Cell-extracellular matrix interaction and physico-chemical characteristics of titanium surfaces depend on the roughness of the material

The interaction of cells with the extracellular matrix at the interface of an implant determines the biology of cells and tissues. We analysed components of cell adhesion and measured physico-chemical characteristics of structural modifications of titanium surfaces: polished, machined, glass particle-blasted, corundum-blasted, vacuum plasma-sprayed. Scanning electron microscopy and profilometry revealed a differentiated topography from smooth to rough surfaces, respectively. Osteoblastic MG-63 cells showed an increased spreading on surfaces with low roughness, although without a straight correlation with the surface topography. Integrin expression was increased on structured surfaces compared with polished material, and the organization of the actin cytoskeleton and fibronectin was impaired on extremely rough surfaces. Electrochemical methods, especially the electrochemical impedance spectroscopy (EIS) was used to evaluate physico-chemical characteristics, and the impedance curves revealed a dependence on the roughness of the material surfaces. Further analyses of the EIS results were performed using equivalent circuits which model the electrical flow through the interface. First indications for a correlation between parameters from the equivalent circuits with surface properties were obtained which promise a relevance for the biological response of the cells.     

钛及钛合金种植体表面处理的研究进展

钛的优良特性早已被公认为首选牙用种植材料,其表面氧化膜是形成骨结合的关建。为了进一步增加其耐腐蚀性和生物相容性,人们采取了多种方式进行表面处理,最终达到在植入体的不同部位产生不同的粗糙表面,以利于不同细胞在不同地方的附着,增加其生物活性。同时,达到避免各种污染物对纯钛种植体影响的目的。近年来钛种植体材料表面处理已经成为研究热点,本文就表面处理的方法进行综述。     

The influence of different surface treatments on the primary stability of cementless acetabular cups: an in vitro study]

INTRODUCTION: Long-term stability of cementless acetabular cups depends on osseointegration, which requires primary stability of the implant. The aim of this study was to determine the influence of different surface treatments on the primary stability of press-fit acetabular cups. Mechanical lever-out tests were performed to quantify the stability in vitro. MATERIALS AND METHODS: A hemispherical press-fit cup design with a flattened pole was used and different surface modifications were applied: smooth, corundum-blasted, titanium plasma spray, rough titan plasma spray, and titanium plasma spray with a rim. The outer diameter of all cups was kept constant. Polyurethane foam was selected as the test material and cup insertion was performed with a maximal force of 6000 N. The excess length between the cup and the surface of the foam blocks was measured. The maximum lever-out force was measured and the lever-out torque was calculated. RESULTS: The excess length of cups with a smooth surface was significantly less (p<0.001) than for the other cups, with no significant differences among the other surface modifications. The lever-out torque for cups with a smooth surface was significantly less (p<0.001) than for the other cups, with no significant differences among the other surface modifications. CONCLUSION: Only the cup with a smooth surface showed significant differences for excess length and lever-out torque. The other surface modifications exhibited the same stability. As long as a rough surface is chosen, cup design seems to have a greater influence on stability than surface modification. Although the study did not mimic real in vivo conditions and the lever-out-torques cannot be transferred to clinical situations, initial stability before bony ingrowth occurred could be clearly analysed.     

Electrochemical properties of biocompatible material hardness modifications on titanium and steel under mechanical loads]

Friction corrosion may appear between different implant components or between implant and hard tissue. The sliding micro movements induce fretting wear corrosion and have been recently reported as a cause of joint prostheses failure. A surface coating is desirable, that retains the mechanical properties of the substrate, offers good biocompatibility and improves the fretting corrosion resistance. In this study it could be demonstrated that tantalum and niobium coatings fulfill the requirements. On titanium substrates the coating decreases the abrasion against PMMA, an orthopedic relevant material. Furthermore, in the case of medical steel substrates the biocompatibility and the corrosion properties are improved. The better abrasion-resistance is minimizing the release of allergological critical particles like nickel and chromium.     

