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.     

Assesment of the Biological Activity of Chemically Immobilized rhBMP‐2 on Titanium surfaces in vivo

Previously it has been shown that recombinant human bone morphogenetic protein (rhBMP‐2) can be chemically immobilized by “anchor molecules” on titanium surfaces for serving as a drug delivery device. This opened the question of whether the insoluble immobilized rhBMP‐2 retained its activity in comparison to the same amount of soluble rhBMP‐2 included with the implant samples. Electropolished titanium miniplates (10 × 6 × 0.8 mm) were “surface‐enhanced” by a novel treatment with chromosulfuric acid and then coated with a total amount of 150–200 ng rhBMP‐2 prepared by recombinant technology. Periosteal flaps (7 × 20 mm) were detached and isolated from the anterior surface of the tibiae of adult rabbits and wrapped around the titanium sample plates which were then implanted in the M. gastrocnemius. In the first experimental group various controls without rhBMP‐2 were combined (n = 12). In the second experimental group implants with chemically immobilized rhBMP‐2 (n = 8) were compared with implants to which non‐immobilized soluble rhBMP‐2 was added (n = 8). Animals were sacrificed after 28 days and a quantitative evaluation was carried out by means of serial sections. Untreated control plates showed bone formation in 2/12 implants, rhBMP‐2 coated implants in 6/8 and implants with free rhBMP‐2 administered subperiostally in 8/8 cases. In the case of rhBMP‐2 coated implants the induced bone had direct contact to the implant in all cases while in the group with free administered rhBMP‐2 the bone had no contact to the implant in two cases, but was separated by a fibrous capsule. Bone volume, bone surface area, and trabecular number displayed no difference between the two rhBMP‐2‐groups. However, in the biocoated group a tendency to an increase in the bone‐implant contact area was evident. No differences in osteoid area, osteoid perimeter and eroded perimeter were detected. We conclude that in the case of non‐immobilized rhBMP‐2 there is the danger for formation of fibrous tissue between the implant and the newly formed bone and in addition the generation of ectopic bone at inappropriate places. In contrast chemically immobilized rhBMP‐2 does not have these drawbacks and at the same time displays a biological activity on surfaces similar to that of soluble rhBMP‐2 demonstrating that biomaterial surfaces can be tailored for a selective and specific interaction with the target tissue.     

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.     

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.     

Development of an universal affinity fusion tag (Poly‐DOPA) for immobilizing recombinant proteins on biomaterials

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.     

Osseointegration of Titanium Implants by Addition of Recombinant Bone Morphogenetic Protein 2 (rhBMP‐2)

The osseointegration of long‐term implants is often incomplete such that gaps remain between the implant surface and the surrounding hard tissue. This study examines the effect of soluble recombinant human bone morphogenic protein 2 (rhBMP‐2) on gap healing and osseous integration. The effect of a single, intraoperative application of soluble rhBMP‐2 on the formation of new bone around titanium implants was studied. A total of 8 titanium‐alloy cylinders (Ti‐6Al‐4V) with a plasma spray coating (TPS; 400 μm thickness) were implanted into femoral condyles of mature sheep: rhBMP‐2 solution (1 μg) was pipetted into the 1 mm wide cleft around 4 implants; 4 further implants served as rhBMP‐2‐free controls. Two of these controls exhibited an additional calciumphosphate‐coating. The cleft around the implants served as testing zone to study the formation of new bone by microradiographical and histological analyses. The follow‐up periods were 4 and 9 weeks, respectively. A significant amount of new bone contacting the implants' surface was detected where rhBMP‐2‐solution had been used: In 50% a circumferential osseoinduction occurred within 4 weeks and a nearly complete osseointegration was observed after 9 weeks. In all cases bone formation was exaggerated and filled the spongiosa with compact bone. Time matched TPS‐controls and controls with calciumphosphate coating showed no notable formation of new bone. The results suggest that a single administration of soluble rhBMP‐2 into a bone cavity can augment bone formation and also osseointegration of titanium implants. Further investigations based on these findings are necessary to develop long‐term implants (e. g. joint replacements) with rhBMP‐2‐biocoating for humans.     

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.     

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.     

