Biofunctionalization strategies on tantalum-based materials for osseointegrative applications

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant’s biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO₃ or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO₃-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.     

The impact of the RGD peptide on osteoblast adhesion and spreading on zinc-substituted hydroxyapatite surface

The incorporation of zinc into the hydroxyapatite structure (ZnHA) has been proposed to stimulate osteoblast proliferation and differentiation. Another approach to improve cell adhesion and hydroxyapatite (HA) performance is coating HA with adhesive proteins or peptides such as RGD (arginine–glycine–aspartic acid). The present study investigated the adhesion of murine osteoblastic cells to non-sintered zinc-substituted HA disks before and after the adsorption of RGD. The incorporation of zinc into the HA structure simultaneously changed the topography of disk’s surface on the nanoscale and the disk’s surface chemistry. Fluorescence microscopy analyses using RGD conjugated to a fluorescein derivative demonstrated that ZnHA adsorbed higher amounts of RGD than non-substituted HA. Zinc incorporation into HA promoted cell adhesion and spreading, but no differences in the cell density, adhesion and spreading were detected when RGD was adsorbed onto ZnHA. The pre-treatment of disks with fetal bovine serum (FBS) greatly increased the cell density and cell surface area for all RGD-free groups, overcoming the positive contribution of zinc to cell adhesion. The presence of RGD on the ZnHA surface impaired the effects of FBS pre-treatment possibly due to competition between FBS proteins and RGD for surface binding sites.     

Investigation of effects of Argenine–Glycine–Aspartate (RGD) and nano-scale titanium coatings on cell spreading and adhesion

This paper examines the effects of physical and chemical surface modifications on the biocompatibility of silicon surfaces that are relevant to implantable silicon Bio-micro-electro-mechanical systems (BioMEMS). Two types of surface modifications were explored. The first involved the deposition of nano-scale biocompatible layers of pure titanium on silicon, while the second explored the covalent attachment of the binding peptide Argenine–Glycine–Aspartic acid (RGD) for improved cell adhesion. Improvements in biocompatibility were assessed through examination of cell areas after culture, as well as the measurements of adhesion strengths, as determined by shear assay techniques. The titanium nanolayers and the RGD coating resulted in improvements in biocompatibility. Increased cell spreading areas and improved adhesion strength were obtained from short and long-term studies of Human Osteosarcoma (HOS) cells cultured on the coated surfaces. RGD functionalization resulted in the greatest improvement in cell spreading area and adhesion strength for short culture times. The effects of the titanium, while less than those of RGD for short culture times, appeared to be greater after 48 h of culture.     

RGD修饰钛表面对人牙龈成纤维细胞初期黏附和铺展的影响

用羰基二咪唑(1,1'-carbonyldiimidazole,CDI)将含RGD的短肽共价连接到纯钛表面,研究接枝后的钛表面对原代培养的人牙龈成纤维细胞(human gingival fibroblasts,HGF)初期黏附和铺展的影响.结果表明,RGD修饰的纯钛表面粘附的细胞数比未修饰钛表面多,细胞铺展面积比钛表面的大,应力纤维的形成比钛表面早.RGD接枝钛表面更有利于人牙龈成纤维细胞的粘附,改善了纯钛的生物相容性.     

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.     

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.     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Surface functionalization of materials to initiate auto‐biocompatibilization in vivo