Functionalization of Titanium with Chitosan via Silanation: Evaluation of Biological and Mechanical Performances

Complications in dentistry and orthopaedic surgery are mainly induced by peri-implant bacterial infections and current implant devices do not prevent such infections. The coating of antibacterial molecules such as chitosan on its surface would give the implant bioactive properties. The major challenge of this type of coating is the attachment of chitosan to a metal substrate. In this study, we propose to investigate the functionalization of titanium with chitosan via a silanation. Firstly, the surface chemistry and mechanical properties of such coating were evaluated. We also verified if the coated chitosan retained its biocompatibility with the peri-implant cells, as well as its antibacterial properties. FTIR and Tof-SIMS analyses confirmed the presence of chitosan on the titanium surface. This coating showed great scratch resistance and was strongly adhesive to the substrate. These mechanical properties were consistent with an implantology application. The Chitosan-coated surfaces showed strong inhibition of Actinomyces naeslundii growth; they nonetheless showed a non significant inhibition against Porphyromonas gingivalis after 32 hours in liquid media. The chitosan-coating also demonstrated good biocompatibility to NIH3T3 fibroblasts. Thus this method of covalent coating provides a biocompatible material with improved bioactive properties. These results proved that covalent coating of chitosan has significant potential in biomedical device implantation.     

Physicochemical principles of tissue material interactions

Biocompatibility of a material has to be adapted to the specific properties of the locus of application that are the type of tissue and the composition of extracellular fluid or the blood being in contact with the surface. The biocompatibility is beyond that greatly influenced by the design of the medical device which has to be planned close to the material's properties and the function within the body. Physical chemical reactions at and physical properties of the surface which influence the adsorption behavior for biomacromolecules. Conformational or functional changes of f.i. proteins due to physical forces originating from the surface could be the communication messages to the immunological system. The immersion of a material into an aqueous electrolyte leads generally to a space charge layer on both sides of the interface forming the electrical double layer, physically described by the isoelectric point of the materials surface. A numerical example hints on the importance of the double layer structure for the 'communication' between an implant and the surrounding extracellular fluid including beside ions complex structured proteins as biomacromolecules. Biocompatibility depends on the physical structure of the material and physicochemical properties of the interface to the biosystem. The conductivity of the surface film control reactions across the interface with biomacromolecules of the biological environment. Conformational unchanged macromolecules are the prior condition for biocompatibility and controls the attachment and probably also the degree of attachment via adhesion proteins. Later on, when the cells develop tension through the cytoskeleton on these attachment sites, the strength of the integrin adhesion protein-matrix protein interaction might probably prove decisive in differentiation state of the cell. It has been proved by molecular biological methods that an undestroyed oxide layer of anatase on titanium through passivation leaves for instance albumin conformational unchanged.     

Standardized testing of skeletal implant surfaces with an osteoblast cell culture system. IV. Specific gene expression during differentiation]

Successful osseointegration of an implant depends on the properties of the material of which it is made. A standardized cell culture system for the assessment of the biological effect of material surfaces has already been described. In the present study, this system has been extended to include the quantitative analysis of the material-dependent osteoblast gene expression. Human foetal osteoblasts (hFOB 1.19) were cultured for 3 weeks on titanium surfaces of varying roughness, and on surfaces of chromium-cobalt-molybdenum alloy (CrCoMo). Using a real time RT-PCR technique, expressions of alkaline phosphatase, collagen 1 and osteocalcin were determined as parameters of osteoblast differentiation. In comparison with CrCoMo, differentiation was accelerated on titanium. While the smooth titanium surface leads to earlier cell growth, the rough surface induces more prolonged and stronger cell proliferation. Our results confirm at the molecular level the excellent clinical biocompatibility of titanium surfaces. The real-time RT-PCR provides a new method for the quantitative assessment of material-dependent osteoblastic differentiation.     