Layer‐By‐Layer Films as a Biomimetic Reservoir for rhBMP‐2 Delivery: Controlled Differentiation of Myoblasts to Osteoblasts

Efficient delivery of growth or survival factors to cells is one of the most important long‐term challenges of current cell‐based tissue engineering strategies. The extracellular matrix acts as a reservoir for a number of growth factors through interactions with its components. In the matrix, growth factors are protected against circulating proteases and locally concentrated. Thus, the localized and long‐lasting delivery of a matrix‐bound recombinant human bone morphogenetic protein 2 (rhBMP‐2) from a biomaterial surface would mimic in vivo conditions and increase BMP‐2 efficiency by limiting its degradation. Herein, it is shown that crosslinked poly(L ‐lysine)/hyaluronan (HA) layer‐by‐layer films can serve as a reservoir for rhBMP‐2 delivery to myoblasts and induce their differentiation into osteoblasts in a dose‐dependent manner. The amount of rhBMP‐2 loaded in the films is controlled by varying the deposition conditions and the film thickness. Its local concentration in the film is increased up to ≈500‐fold when compared to its initial solution concentration. Its adsorption on the films, as well as its diffusion within the films, is evidenced by microfluorimetry and confocal microscopy observations. A direct interaction of rhBMP‐2 with HA is demonstrated by size‐exclusion chromatography, which could be at the origin of the rhBMP‐2 “trapping” in the film and of its low release from the films. The bioactivity of rhBMP‐2‐loaded films is due neither to film degradation nor to rhBMP‐2 release. The rhBMP‐2‐containing films are extremely resistant and could sustain three successive culture sequences while remaining bioactive, thus confirming the important and protective effect of rhBMP‐2 immobilization. These films may find applications in the local delivery of immobilized growth factors for tissue‐engineered constructs and for metallic biomaterial surfaces, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition.     

Measuring bone cell adhesion on implant surfaces using a spinning disc device

The adhesion behaviour of osteoblastic cells on implant surfaces is a main focus during the development of osteoconductive implant surfaces. Therefore, besides cell spreading and proliferation on surfaces the adhesion strength of cells to the substrate is of high interest. There are different approaches to determine cell adhesion but only few quantitative methods. For this purpose, we have developed an adhesion device based on the spinning disc principle in conjunction with an inverse confocal laser scanning microscope (LSM). Mirror polished disc‐shaped test samples made of titanium‐ (Ti6Al4V) and cobalt‐alloys (Co28Cr6Mo), as well as stainless steel (316L), were seeded with osteoblasts, stained with a fluorescent dye, at defined radial positions and were incubated for 18 h in cell culture medium (DMEM). After incubation the test samples were placed into the adhesion chamber filled with DMEM. By means of a computer controlled motor the test samples were rotated for 3 min. Using the LSM the detachment of the cells at defined radial positions was determined and the cell count was recorded before and after rotation with the help of imaging software. An average shear stress of 47.1 N/m², 53.2 N/m² and 49.4 N/m² was assessed for the mirror polished Ti6Al4V, Co28Cr6Mo and 316L surfaces respectively. The technique is suitable for studying bone cell adhesion strength on orthopaedic implant materials. Future investigations will focus on different bioactive and anti‐infectious implant surfaces, as well as soluble bioactive factors.     

Biological Multi‐layer Systems as Implant Surface Modification

In living organisms the natural contact areas between cells are the cell membranes. These membranes separate the individual cells or build reaction compartments in an aqueous environment. Beside their structural role they also have specific functions that are due to the great variety of their components which are lipids (about 40 %) and proteins (about 60 %). In terms of implant development the next neighbors of the cells are artificial materials which do not belong to the natural cellular environment. Therefore, a biocompatible implant surface is needed which is achieved by either the correct choice of the material and surface roughness or a functionalization of the surface. To date little is known of the role lipids could play in this context. However, from literature we know that phospholipids can cause calcification and that modified phosphorylcholine polymers ('MPC polymers') are used to decrease cell adhesion and to improve blood compatibility. In the last few years it became obvious that the lipid contribution in the membrane is not only important as support for proteins but that the lipid membrane itself can also be a target for drug design and its structure can influence the function of the proteins. We therefore focused our interest on this class of amphiphilic molecules. In this work we present initial observations on the modification of metallic implant surfaces of Ti‐6Al‐7Nb (in mass percent) by phospholipid multilayers, using contact angle measurements and surface sensitive characterization techniques such as scanning electron microscopy (SEM) and scanning force microscopy (SFM). Preliminary data concerning cell adhesion experiments are also presented.     

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.     

Bioactive TiOB‐Coating on Titanium Alloy Implants Enhances Osseointegration in a Rat Model

The surface properties of titanium alloy implants for improved osseointegration in orthopaedic and dental surgery have been modified by many technologies. Hydroxyapatite coatings with a facultative integration of growth factors deposited by plasma spraying showed improved osseointegration. Our approach in order to enhance osseointegration was carried out by a surface modification method of titanium alloy implants called plasma chemical oxidation (PCO). PCO is an electrochemical procedure that converts the nm‐thin natural occurring titanium‐oxide layer on an implant to a 5 µm thick ceramic coating (TiOB‐surface). Bioactive TiOB‐surfaces have a porous microstructure and were loaded with calcium and phosphorous, while bioinert TiOB‐surfaces with less calcium and phosphorous loadings are smooth. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bone response to TiOB‐surfaces in vivo. 64 rats were randomly assigned to four groups of implants: (i) pure titanium alloy (control), ii) titanium alloy, type III anodization, (iii) bioinert TiOB‐surface, and (iv) bioactive TiOB‐surface. Mechanical fixation was evaluated by pull out tests at 3 and 8 weeks. The bioactive TiOB‐surface showed significantly increased shear strength at 8 weeks compared to all other groups.     

