Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN)... more
Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces.
Research Interests: Genetics, Chemistry, Titanium, Wear, Medicine, and 13 moreImplants, Model, Humans, Male, Solubility, Alendronate, Molecular sciences, RANKL, Implant, OPG, Ligand, Bone Resorption, and Denosumab
Bone tissue defects resulting from periodontal disease are often treated using guided tissue regeneration (GTR). The barrier membranes utilized here should prevent soft tissue infiltration into the bony defect and simultaneously support... more
Bone tissue defects resulting from periodontal disease are often treated using guided tissue regeneration (GTR). The barrier membranes utilized here should prevent soft tissue infiltration into the bony defect and simultaneously support bone regeneration. In this study, we designed a degradable poly(l-lactide-co-glycolide) (PLGA) membrane that was surface-modified with cell adhesive arginine-glycine-aspartic acid (RGD) motifs. For a novel method of membrane manufacture, the RGD motifs were coupled with the non-ionic amphiphilic polymer poly(2-oxazoline) (POx). The RGD-containing membranes were then prepared by solvent casting of PLGA, POx coupled with RGD (POx_RGD), and poly(ethylene glycol) (PEG) solution in methylene chloride (DCM), followed by DCM evaporation and PEG leaching. Successful coupling of RGD to POx was confirmed spectroscopically by Raman, Fourier transform infrared in attenuated reflection mode (FTIR-ATR), and X-ray photoelectron (XPS) spectroscopy, while successful ...
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1 AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Krakow, Poland. 2 Technische Universität Dresden, Faculty of Chemistry and Food Chemistry, Chair of Macromolecular Chemistry, Dresden, Germany. 3... more
1 AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Krakow, Poland. 2 Technische Universität Dresden, Faculty of Chemistry and Food Chemistry, Chair of Macromolecular Chemistry, Dresden, Germany. 3 Center of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland. 4 Technische Universität Dresden, Institut für Werkstoffwissenschaft, Max-Bergmann-Zentrum für Biomaterialien, Dresden, Germany. 5 Leibniz-Institut für Polymerforschung Dresden e.V., Institut für Makromolekulare Chemie, Polymer Separation Group, Dresden and Technische Universität Dresden, Dresden, Germany.
PURPOSE Existing knowledge about the appearance, thickness, and chemical composition of phosphate coatings on titanium inside porous structures is insufficient. Such knowledge is important for the design and fabrication of porous... more
PURPOSE Existing knowledge about the appearance, thickness, and chemical composition of phosphate coatings on titanium inside porous structures is insufficient. Such knowledge is important for the design and fabrication of porous implants. METHODS Metallic scaffolds were fabricated by selective laser melting of 316L stainless steel powder. Phosphate coatings were deposited on Ti sensors placed either outside the scaffolds or in the holes in the scaffolds. The electrochemically-assisted cathodic deposition of phosphate coatings was performed under galvanostatic conditions in an electrolyte containing the calcium and phosphate ions. The phosphate deposits were microscopically investigated; this included the performance of mass weight measurements and chemical analyses of the content of Ca2+ and 24 PO ions after the dissolution of deposits. RESULTS The thicknesses of the calcium phosphate coatings were about 140 and 200 nm for isolated titanium sensors and 170 and 300 nm for titanium...
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their surface properties. A main challenge in this area is the development of processing routes enabling for a simple but efficient surface design of complex shaped geometries. Against this background, this work aimed at the... more
their surface properties. A main challenge in this area is the development of processing routes enabling for a simple but efficient surface design of complex shaped geometries. Against this background, this work aimed at the implementation of self-assembly principles for surface functionalization of 3D-printed poly(lactic-co-glycolic acid) (PLGA)-based constructs with macro- and microporous geometries via precision extruding deposition. METHODS Three-component melts from PLGA, CaCO3 and amphiphilic polymers (poly(2-oxazoline) block copolymer) were printed and their bulk and surface properties were studied. RESULTS Melts with up to 30 mass % of CaCO3 could be successfully printed with homogeneously distributed mineral particles. PLGA degradation during the printing process was temperature and time dependent: the molecular weight reached 10 to 15% of the initial values after ca. 120 min of heat exposure. Filament surfaces from melts containing CaCO3 show an increasing microroughness a...