Ceramic modifications of porous titanium: Effects on macrophage activation

Porous titanium is one of the most widely used implant materials because of its mechanical properties, however, it is also characterised by low bioactivity. To improve the above parameter we prepared three modifications of the porous (30 wt%) titanium (Ti) surface by covering it with bioactive hydroxyapatite (HA), bioglass (BG) and calcium silicate (CS). Subsequently we tested the impact of the modifications on macrophages directing the inflammatory response that might compromise the implant bioactivity. In the study we investigated the in vitro effects of the materials on murine cell line RAW 264.7 macrophage adherence, morphology and activation (production/release of metalloproteinase MMP-9 and pro- and anti-inflammatory cytokines). CS Ti decreased the macrophage adherence and up-regulated the release of several pro-inflammatory mediators, including TNF-α, IL-6, IL-12. Also HA Ti reduced the cell adherence but other parameters were generally not increased, except of TNF-α. In contrast, BG Ti improved macrophage adherence and either decreased production of multiple mediators (MMP-9, TNF-α, IFN-γ, MCP-1) or did not change it in comparison to the porous titanium. We can conclude that analyzing the effects on the inflammatory response initiated by macrophages in vitro, calcium silicate did not improve the biological properties of the porous titanium. The improved bioactivity of titanium was, however, achieved by the application of the hydroxyapatite and bioglass layers. The present in vitro results suggest that these materials, HA Ti and especially BG Ti, may be suitable for in vivo application and thus justify their further investigation.     

医用多孔钛的研究进展

钛及钛合金由于其优良的抗腐蚀性、生物相容性、低密度和高强度等特点，已广泛应用于承力部位的骨修复，但是如何促进 植入体与骨组织界面的有效结合仍是技术瓶颈，一方面由于植入体的弹性模量与骨组织不匹配，由此产生的应力屏蔽易导致植 入体松动，另一方面植入体缺乏骨诱导作用，导致材料骨界面之间不能形成有效的生物化学结合。近来，具有表面优化处理的新 型生物医用多孔钛材料通过引入孔隙的方法，使其与骨组织的力学性能相匹配，并且应用活化改性技术，使其具有生物活性，成 为目前骨替代材料的研究热点和发展方向。本文简要总结了多孔钛材料在力学性能、生物相容性、制备方法和表面改性等方面的 研究进展，强调在保证其多孔优势性能的前提下，通过生物活性因子的引入，进一步改善其生物相容性，提高结合力，延长植入体 的寿命，使其具有诱导成骨功能，是新型多孔钛材料的发展趋势。     

In vitro assays for adhesion and migration of osteoblastic cells (Saos-2) on titanium surfaces

The first event occurring at the boundary between a metal implant and living tissue is the attachment of cells onto the metal surface of the implant. The attachment characteristics of the metal in this situation are critical in determining its biocompatibility and usefulness as artificial bone and tooth implants. Using the human osteosarcoma cell line Saos-2, we attempted to establish simple and reliable methods for evaluating the attachment of cultured osteoblastic cells onto titanium samples that had been subjected to various surface treatments. Fluorescence actin imaging showed that cells cultured on titanium with hydrofluoric acid etching (HF-Ti) exhibited delayed spreading of their cytoplasm, as compared to cells cultured for the same length of time on nitrided titanium or physically polished titanium. The HF-Ti-cultured cells also exhibited poor assembly of focal contacts, as visualized by vinculin immunofluorescence. Furthermore, in motility assays based on an in vitro wound model, cells cultured on HF-Ti migrated more slowly than cells cultured on other titanium surfaces. These data suggest that Saos-2 cells attach less effectively to the HF-Ti surface. The methods described in this study should be useful for assessing the initial interactions of cultured cells with various materials, including metals.S.G. is the recipient of a grant awarded to foreign students by the government of Japan. This study was supported by the Integrated Center for Science (INCS) at Ehime University.     

Cytotoxicity testing of a polyurethane nanofiber membrane modified with chitosan/β-cyclodextrin/berberine suitable for wound dressing application: evaluation of biocompatibility

In this study we evaluated the biocompatibility of a modified polyurethane nanofiber membrane on a polypropylene spunbond substrate. This material was treated with plasma using diffuse coplanar surface barrier discharge, and subsequent modification was done by continuous spraying of a biologically active chitosan solution (CHIT) containing an inclusion complex of β-cyclodextrin (β-CD) encapsulating berberine (BRB). Biocompatibility was evaluated using several in vitro assays. Human dermal fibroblasts (HDFs) and 3T3 murine fibroblasts were used as biological models. The results of these assays showed that a polyurethane nanofiber membrane modified by CHIT/β-CD/BRB appears to be non-toxic and biocompatible; potentially, it could be used as a wound dressing after further testing.     

