Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography
Introduction
Events leading to integration of an implant into bone, and hence determining the long-term performance of the device, take place largely at the interface formed between the tissue and the implant. The development of this interface is complex and is influenced by numerous factors, including surface chemistry and surface topography of the foreign material [1], [2], [3], [4], [5]. To improve bone-tissue integration, various techniques have been used to increase the surface roughness of titanium implants, including machining/micromachining, particle blasting, titanium plasma-spraying, chemical/electrochemical etching, particle blasting and chemical etching, electrochemical anodization, or pulsed laser ablation [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] . Topographic features on implants range from millimeters to nanometers and are all believed to be relevant to the biological response of the host [2], [13], [14], [15]. More specifically, micrometer and/or nanometer scale topographies affect different aspects of cell behavior such as cell adhesion, cell proliferation, cell differentiation, cell morphology, cell orientation, contact guidance, tissue organization, mechanical interlocking, production of local factors and microenvironments, and also cell selection [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. In addition to producing the desired topographies, the surface roughening processes can also alter surface composition, surface charge and/or surface energy of the implant [13], [14], [35], [36], [37]. Many in vivo studies have compared the effects of the surface structure on the biomechanical stability of the device, on the morphology of the bone surrounding it and/or on the extent of the bone-implant interface [6], [8], [10], [11], [12], [16], [17], [18], [38]. Various results have been obtained, depending on the roughness amplitude but also on the method used to produce the surface topography [8], [9], [10], [11], [12], [16], [17], [18], [39].
Cell adhesion is one of the initial events essential to subsequent proliferation and differentiation of bone cells before bone tissue formation [40], [41]. Many in vitro evaluations of cell adhesion on substrates with various and usually complex topographies have been performed [19], [23], [29], [33], [40], [42], [43], [44], [45], [46], [47], [48]. However, up to now little is known concerning the main topographical properties influencing the cell response to implant surfaces. In order to obtain precise information about the effects of the surface topography on cell behavior, one should be able to study independently the micro topography (structures larger than 1 μm), the nanotopography (structures smaller than 1 μm) and the superposition of both, all this without changing the chemical characteristics of the surfaces. This requires the use of model surfaces. Recently, scale-resolved electrochemical surface structuring of bulk titanium has been reported [49]. Optimized through-mask electrochemical micromachining (EMM) of mechanically polished titanium led to well-defined microstructures comparable to cell size. Hexagonal arrays of closely spaced smooth hemispherical cavities of 10–100 μm diameter were made reproducibly using computer-controlled dissolution and precise charge control. Porous anodization and chemical etching, respectively, permitted to generate nanotopography on flat and on previously microstructured surfaces.
The aim of the present study is to use such well defined titanium model surfaces for the investigation how microscale and nanoscale roughness separately and in combination affect the early phase of cell-surface interactions that occur between some hours and some days. For this a kinetic morphological analysis of adhesion, spreading and proliferation of bone-derived MG63 cells was carried out. These cells exhibit the characteristics of immature human osteoblasts and have been widely used in the literature for the evaluation of implant surfaces.
Section snippets
Surface design
Hemispherical cavities with a diameter comparable to cell size and arranged in hexagonal patterns were chosen to study the role of the micro topography on cell behavior. The sample surface outside the cavities is the original mechanically polished surface, whereas the inside of the cavities has an electropolished surface finish [49]. For a given ratio RS=Sc/Sf, where Sc is the area of the electropolished cavities and Sf is the area of the flat region between the cavities, the diameter of the
Effect of nanotopography
The SEM micrographs of Fig. 2 for different nanostructured surfaces show the evolution of the morphology of the cells and their proliferation from 4 h to 7 days. Confluence was reached on all surfaces at 7 days. The cells were flat and spread on the polished (1) and electro. (2) surfaces. On anodized (4) and etched (3) surfaces, notably after 4 h, the cells appeared less spread and thicker than on the other surfaces. After 3 days, the cells on polished (1), electro. (2) and anodized (4) surfaces
Discussion
In the present paper titanium disks with electrochemically produced scale-resolved surface topography were used to study the effect on cell-surface interactions of nanoscale and microscale surface roughness separately and in combination of both. In particular, the initial phase of adhesion, spreading and proliferation was investigated by SEM and immunolabeling using MG63 human osteoblast like cells because these cells have been widely used in the past for immuno- and bio-chemical studies of
Conclusions
The present study of adhesion, morphology and proliferation of osteoblast-like MG63 cells on titanium model surfaces with well-characterized scale-resolved surface topography leads to the following conclusions:
- •
On flat titanium surfaces containing submicrometer scale roughness produced by chemical etching or porous anodization, the cells exhibit a higher thickness and delayed apparition of the focal contacts compared to smooth polished and electropolished surfaces. Submicrometer scale roughness
Acknowledgements
This work was funded by CTI (Commission for Technology and Innovation, grant number 4719.1) and European Funds for Regional Development (FEDER, Obj.2-99.2-01b-no. 78). The authors thank R. Christ (Institut Straumann AG) for SEM pictures, N. Xanthopoulos (IMX-LMCH-EPFL) for surface analysis, and I. Loison (LR2B, University of Littoral Côte d’Opale) for technical assistance in the cell cultures. Institut Straumann AG (Waldenburg, Switzerland) is gratefully acknowledged for supplying the
References (59)
- et al.