Cellular growth and differentiation in contact with artificial materials is controlled by exchange of information between solid and components of the extracellular matrix. Usually cells are connected with the extracellular matrix via adhesive proteins, f. i. fibronectin or laminin. Fibronectin is a large glycoprotein consisting of a dimer of two subunits which are connected via two disulfide‐bonds at their carboxy terminal ends. Each subunit contains binding sites for heparin, collagen and the cell itself. Fibronectin is bound via sequences of type III to cellular integrins. The cell‐specifity of the implant‐surface can be realized by physical, chemical or biological functionalization. The RGD type III‐sequence arg‐gly‐asp of fibronectin can be connected to the implant surface via metal organic spacers. Already experimentally performed was the reaction between short‐chain III sequences with titanium alcoxides. The metal‐organic compound carries the sequence as a functional group. The coupling to the metal(oxide)‐surface takes place with a sol‐gel‐process via hydrolysis and condensation of the alkoxide groups. In appropriate preparatory work mono amino acids, polycarbocylic acids and other short chain biological effective molecules have already been bound to titanium. The free functionalities: ‐NH₂, ‐COOH, ‐SO₃ were prooved by FT‐IR‐ or surface enhanced raman spectroscopy (SERS). The provided surfaces show differently strong mineralization after incubation in simulated body fluids containing calcium phosphates; a sterilization in an autoclave does not modify the chemical composition and the morphology of the surface modification. The goal for implants with contact to hard tissue is to prepare a surface with free functionalities which adsorb preferently adhesive proteins from the host extracellular matrix as an auto‐biocompatibilization.     

磷灰石-硅灰石生物活性玻璃陶瓷表面接枝多肽改性研究

为在磷灰石-硅灰石生物活性玻璃陶瓷(Apatite-Wollastonite Bioactive Glass-Ceramic, AW)表面引入能够促进细胞粘附的RGD(精氨酸-甘氨酸-天冬氨酸)多肽以提高其生物活性, 采用低温等离子法在材料的表面引入活性氨基基团, 并通过浸渍法使氨基基团与多肽发生反应。采用XRD、XPS、ATR-FTIR对AW的相组成及表面改性特性进行表征, 确认通过低温等离子法在AW表面接上氨基, RGD多肽分子与氨基反应以化学键合的形式接枝到材料表面(RGD-AW), 实现了在AW表面接枝生物大分子的改性。将改性前后的材料分别与类成骨细胞(MG63细胞)混合培养并使用荧光显微镜、SEM及MTT等测试方法对材料的细胞生物学性能进行了表征。细胞实验结果表明: 接枝RGD多肽分子的材料在细胞培养的早期阶段比AW更有利于细胞的粘附及铺展。     

Rationally Designed Peptide Interface for Potential Modulated Cell Adhesion and Migration

Conformational changes of peptides are critically important in the control of their biological activities. Here, a quaternary ammonium group‐terminated RGD‐containing peptide (RGD‐NMe₃) is designed, which may undergo reversible conformational switch upon different electrochemical potentials. Potential responsive peptide interfaces are constructed on gold substrates with RGD‐NMe₃ in a tetra (ethylene glycol) background. It is demonstrated that by applying positive and negative potentials, the RGD peptide can be reversibly switched between linear and cyclic conformation, which can be used in reversible controlling of cell adhesion/migration on the interface. Furthermore, by combining microfluidics, adhesion of the cells in specific areas on the surface and subsequent directional migration of the cells can be controlled. It is believed that this straightforward potential modulation mechanism for peptide conformation control may find a wide use in design responsive peptide interfaces.     

生物材料细胞粘附肽仿生修饰的研究进展

RGD(Arg-G1y-Asp)短肽序列是一种细胞粘附肽,能被细胞膜上的整合素识别,参与细胞与基质间的粘附.为改善合成生物材料缺乏细胞识别信号的缺点,可将含RGD序列肽经本体或表面修饰引入材料,使材料具有良好的细胞亲和性.综述了采用含RGD肽对各种合成生物材料进行仿生修饰的研究进展.     