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Tissue regeneration is regulated by the cellular microenvironment, e.g. the extracellular matrix. Here, sulfated glycosaminoglycans (GAG), are of vital importance interacting with mediator proteins and influencing their biological... more
Tissue regeneration is regulated by the cellular microenvironment, e.g. the extracellular matrix. Here, sulfated glycosaminoglycans (GAG), are of vital importance interacting with mediator proteins and influencing their biological activity. Hence, they are promising candidates for controlling tissue regeneration. This review addresses recent achievements regarding chemically modified GAG as well as collagen/GAG-based coatings and hydrogels including (i) chemical functionalization strategies for native GAG, (ii) GAG-based biomaterial strategies for controlling cellular responses, (iii) (bio)chemical methods for characterization and iv) protein interaction profiles and attained tissue regeneration in vitro and in vivo. The potential of GAG for bioinspired, functional biomaterials is highlighted.
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The objective of this study was to investigate the reproducibility, mechanical integrity, surface characteristics and corrosion behavior of nanotubular (NT) titanium oxide arrays in comparison with a novel nano-pitted (NP) anodic film.... more
The objective of this study was to investigate the reproducibility, mechanical integrity, surface characteristics and corrosion behavior of nanotubular (NT) titanium oxide arrays in comparison with a novel nano-pitted (NP) anodic film. Surface treatment processes were developed to grow homogenous NT and NP anodic films on the surface of grade 2 titanium discs and dental implants. The effect of process parameters on the surface characteristics and reproducibility of the anodic films was investigated and optimized. The mechanical integrity of the NT and NP anodic films were investigated by scanning electron microscopy, surface roughness measurement, scratch resistance and screwing tests, while the chemical and physicochemical properties were investigated in corrosion tests, contact angle measurement and X-ray photoelectron spectroscopy (XPS). The growth of NT anodic films was highly affected by process parameters, especially by temperature, and they were apt to corrosion and exfoliati...
Research Interests: Materials Science, Biomedical Engineering, Biomaterials, Scanning Electron Microscopy, Titanium, and 13 moreNanotechnology, Dental Materials, Dental Implants, Medicine, Corrosion, Surface Roughness, Anodization, Nanotubes, Titania Nanotubes, Surface treatment, Surface Properties, Reproducibility of Results, and X Ray Photoelectron Spectroscopy
Poly(L-lactide-co-glycolide) (PLGA) scaffolds of pore size within the range of 250-320 μm were produced by solvent casting/ porogen leaching method. Afterwards, they were modified through adsorption of collagen type I and incubation in... more
Poly(L-lactide-co-glycolide) (PLGA) scaffolds of pore size within the range of 250-320 μm were produced by solvent casting/ porogen leaching method. Afterwards, they were modified through adsorption of collagen type I and incubation in simulated body fluid (SBF) to allow deposition of hydroxyapatite (HAp). The wettability of the scaffolds was measured by sessile drop test. Scanning electron microscopy (SEM) evaluation and energy dispersive X-ray analysis (EDX) were also performed. SEM evaluation and EDX analysis depicted the presence of HAp deposits and a collagen layer on the pore walls on the surface and in the bulk of the scaffolds. Wettability and water droplets penetration time within the scaffolds decreased considerably after applying modifications. Human mesenchymal stem cells (hMSC) were cultured on the scaffolds for 28 days and cell morphology, proliferation and differentiation as well as calcium deposition were evaluated. Lactate dehydrogenase (LDH) activity results reveal...