BMP‐7 (OP‐1) in human orthopaedic surgery: Local and systemic effects of application

Six patients with nonunion of long bones, one with delayed bone formation during distraction osteogenesis, and one with nonunion of the neck of the femur were enrolled in a clinical trial. Duration of nonunion was from 8 to 94 months (mean 29,4 months). Recombinant human OP‐1 (BMP‐7) was purchased from the manufacturer. One unit (3,5 mg of active substance mixed with 1 gram of type I bovine bone‐derived collagen) was used in each patient. In four patients OP‐1 implant was delivered by standard open operative procedure, in 3 percutaneously. In one patient with femoral neck nonunion OP‐1 implant was placed intramedullary. No patient had serious adverse reactions related to the implant. At 5 to 8 weeks following the operative procedure five out of 6 patients with nonunion of long bones and one patient with delayed bone formation in distraction osteogenesis were judged by clinical and radiological criteria to have been treated successfully. In one patient with femoral neck nonunion bone union was achieved after 22 weeks. To determine whether the local stimulation with OP‐1 might induce prolonged changes in systemic levels of growth and angiogenic factors, the serum levels of the angiogenin (ANG), interleukin‐8 (IL‐8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin‐like growth factor‐1 (IGF‐1) were assessed prior, and 1, 2, 6 and 12 weeks after operative treatment with OP‐1 device. There is no evidence for a potentially harmful systemic rise in growth and angiogenesis factors after local administration of the amount of BMP‐7 used in our study.     

Soybean Lecithin‐Mediated Nanoporous PLGA Microspheres with Highly Entrapped and Controlled Released BMP‐2 as a Stem Cell Platform

Injectable polymer microsphere‐based stem cell delivery systems have a severe problem that they do not offer a desirable environment for stem cell adhesion, proliferation, and differentiation because it is difficult to entrap a large number of hydrophilic functional protein molecules into the core of hydrophobic polymer microspheres. In this work, soybean lecithin (SL) is applied to entrap hydrophilic bone morphogenic protein‐2 (BMP‐2) into nanoporous poly(lactide‐co‐glycolide) (PLGA)‐based microspheres by a two‐step method: SL/BMP‐2 complexes preparation and PLGA/SL/BMP‐2 microsphere preparation. The measurements of their physicochemical properties show that PLGA/SL/BMP‐2 microspheres had significantly higher BMP‐2 entrapment efficiency and controlled triphasic BMP‐2 release behavior compared with PLGA/BMP‐2 microspheres. Furthermore, the in vitro and in vivo stem cell behaviors on PLGA/SL/BMP‐2 microspheres are analyzed. Compared with PLGA/BMP‐2 microspheres, PLGA/SL/BMP‐2 microspheres have significantly higher in vitro and in vivo stem cell attachment, proliferation, differentiation, and matrix mineralization abilities. Therefore, injectable nanoporous PLGA/SL/BMP‐2 microspheres can be potentially used as a stem cell platform for bone tissue regeneration. In addition, SL can be potentially used to prepare hydrophilic protein‐loaded hydrophobic polymer microspheres with highly entrapped and controlled release of proteins.     

Free form fabricated features on CoCr implants with and without hydroxyapatite coating in vivo: a comparative study of bone contact and bone growth induction

The current study evaluates the in vivo response to free form fabricated cobalt chromium (CoCr) implants with and without hydroxyapatite (HA) plasma sprayed coatings. The free form fabrication method allowed for integration of complicated pyramidal surface structures on the cylindrical implant. Implants were press fit into the tibial metaphysis of nine New Zealand white rabbits. Animals were sacrificed and implants were removed and embedded. Histological analysis, histomorphometry and electron microscopy studies were performed. Focused ion beam was used to prepare thin sections for high-resolution transmission electron microscopy examination. The fabricated features allowed for effective bone in-growth and firm fixation after 6 weeks. Transmission electron microscopy investigations revealed intimate bone-implant integration at the nanometre scale for the HA coated samples. In addition, histomorphometry revealed a significantly higher bone contact on HA coated implants compared to native CoCr implants. It is concluded that free form fabrication in combination with HA coating improves the early fixation in bone under experimental conditions.     