Both Enhanced Biocompatibility and Antibacterial Activity in Ag-Decorated TiO2 Nanotubes

In this study, Ag is electron-beam evaporated to modify the topography of anodic TiO₂ nanotubes of different diameters to obtain an implant with enhanced antibacterial activity and biocompatibility. We found that highly hydrophilic as-grown TiO₂ nanotubes became poorly hydrophilic with Ag incorporation; however they could effectively recover their wettability to some extent under ultraviolet light irradiation. The results obtained from antibacterial tests suggested that the Ag-decorated TiO₂ nanotubes could greatly inhibit the growth of Staphylococcus aureus. In vitro biocompatibility evaluation indicated that fibroblast cells exhibited an obvious diameter-dependent behavior on both as-grown and Ag-decorated TiO₂ nanotubes. Most importantly, of all samples, the smallest diameter (25-nm-diameter) Ag-decorated nanotubes exhibited the most obvious biological activity in promoting adhesion and proliferation of human fibroblasts, and this activity could be attributed to the highly irregular topography on a nanometric scale of the Ag-decorated nanotube surface. These experimental results demonstrate that by properly controlling the structural parameters of Ag-decorated TiO₂ nanotubes, an implant surface can be produced that enhances biocompatibility and simultaneously boosts antibacterial activity.     

Cells,growth factors and bioactive surface properties in a mechanobiological model of implant healing

Interface conditions are of prime importance for implant fixation in the early post-operative period and modelling of specific biochemical interactions at implant surface is still missing. We hypothesized that updating osteoblast adhesion properties and growth factor source in an active zone located at the implant surface was relevant to model biochemical interactions of implant with its environment. We proposed an innovative set of diffusive–convective–reactive equations which relevant parameters were the cell decay factor, the cell motility and the growth factor balance.Initial comparison with histomorphometic results from a stable PMMA canine implant model provided an encouraging base to implement a numerical sensitivity analysis to evaluate the role of three types of bioactive surfaces: acid-etched titanium, coarse grit-blasted acid-etched titanium and coarse grit-blasted acid-etched titanium with RGDS peptide. We found that cell diffusion decrease (acid-etched+RGDS peptide vs. PMMA), and increase of local growth factor fraction (PMMA vs. acid-etched+RGDS peptide), significantly improved the amount of mineralized tissue on the implant surface. When the variation of structural fraction to cell motility and growth factor synthesis was investigated, an envelope pattern with an optimum was obtained but this could be exceeded for strong surface modifications and/or for high growth factor concentrations. The model also confirmed that implant bioactive properties should play a limited role to reduce heterogeneity of new-formed tissue. In conclusion, we suggested that our innovative theoretical approach was relevant to investigate implant fixation and could potentially help in reduction of implant revision.     

Cell cultivation on porous titanium implants with various structures

The paper presents data on the cultivation of human dermal fibroblasts and rabbit mesenchymal stromal cells on two types of porous titanium implants, i.e., those with irregular pores formed by pressed titanium particles and those with regular pores formed by the cohesion of one-size titanium particles inside the implant. The goal of this study was to determine what type of titanium implant porosity ensured its strongest interaction with cells. Cells were cultivated on implants for 7 days. During this period, they formed a confluent monolayer on the implant surface. Cells grown on titanium implants were monitored by scanning electron microscopy. Fibroblasts interaction with implants depended on the implant porosity structure. On implants with irregular pores cells were more spread. On implants with regular pores fibroblasts enveloped particles and were only occasionally bound with neighboring particles by small outgrowths. There was no tight interaction of particles inside the implant. In implants formed by pressed particles, cells grow not only on surface, but also in the depth of the implant. Thus, we suppose that a tighter interaction of cells with the titanium implant and, supposedly, tissues with the implant in the organism will take place in the variant when the implant structure is formed by pressed titanium particles, i.e., cellular interaction was observed inside the implant. In implants with irregular pores, cells grew both on the surface and in the depth. Thus, cells exhibited more adequate interactions with irregular pore titanium implants in vitro and hopefully the same interaction will be true in tissues after the implantation of the prosthesis into the organism.     

