeitsgruppe Theoretische Chemie, TU Dresden, Germany Institut für Ionenstrahlphysik und Materialforschung, FZ Dresden-Rossendorf, Germany Unfallchirurgie der Chirurgischen Universitätsklinik, Knappschaftskrankenhaus Bochum-Langendreer, Germany Corresponding Author e-mail: hp.jennissen@uni-due.de

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a growth factor of the transforming growth factor-beta superfamily. Members of this protein family are involved in the development of various mammalian tissues, including the inner ear. As their notations indicate, they also have well-known effects on bone formation and regeneration. In this study, we examined the influence of rhBMP-2 on spiral ganglion (SG) neurite growth in vitro and showed the presence of its most preferred receptor BMPR-IB in spiral ganglion cells both in vitro and in vivo. SG explants of postnatal day 4 rats were analysed for neurite length and number after organotypical cell culture for 72 h, fixation and immunolabeling. Different concentrations of rhBMP-2 were used in a serum-free culture media. Neurite growth was compared with control groups that lacked stimulative effects; with neutrophin-3 (NT-3), which is a well-established positive stimulus on neurite length and number; and with combinations of these parameters. The results display that neurite number and total neurite length per explant in particular concentrations of rhBMP-2 increased by a maximum factor of two, while the mean neurite length was not affected. NT-3 demonstrated a much more potent effect, delivering a maximum increase of a factor of five. Furthermore, a combination of both growth factors shows a predominant effect on NT-3. Immunohistological detection of BMPR-IB was successful both in cell culture explants and in paraffin-embedded sections of animals of different ages. The results show that rhBMP-2 is, among other growth factors, a positive stimulus for SG neurite growth in vitro. Most growth factors are unstable and cannot be attached to surfaces without loss of their biological function. In contrast, rhBMP-2 can be attached to metal surfaces without loss of activity. Our findings suggest in vivo studies and a future clinical application of rhBMP-2 in cochlear implant technology to improve the tissue/electrode interface.

The covalent and non‐covalent immobilization of growth factors such as recombinant human bone morphogenetic protein 2 (rhBMP‐2) on metals and bone replacement materials in bioactive form is a recent development. Up to now the immobilization technology usually involved the chemical modification and activation of the biomaterial surface followed by attachment of the bioactive protein. Here we suggest an alternative method in which an affinity tag fused to an active protein will allow immobilization without additional chemistry. For biomaterials such as minerals, metals (titanium, steel, CoCrMo), glass ceramics, teflon and possibly bone and teeth ideal adhesion molecules would be the foot proteins (Mefps) of the mussel M. edulis which contain the rare amino acid dihydroxy phenylalanine (DOPA). Recently it could be shown by Messersmith's group that a single DOPA‐molecule can be non‐covalently bound to titanium dioxide surface with a dissociation energy of 22.2 kcal/mol (Lee, H.; Scherer, N. F.; Messersmith, P. B. Proc. Natl. Acad. Sci. U. S. A 2006, 103, 12999–13003).We therefore propose the DOPA‐tag as a general and versatile affinity tag for the immobilization of proteins on biomaterials.