Research Interests: Chemistry, Biomedical Engineering, Medicine, Biomaterials and Tissue Engineering, Humans, and 14 morePubmed, Animals, Male, Mesenchymal Stem Cell, Alkaline phosphatase, Clinical Sciences, Cattle, In Vivo, Adult, Durapatite, Lactic Acid, Mesenchymal Stromal Cells, Tissue Scaffolds, and Collagen type I
A satisfactory clinical outcome in dental implant treatment relies on primary stability for immediate load bearing. While the geometric design of an implant contributes to mechanical stability, the nature of the implant surface itself is... more
A satisfactory clinical outcome in dental implant treatment relies on primary stability for immediate load bearing. While the geometric design of an implant contributes to mechanical stability, the nature of the implant surface itself is also critically important. Biomechanical and microcomputerized tomographic evaluation of implant osseointegration was performed to compare alternative structural, chemical and biochemical, and/or pharmaceutical surface treatments applied to an identical established implant design. Dental implants with the same geometry but with 6 different surface treatments were tested in vivo in a sheep model (pelvis). Peri-implant bone density and removal torque were compared at 2, 4, and 8 weeks after implantation. Implant surfaces tested were: sandblasted and acid-etched titanium (Ti), sandblasted and etched zirconia, Ti coated with calcium phosphate (CaP), Ti modified via anodic plasma-chemical treatment (APC), bisphosphonate-coated Ti (Ti + Bisphos), and Ti c...
Research Interests: Materials Science, Dentistry, Biomedical Engineering, Osseointegration, Agriculture, and 15 moreDental Materials, Dental Implants, Medicine, Implants, Collagen, Animals, Calcium Phosphates, Bone Density, Ilium, Implant, Dental Prosthesis Design, Osteogenesis, Dental Implant, Materials Testing, and Biomechanical Phenomena
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Background: The functionalization of metallic surfaces aims at promoting the cellular response at the biomaterial–tissue interface. This study investigates the effects of the functionalization of titanium (Ti) microtopography with a... more
Background: The functionalization of metallic surfaces aims at promoting the cellular response at the biomaterial–tissue interface. This study investigates the effects of the functionalization of titanium (Ti) microtopography with a calcium phosphate (CaP) coating with and without peptide 15 (P‐15), a synthetic peptide analog of the cell‐binding domain of collagen I, on the in vitro progression of osteogenic cells.Methods: Sandblasting and acid etching (SBAE; control) Ti microtopography was coated with CaP, enabling the loading of two concentrations of P‐15: 20 or 200 μg/mL. A machined Ti was also examined. Rat calvarial osteogenic cells were cultured on Ti disks with the surfaces mentioned above for periods up to 21 days (n = 180 per group).Results: CaP coating exhibited a submicron‐scale needle‐shaped structure. Although all surfaces were hydrophobic at time zero, functionalization increased hydrophilicity at equilibrium. Microtopographies exhibited a lower proportion of well‐spre...
Research Interests: Chemistry, Dentistry, Calcium, Cell Adhesion, Medicine, and 15 morePeriodontology, Surface modification, Collagen, Animals, Calcium Phosphates, Osteopontin, Alkaline phosphatase, Rats, Surface Properties, Cell Proliferation, Osteoblasts, Cell Survival, Cytoplasm, Cell count, and Cell culture techniques
Research Interests: Materials Engineering, Chemical Engineering, Materials Science, Biomedical Engineering, Conservation, and 15 moreMaterials, Titanium, Crystallization, Comparative Study, Medicine, Porosity, Nanostructures, Nanostructure, Contact angle, Biomaterial, Etching, Rock blasting, Surface Properties, Materials Testing, and Biocompatible Materials
Biological implant surface coatings are an emerging technology to increase bone formation. Such an approach is of special interest in anatomical regions like the maxilla. In the present study, we hypothesized that the coating of titanium... more
Biological implant surface coatings are an emerging technology to increase bone formation. Such an approach is of special interest in anatomical regions like the maxilla. In the present study, we hypothesized that the coating of titanium implants with components of the organic extracellular matrix increases bone formation and implant stability compared to an uncoated reference. The implants were coated using collagen‐I with either two different concentrations of chondroitin sulfate (CS) or two differentially sulfated hyaluronans. Implant coatings were characterized biochemically and with atomic force microscopy. Histomorphometry was used to assess bone‐implant contact (BIC) and bone‐volume density (BVD) after 4 and 8 weeks of submerged healing in the maxilla of 20 minipigs. Further, implant stability was measured by resonance frequency analysis (RFA). Implants containing the lower CS concentration had significantly more BIC, compared to the uncoated reference at both times of intere...