Adhesion, growth and differentiation of osteoblasts on surface-modified materials developed for bone implants

This review briefly outlines the history and possibilities of bone reconstruction using various types of artificial materials, which allow interaction with cells only on the surface of the implant or enable ingrowth of cells inside the material. Information is also provided on the most important properties of bone cells taking part in bone tissue development, and on diseases and regeneration. The most common cell types used for testing cell-material interaction in vitro are listed, and the most commonly used approaches to this testing are also mentioned. A considerable part of this review is dedicated to the physical and chemical properties of the material surface, which are decisive for the cell-material interaction, and also to modifications to the surface of the material aimed at integrating it better with the surrounding bone tissue. Special attention is paid to the effects of nanoscale and microscale surface roughness on cell behaviour, to material surface patterning, which allows regionally-selective adhesion and growth of cells, and also to the surface chemistry. In addition, coating the materials with bioactive layers is examined, particularly those created by deposition of fullerenes, hybrid metal-fullerene composites, carbon nanotubes, nanocrystalline diamond films, diamond-like carbon, and nanocomposite hydrocarbon plasma polymer films enriched with metals.     

Model for immunologic testing of biomaterials]

Corrosion products and electric fields are capable of changing proteins to antigens, thus permitting the immunological system to identify the biomaterial as foreign. The reaction between corrosion products and a macro-molecule also leads to an antigen (carrier antigen), such as conformational changes of a macro-molecule, e.g. a protein, caused by the electric field at the implant surface (modified macro-molecule antigen). While the sensitivity to corrosion and the effectiveness of galvanic elements is measurable by electrochemical methods, suitable methods of determining the field strength in the vicinity of biomaterial surfaces are still unavailable. The influence of the double layer of uncoated and coated titanium surfaces on the conformation of proteins and their conversion to antigens are investigated with polyclonal antibodies capable of identifying the unchanged protein despite adsorption to the surface. 14C-marked Bovine Serum Albumin serves as a model protein. Determination of the total number of protein molecules adsorbed is effected via the detection of the emitted electrons. The quotient of the concentration of natural proteins to the concentration of adsorbed molecules gives the biocompatibility index, which is independent of the surface area, and gives an indication of the expected biocompatibility of the material. The results of the biological tests of titanium and two coating materials on titanium were confirmed in an animal experiment. It is possible that in the future immunological tests may replace experiments in animals.     

Morphology and chemical characterization of Ti surfaces modified for biomedical applications

The aim of the present work is to characterize in detail the chemical composition and morphology of titanium surfaces subjected to various environments. Modifications consisted of exposure of Ti to acidic, alkaline or polymer solutions. Such modifications result in chemical and/or morphological changes in the Ti surface. Special attention has been given to identifying the factors influencing cell adhesion and growth. SEM examinations provided morphological characterization of the Ti samples. Surface analytical techniques such as AES or XPS combined with Ar(+) ion sputtering allowed examination of the chemical properties of the Ti surface after chemical pretreatments and investigating the chemical composition of the Ti oxide layer. Raman spectroscopy investigations allowed determination of the crystalline phases of the Ti-oxide layers and characterization of the dextran-modified surface. The results show large differences in the morphology of Ti pretreated with different procedures whereas only minor differences in the chemistry of the surfaces were found. High-resolution Auger investigations have revealed that all the chemical modifications of Ti surfaces resulted in the formation of a titanium oxide layer. XPS confirmed that TiO(2) is the main component of the chemically modified Ti surface. The Raman spectroscopy investigations showed that the titanium surface with a dextran coating is rich in hydroxyl groups. All the surfaces investigated exhibit a hydrophilic character. The possible influence of various surface features on surface biocompatibility is discussed.     