Hydrophobic,Electrostatic, and Dynamic Polymer Forces at Silicone Surfaces Modified with Long‐Chain Bolaform Surfactants

Surfactant self‐assembly on surfaces is an effective way to tailor the complex forces at and between hydrophobic‐water interfaces. Here, the range of structures and forces that are possible at surfactant‐adsorbed hydrophobic surfaces are demonstrated: certain long‐chain bolaform surfactants—containing a polydimethylsiloxane (PDMS) mid‐block domain and two cationic α, ω‐quarternary ammonium end‐groups—readily adsorb onto thin PDMS films and form dynamically fluctuating nanostructures. Through measurements with the surface forces apparatus (SFA), it is found that these soft protruding nanostructures display polymer‐like exploration behavior at the PDMS surface and give rise to a long‐ranged, temperature‐ and rate‐dependent attractive bridging force (not due to viscous forces) on approach to a hydrophilic bare mica surface. Coulombic interactions between the cationic surfactant end‐groups and negatively‐charged mica result in a rate‐dependent polymer bridging force during separation as the hydrophobic surfactant mid‐blocks are pulled out from the PDMS interface, yielding strong adhesion energies. Thus, (i) the versatile array of surfactant structures that may form at hydrophobic surfaces is highlighted, (ii) the need to consider the interaction dynamics of such self‐assembled polymer layers is emphasized, and (iii) it is shown that long‐chain surfactants can promote robust adhesion in aqueous solutions.     

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

Bone morphogenetic proteins (BMP) 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. Recently BMP‐2 has been chemically immobilized on implant surfaces leading to enhanced bone growth and accelerated integration in sheep. Although the 3D‐structure of BMP‐2 is known the surface topography has not been the subject of a detailed analysis. Therefore we have begun implementing the technique of 3D‐rapid prototyping as a novel method for gaining topographical information on the structure‐function relationship of proteins (Laub et al., 2001, FASEB J. 15, A543). 3D‐rapid prototyping allows the construction of accurate three‐dimensional models of proteins based on their x‐ray crystallographic data. In this way we constructed a 3D scale image of BMP‐2 of the size 140 mm × 70 mm × 50 mm corresponding to a ca. 20 × 10⁶ fold magnification (scale 1 nm = 2 cm). BMP‐2 is a twisted banana‐shaped molecule consisting of a convex and a concave face and has a horn‐like protuberance cross‐turned at 180° (long axis) at each end. In the center of the convex face there is a ca. 1 nm deep crater like pit ca. 1.8 nm in diameter. The concave face is characterized by a 6–7 nm long helical groove 0.8–1.6 nm wide and ca 0.8 nm deep, into which a left‐handed helix with a pitch of 8–9 nm and a helical radius of 0.35–0.45 nm can be fitted. The concave face of BMP‐2 therefore corresponds to an imprint (groove) of a left‐handed helix i. e. to an anti‐helix or anthelix. The possible endogenous ligands and functions of these structures are unknown. These results demonstrate that full scale 3D molecular models of proteins can lead to new perceptions in understanding the interactions between ligands and proteins by macroscopic viewing and in‐hand fitting of the molecules without the aid of a computer.     

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.     

PLGA film/Titanium nanotubues as a sustained growth factor releasing system for dental implants

Ti-based implants sometimes fail to integrate with surrounding bone tissue due to insufficiency of new bone formation and surface bonding. To overcome this problem, this research focused on establishing a sustained bone growth factor delivery system by applying anodized TiO2 nanotube arrays and PLGA film on the titanium implant surface. TiO2 nanotube arrays were made by anodic oxidation method, and were then filled with rhBMP2 by vacuum freeze-drying. Next, PLGA was deposition on the surface of this material. The designed system was characterized, pharmacokinetic release rate of rhBMP2 was determined. Adhesion, proliferation, and differentiation activity of osteoblasts cultured on the new surfaces and traditional titanium surfaced were compared. SEM showed that a surface of TiO2 nanotube arrays were successfully generated. PLGA membranes of 50?nm, 250?nm, 800?nm thickness were successfully deposited on the surfaces of TiO2 nanotube layers by using 1%, 3%, 10% PLGA solutions. PLGA film of 250?nm thickness showed ideally controlled release of rhBMP2, lasting for 4 weeks. Furthermore, 250?nm thickness PLGA film improved osteoblast adhesion, proliferation, and levels of alkaline phosphatase. In conclusion, the PLGA film / TiO2 nanotube growth factor delivery system can effectively sustain the release of rhBMP-2, and promote proliferation and differentiation of MC3T3-E1 osteoblasts.

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