Research Interests: Materials Science, Biomedical Engineering, Osseointegration, Dental Implants, Medicine, and 12 moreExtracellular Matrix, Surface modification, Female, Animals, Glycosaminoglycan, Implant, Swine, Resonance Frequency Analysis, Chondroitin Sulfate, Medicine and Health Sciences, Materials Testing, and Collagen type I
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Research Interests: Chemistry, Tissue Engineering, Regeneration, Regeneration (Biology), Medicine, and 15 moreExtracellular Matrix, Cartilage, Animals, Connective tissue, Proteins, Glycosaminoglycan, Hyaluronic Acid, Glycosaminoglycans, Chondroitin Sulfate, Protein Binding, Sulfates, Dermatan sulfate, Pharmacology and pharmaceutical sciences, stereoisomerism, and Sulfation
Research Interests: Engineering, Chemistry, Kinetics, Comparative Study, Medicine, and 15 moreBiological Sciences, Humans, Recombinant DNA, CHEMICAL SCIENCES, Hyaluronic Acid, Chondroitin Sulfate, Aqueous Solution, Recombinant Proteins, Enzyme Linked Immunosorbent Assay, Polyelectrolyte, Biomacromolecules, Biosensing Techniques, Chemical Modification, Regioselectivity, and Sulfation
Research Interests: Materials Science, Cell Biology, Medicine, Multidisciplinary, Extracellular Matrix, and 15 moreSignal Transduction, Cell Differentiation, Humans, Myofibroblasts, Clinical Sciences, Hyaluronic Acid, Fibronectin, Biomimetic materials, Surface Properties, Transforming Growth Factor Beta, Materials Testing, Sulfates, Myofibroblast, Transforming Growth Factor, and Collagen type I
Research Interests: Materials Science, Chemistry, Cell Biology, Medicine, Extracellular Matrix, and 15 moreDexamethasone, Cell Differentiation, Humans, Animals, Male, Calcium Phosphates, Mesenchymal Stem Cell, Glycosaminoglycan, Alkaline phosphatase, Hyaluronic Acid, Glycosaminoglycans, Adult, Mesenchymal Stromal Cells, Gene Expression Regulation, and Collagen type I
Research Interests: Materials Science, Inflammation, Cell Biology, Macrophages, Medicine, and 15 moreMultidisciplinary, Extracellular Matrix, Cell Differentiation, Humans, Macrophage, Animals, Phenotype, Hyaluronic Acid, Rats, Biological markers, Macrophage Polarization, Interleukin, Interferon regulatory factors, Collagen type I, and Inflammation Mediators
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Surface functionalization with bioactive molecules (BAMs) on a nanometre scale is a main field in current biomaterial research. The immobilization of a vast number of substances and molecules, ranging from inorganic calcium phosphate... more
Surface functionalization with bioactive molecules (BAMs) on a nanometre scale is a main field in current biomaterial research. The immobilization of a vast number of substances and molecules, ranging from inorganic calcium phosphate phases up to peptides and proteins, has been investigated throughout recent decades. However, in vitro and in vivo results are heterogeneous. This may be at least partially attributed to the limits of the applied immobilization methods. Therefore, this paper highlights, in the first part, advantages and limits of the currently applied methods for the biological nano-functionalization of titanium-based biomaterial surfaces. The second part describes a new immobilization system recently developed in our groups. It uses the nanomechanical fixation of at least partially single-stranded nucleic acids (NAs) into an anodic titanium oxide layer as an immobilization principle and their hybridization ability for the functionalization of the surface with BAMs conj...