SPA用于丝素膜的生物改性研究

用生长因子RGD半抗原（GLY-ARG-GLY-ASP-SER-PRO-LYS)连接到卵清蛋白Ovalbumin(OVA)载体上诱发出了抗RGD抗体IgG，并用丝素溶液包埋SPA(Staphylo-coccal protein A，简称A蛋白或SPA)制成不溶性SPA丝素膜为材料，然后用诱发出的RGD抗体IgG结合到不溶性SPA丝素膜的表面，制成IgG-SPA丝素膜，再在其上结合粘附生长因子RGD，制成RGD-IgG-SPA丝素膜。利用这一丝素膜培养血管内皮细胞(Vas-cular Endothelial Cell，简称EC细胞)，用四甲基偶氮些盐比色方法(MTT法)检测细胞的生长增殖情况。结果表明，RGD-IgG-SPA丝素膜能有效促进EC细胞的生长。对不溶性SPA丝素膜和IgG以及IgG和RGD之间的生物结合力测定，表明其结合力远大于离解力。同时在细胞培养液中没有检测到丝素膜中SPA的渗漏。RGD-IgG-SPA丝素膜为其作具有的这些优良性质为血管支架打下了基础。     

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.     

Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration

To control specific endothelial cell (EC) functions, cell adhesive RGDS, EC specific REDV and YIGSR peptides, and angiogenic SVVYGLR sequences were covalently immobilized onto polyethylene terephthalate (PET) surfaces for the purpose of cell culture. X-ray photoelectron spectroscopy, atomic force microscopy, fluorescence microscopy and contact angle measurement were employed for characterization of surface modifications. The peptide density on PET surfaces was evaluated by fluorescence microscopy. The surfaces immobilized with peptides were exposed to human umbilical vein endothelial cells to study their specific effects onto EC functions. The results showed that the surface functionalized by these peptides enhanced the EC adhesion, spreading and migration as compared with native PET surfaces. Specifically, the RGDS peptides induced more cell adhesion than other peptides. The YIGSR and SVVYGLR sequences induced more cell spreading and cell migration, represented by intense focal adhesion at the leading edges of cell spreading and migration. The bi-functionalization of RGDS and SVVYGLR peptides (MIX) combined the advantages of both peptides and induced significant EC adhesion, spreading and migration. Our study indicates that the surface functionalization by peptides specific for ECs, especially the combination of RGDS with SVVYGLR or YIGSR peptides, has potential applications in promoting endothelialization of vascular prostheses and for construction of vascularized tissues in tissue engineering.     

Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing

Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability, and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and the signaling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 mum when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization and initiate cell migration and signaling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.     

Biomimetic coatings functionalized with adhesion peptides for dental implants

A complete biological integration into the surrounding tissues (bone, gingiva) is a critical step for clinical success of a dental implant. In this work biomimetic coatings consisting either of collagen type I (for the gingiva region) and hydroxyapatite (HAP) or mineralized collagen (for the bone interface) have been developed as suitable surfaces regarding the interfaces. Additionally, using these biomimetic coatings as a matrix, adhesion peptides were bound to further increase the specificity of titanium implant surfaces. To enhance cell attachment in the gingiva region, a linear adhesion peptide developed from a laminin sequence (TWYKIAFQRNRK) was bound to collagen, whereas for the bone interface, a cyclic RGD peptide was bound to HAP and mineralized collagen using adequate anchor systems. The biological potential of these coatings deduced from cell attachment experiments with HaCaT human keratinocytes and MC3T3-E1 mouse osteoblasts showed the best results for collagen and laminin sequence coating for the gingiva region and mineralized collagen and RGD peptide coatings for regions with bone contact. Our concept opens promising approaches to improve the biological integration of dental implants.     

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.     

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.     

Inside Front Cover: Tailoring Cell Adhesion Using Surface‐Grafted Polymer Gradient Assemblies (Adv. Mater. 23/2005)

A three‐step approach to tailor cell adhesion via surface‐grafted polymer gradients is shown in this image from the work of Genzer and co‐workers on p. 2802. Surface‐anchored polymer assemblies with gradients in polymer molecular weight and/or grafting density are first employed to tailor adsorption of the protein, which in turn governs the number density as well as the extent of spreading of osteoblastic cells. Increasing the surface coverage of the polymer results in a decrease in the amount of protein adsorbed, which causes a decrease in the number of cells adhered and a change in cell morphology.