Polysaccharide-coated thermosets for orthopedic applications: from material characterization to in vivo tests

The long-term stability and success of orthopedic implants depend on the osseointegration process, which is strongly influenced by the biomaterial surface. A promising approach to enhance implant integration involves the modification of the surface of the implant by means of polymers that mimic the natural components of the extracellular matrix, for example, polysaccharides. In this study, methacrylate thermosets (bisphenol A glycidylmethacrylate/triethyleneglycol dimethacrylate), a widely used composition for orthopedic and dental applications, have been coated by electrostatic deposition of a bioactive chitosan-derivative. This polysaccharide was shown to induce osteoblasts aggregation in vitro, to stimulate cell proliferation and to enhance alkaline phosphatase activity. The coating deposition was studied by analyzing the effect of pH and ionic strength on the grafting of the polysaccharide. Contact angle studies show that the functionalized material displays a higher hydrophilic character owing to the increase of surface polar groups. The mechanical properties of the coating were evaluated by nanoindentation studies which point to higher values of indentation hardness and modulus (E) of the polysaccharide surface layer, while the influence of cyclic stress on the construct was assessed by fatigue tests. Finally, in vivo tests in minipigs showed that the polysaccharide-based implant showed a good biocompatibility and an ability for osseointegration at least similar to that of the titanium Ti6Al4V alloy with roughened surface.     

Hybrid titanium/biodegradable polymer implants with an hierarchical pore structure as a means to control selective cell movement

In order to improve implant success rate, it is important to enhance their responsiveness to the prevailing conditions following implantation. Uncontrolled movement of inflammatory cells and fibroblasts is one of these in vivo problems and the porosity properties of the implant have a strong effect on these. Here, we describe a hybrid system composed of a macroporous titanium structure filled with a microporous biodegradable polymer. This polymer matrix has a distinct porosity gradient to accommodate different cell types (fibroblasts and epithelial cells). The main clinical application of this system will be the prevention of restenosis due to excessive fibroblast migration and proliferation in the case of tracheal implants. METHODOLOGY/PRINCIPAL FINDINGS: A microbead-based titanium template was filled with a porous Poly (L-lactic acid) (PLLA) body by freeze-extraction method. A distinct porosity difference was obtained between the inner and outer surfaces of the implant as characterized by image analysis and Mercury porosimetry (9.8±2.2 μm vs. 36.7±11.4 μm, p≤0.05). On top, a thin PLLA film was added to optimize the growth of epithelial cells, which was confirmed by using human respiratory epithelial cells. To check the control of fibroblast movement, PKH26 labeled fibroblasts were seeded onto Titanium and Titanium/PLLA implants. The cell movement was quantified by confocal microscopy: in one week cells moved deeper in Ti samples compared to Ti/PLLA. CONCLUSIONS: In vitro experiments showed that this new implant is effective for guiding different kind of cells it will contact upon implantation. Overall, this system would enable spatial and temporal control over cell migration by a gradient ranging from macroporosity to nanoporosity within a tracheal implant. Moreover, mechanical properties will be dependent mainly on the titanium frame. This will make it possible to create a polymeric environment which is suitable for cells without the need to meet mechanical requirements with the polymeric structure.     

Preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering

In this study, we report the physico-chemical and biological properties of a novel biodegradable composite scaffold made of nano-hydroxyapatite and natural derived polymers of chitosan and carboxymethyl cellulose, namely, n-HA/CS/CMC, which was prepared by freeze-drying method. The physico-chemical properties of n-HA/CS/CMC scaffold were tested by infrared absorption spectra (IR), transmission electron microscope(TEM), scanning electron microscope(SEM), universal material testing machine and phosphate buffer solution (PBS) soaking experiment. Besides, the biological properties were evaluated by MG63 cells and Mesenchymal stem cells (MSCs) culture experiment in vitro and a short period implantation study in vivo. The results show that the composite scaffold is mainly formed through the ionic crossing-linking of the two polyions between CS and CMC, and n-HA is incorporated into the polyelectrolyte matrix of CS-CMC without agglomeration, which endows the scaffold with good physico-chemical properties such as highly interconnected porous structure, high compressive strength and good structural stability and degradation. More important, the results of cells attached, proliferated on the scaffold indicate that the scaffold is non-toxic and has good cell biocompatibility, and the results of implantation experiment in vivo further confirm that the scaffold has good tissue biocompatibility. All the above results suggest that the novel degradable n-HA/CS/CMC composite scaffold has a great potential to be used as bone tissue engineering material.