Effects of surface roughness of titanium implants on bone remodeling activity of femur in rabbits
Bone
(1997) The importance of surface roughness for implant incorporation
Int J Mach Tool Manufact
(1998)A role for surface topography in creating and maintaining bone at titanium endosseous implants
J Prosthetic Dentist
(2000)- et al.
Topographical control of cells
Biomaterials
(1997) - et al.
Nantotechniques and approaches in biotechnology
Trend Biotechnol
(2001) - et al.
Role of material surfaces in regulating bone and cartilage cell response
Biomaterials
(1996) - et al.
Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition
Biomaterials
(1998) - et al.
The relative influence of the topography and chemistry of TiAl6V4 surfaces on osteoblastic cell behaviour
Biomaterials
(2000) Osteoblast adhesion on biomaterials
Biomaterials
(2000)- et al.
Influence of the surface structure of titanium materials on the adhesion of fibroblasts
Biomaterials
(1996)
Improvement in the morphology of Ti-based surfacesa new process to increase in vitro human osteoblast response
Biomaterials
Variable length scale analysis of surface topographycharacterization of titanium surfaces for biomedical applications
Surf Coat Technol
Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein-and cell-interactions. IIprotein adsorption and early cell interactions
Biomaterials
The interface zone of inorganic implants invivo-titanium implants in bone
Ann Biomed Eng
Osseointegrationa reality
Periodontology 2000
Surface science aspects on inorganic biomaterials
CRC Crit Rev in Biocompat
The titanium-bone interface in vivo
Generalizations regarding the process and phenomenon of osseointegration. Part I. In vivo studies
Int J Oral Maxillofacial Implant
Influence of surface characteristics on bone integration of titanium implants-a histomorphometric study in miniature pigs
J Biomed Mater Res
The effects of micromachined surfaces on formation of bonelike tissue on subcutaneous implants as assessed by radiography and computer image processing
J Biomed Mater Res
Osseointegration enhanced by chemical etching of the titanium surface-A torque removal study in the rabbit
Clin Oral Implant Res
Bone healing capacity of titanium plasma-sprayed and hydroxylapatite-coated oral implants
Clin Oral Implant Res
Surface characterization and biological evaluation of spark-eroded surfaces
J Mater Sci Mater Med
Measurement and evaluation of the chemical composition and topography of titanium implant surfaces
Wavelength-dependent roughnessa quantitative approach to characterizing the topography of rough titanium surfaces
Int J Oral Maxillofacial Implant
Oxidized implants and their influence on the bone response
J Mater Sci Mater Med
Laser microtexturing of implant surfaces for enhanced tissue integration
Key Eng Mater
Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses
J Biomed Mater Res
Cited by (307)
Ultra-high strength TiZrNbTa high entropy alloy substrate coated by coral-like metal oxide nanotubes to enhance biocompatibility
2022, Journal of Alloys and CompoundsDesign of Ti-6Al-4V alloy surface properties by galvanostatic electrochemical treatment in a deep eutectic solvent Ethaline
2022, Surface and Coatings TechnologySubtractive Nanopore Engineered MXene Photonic Nanomedicine with Enhanced Capability of Photothermia and Drug Delivery for Synergistic Treatment of Osteosarcoma
2023, ACS Applied Materials and Interfaces