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Specific surface binding peptides offer a versatile and interesting possibility for the development of biocompatible implant materials. Therefore, eight peptide sequences were examined in regard to their adsorption on zirconium oxide... more
Specific surface binding peptides offer a versatile and interesting possibility for the development of biocompatible implant materials. Therefore, eight peptide sequences were examined in regard to their adsorption on zirconium oxide (ZrO2), titanium zircon (TiZr), and titanium (c.p. Ti). Surface plasmon resonance (SPR) measurements were performed on Ti coated sensor chips to determine the kinetics of the interactions and kinetic rate constants (kon, koff, KD, and Rmax). We also investigated the interactions which are present in our system. Electrostatic and coordinative interactions were found to play a major role in the adsorption process. Four of the eight examined peptide sequences showed a significant adsorption on all investigated materials. Moreover, the two peptides with the highest adsorption could be quantified (up to 370 pmol/cm(2)). For potential biomaterials applications, we proved the stability of the adsorption of selected peptides in cell culture media, under competition with proteins and at body temperature (37 °C), and their biocompatibility via their effects on the adhesion and proliferation of human mesenchymal stem cells (hMSCs). The results qualify the peptides as anchor peptides for the biofunctionalization of implants.
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Due to their outstanding properties nanodiamonds are a promising nanoscale material in various applications such as microelectronics, polishing, optical monitoring, medicine and biotechnology. Beyond the typical diamond characteristics... more
Due to their outstanding properties nanodiamonds are a promising nanoscale material in various applications such as microelectronics, polishing, optical monitoring, medicine and biotechnology. Beyond the typical diamond characteristics like extreme hardness or high thermal conductivity, they have additional benefits as intrinsic fluorescence due to lattice defects without photobleaching, obtained during the high pressure high temperature process. Further the carbon surface and its various functional groups in consequence of the synthesis, facilitate additional chemical and biological modification. In this work we present our recent results on chemical modification of the nanodiamond surface with phosphate groups and their electrochemically assisted immobilization on titanium-based materials to increase adhesion at biomaterial surfaces. The starting material is detonation nanodiamond, which exhibits a heterogeneous surface due to the functional groups resulting from the nitrogen-rich...
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The quantitative analysis of bone formation around biofunctionalised metallic implants is an important tool for the further development of implants with higher success rates. This is, nowadays, especially important in cases of additional... more
The quantitative analysis of bone formation around biofunctionalised metallic implants is an important tool for the further development of implants with higher success rates. This is, nowadays, especially important in cases of additional diseases like diabetes or osteoporosis. Micro computed tomography (muCT), as non-destructive technique, offers the possibility for quantitative three-dimensional recording of bone close to the implant's surface with
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Research Interests: Scanning Electron Microscopy, Titanium, Multidisciplinary, Osteoporosis, Humans, and 14 moreTibia, Female, Animals, Orthopedics, Bone Density, PLoS one, Rats, Surface Properties, Transforming Growth Factor Beta, Wistar Rats, Recombinant Proteins, X Ray Microtomography, Oligonucleotides, and Biomechanical Phenomena
Research Interests: Technology, Tissue Engineering, Image Analysis, Titanium, Polymers, and 19 moreSynchrotron Radiation, Quantitative analysis, Biological Sciences, X Rays, Porosity, Biomolecular Engineering, Three Dimensional Imaging, Materials Design, Cross Section, Three Dimensional, Spatial resolution, Quantitative Analysis, Biomolecular, Bone Formation, Bone Substitutes, Materials Testing, X ray Computed Tomography, X ray Absorption, and Lactic Acid
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Research Interests: Mechanical Engineering, Biomechanical Engineering, Biomedical Engineering, Tissue Engineering, Extracellular Matrix, and 15 moreCell Differentiation, Humans, Collagen, Bone Tissue Engineering, Bone marrow, Alkaline phosphatase, Chondroitin Sulfate, Biomechanical, Osteoblasts, Durapatite, Polyesters, Bone and Bones, Hydrostatic Pressure, Mesenchymal Stromal Cells, and Human Mesenchymal Stem Cells
Inflammation is an important step in the early phase of tissue regeneration around an implanted metallic orthopaedic device. However, prolonged inflammation, which can be induced by metallic corrosion products, can lead to aseptic... more
Inflammation is an important step in the early phase of tissue regeneration around an implanted metallic orthopaedic device. However, prolonged inflammation, which can be induced by metallic corrosion products, can lead to aseptic loosening and implant failure. Cells in peri-implant tissue as well as metal corrosion can induce reactive oxygen species (ROS) formation, thus contributing to an oxidative microenvironment around an implant. Understanding cellular reactions to implant-induced oxidative stress and inflammatory activation is important to help prevent an adverse response to metallic materials. In an earlier study we have shown that endothelial cells grown on Ti6Al4V alloy are subjected to oxidative stress. Since endothelial cells play a critical role in inflammation, in this study we examined the role of oxidative stress in their response to pro-inflammatory activation. Therefore, we stimulated endothelial cells in contact with Ti6Al4V with tumour necrosis factor-α (TNF-α) and monitored the expression of inflammation-associated molecules, such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8). The induction of these proteins was lower in endothelial cells on Ti6Al4V compared to control tissue culture conditions. There was, however, a discrepancy in pro-inflammatory activation at protein compared to mRNA level in the cells on Ti6Al4V. To examine the role of oxidative stress in this response we utilized different ROS scavengers and showed that ROS depletion improved cellular response to TNF-α on Ti6Al4V. These results could contribute to developing strategies to improve tissue response to metal implants.
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Research Interests: Biomaterials, Kinetics, Minerals, Titanium, Multidisciplinary, and 14 moreExtracellular Matrix, Adsorption, Graphite, Calcium Phosphate, Surface Properties, Protein Conformation, Materials Testing, High Sensitivity, Structure activity Relationship, Simulated Body Fluid, Body Fluids, Conformational Change, Anodic Oxidation, and Highly Oriented Pyrolytic Graphite
Research Interests: Biomaterials, Osseointegration, Cell Adhesion, Scanning Electron Microscopy, Titanium, and 19 moreMultidisciplinary, Extracellular Matrix, Synchrotron Radiation, Bone Regeneration, Femur, Female, Animals, Histomorphometry, Bone Density, Goats, Cattle, Micro Computed Tomography, Time Factors, Surface Properties, Bone Formation, Bone Substitutes, X ray Computed Tomography, Biocompatible Materials, and Bone and Bones
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Coating titanium implants with artificial extracellular matrices based on collagen and chondroitin sulfate (CS) has been shown to enhance bone remodelling and de novo bone formation in vivo. The aim of this study was to evaluate the... more
Coating titanium implants with artificial extracellular matrices based on collagen and chondroitin sulfate (CS) has been shown to enhance bone remodelling and de novo bone formation in vivo. The aim of this study was to evaluate the effect of estrogen deficiency and hormone replacement therapy (HRT) on the osseointegration of CS-modified Ti implants. 30 adult female, ovariectomized Wistar rats were fed either with an ethinyl-estradiol-rich diet (E) to simulate a clinical relevant HRT or with a genistein-rich diet (G) to test an alternative therapy based on nutritionally relevant phytoestrogens. Controls (C) received an estrogen-free diet. Uncoated titanium pins (Ti) or pins coated with type-I collagen and CS (Ti/CS) were inserted 8weeks after ovarectomy into the tibia. Specimens were retrieved 28days after implantation. Both the amount of newly formed bone and the affinity index (P<0.05) were moderately higher around Ti/CS implants as compared to uncoated Ti. The highest values were measured in the G-Ti/CS and E-Ti/CS groups, the lowest values for the E-Ti and G-Ti controls. Quantitative synchrotron radiation micro-computed tomography (SRμCT) revealed the highest increase in total bone formation around G-Ti/CS as compared to C-Ti (P<0.01). The effects with respect to direct bone apposition were less pronounced with SRμCT. Using scanning nanoindentation, both the indentation modulus and the hardness of the newly formed bone were highest in the E-Ti/CS, G-Ti/CS and G-Ti groups as compared to C-Ti (P<0.05). Coatings with collagen and CS appear to improve both the quantity and quality of bone formed around Ti implants in ovarectomized rats. A simultaneous ethinyl estradiol- and genistein-rich diet seems to enhance these effects.