Polysaccharide surface engineering of poly(D, L-lactic acid) via electrostatic self-assembly technique and its effects on osteoblast growth behaviours

The objective of this study was to surface modify the poly (D, L-lactic acid) (PDLLA) films and assess the effects of the modified surfaces on the functions of osteoblasts cultured in vitro. A layer-by-layer (LBL) self assembly technique, was used leading to the formation of multilayers on the PDLLA film surfaces. Chitosan (Chi) and poly (styrene sulfonate, sodium salt) (PSS) were utilized as polycation and polyanion in this study, respectively. The layer structure was investigated by using X-ray photoelectron spectroscopy (XPS) and water contact angle measurement, respectively. XPS analysis displayed the presence of chitosan on PDLLA surface. A full coverage of coating with PSS/Chi layers was achieved on the PDLLA surface only after the deposition layers of PEI/(PSS/Chi)₂. These results showed that PDLLA films could be modified with PSS/Chi pairs which may affect the biocompatibility of the modified PDLLA films. To confirm this hypothesis, cell proliferation, cell viability as well as alkaline phosphtase activity of osteoblasts on layer-by-layer modified PDLLA films as well as control samples were investigated in vitro. The proliferation of osteoblasts on modified PDLLA films was found to be greater than that on control (p < 0.05 and p < 0.01) after 1, 4 and 7 days culture, respectively. Cell viability measurement showed that the PSS/Chi modified PDLLA films have higher cell viability (p < 0.01) than control. Osteoblast differentiation function (ALP) on LBL-modified PDLLA film was found significantly higher (p < 0.01) than that of virgin PDLLA films. These data suggests that PSS/Chi pair was successfully employed to surface modify PDLLA film via a layer-by-layer technique, and enhanced its cell biocompatibility.     

Modulation of poly (d,l-lactic acid) with amniotic fluid for improvement of the cell affinity

The objective of this study was to investigate the effects of naturally occurring amniotic fluid modified poly(d,l-lactic acid) (PDLLA) film on the culture of rat calvaria osteoblast. The characteristics of surfaces (both modified and control) were examined by contact angle measurement and electron spectroscopy for chemical analysis (XPS). Cell adhesion and proliferation were used to assess the cell behavior on modified films and control one. MTT assay was used to determine cell viability and alkaline phosphatase (ALP) activity was taken to evaluate differentiated cell function. Compared with the untreated films, cell adhesion of osteoblast was significantly higher (P < 0.05) than that found on control, and osteoblast proliferation was also greater than control one (P < 0.01) at the time interval of 4 and 7 days. Moreover, the alkaline phosphatase (ALP) activity exhibited statistic difference (P < 0.05) and cell viability demonstrated significant difference (P < 0.01) between amniotic fluid modified PDLLA films and control one. These results suggested that amniotic fluid was a suitable material when used to modify PDLLA in order to improve its biocompatibility.     

Surface modification of titanium thin film with chitosan via electrostatic self-assembly technique and its influence on osteoblast growth behavior

Chitosan (Chi) and poly (styrene sulfonate) (PSS) were employed to surface modify titanium thin film via electrostatic self-assembly (ESA) technique in order to improve its biocompatibility. The surface chemistry, wettability and surface topography of the coated films with different number of deposited layers were investigated by using X-ray photoelectron spectroscopy (XPS), water contact angle measurement and atomic force microscopy (AFM), respectively. The results indicated that a full surface coverage for the outmost layer was achieved at least after deposition of five layers, i.e., PEI/(PSS/Chi)₂ on the titanium films. The formed multi-layered structure of PEI(PSS/Chi) _x (x ≥ 2) on the titanium film was stable in air at room temperature and in phosphate buffered solution (PBS) for at least 3 weeks. Cell proliferation, cell viability, DNA synthesis as well as differentiation function (alkaline phosphatase) of osteoblasts on chitosan-modified titanium film (PEI/(PSS/Chi)₆) and control sample were investigated, respectively. Osteoblasts cultured on chitosan-modified titanium film displayed a higher proliferation tendency than that of control (p < 0.01). Cell viability, alkaline phosphatase as well as DNA synthesis measurements indicated that osteoblasts on chitosan-modified titanium films were greater (p < 0.01) than those for the control, respectively. These results suggest that surface modification of titanium film was successfully achieved via deposition of PEI/(PSS/Chi) _x layers, which is useful to enhance the biocompatibility of the titanium film.     

In vitro behavior of osteoblast-like cells on PLLA films with a biomimetic apatite or apatite/collagen composite coating

To investigate the methods to improve the cell–material interaction of devices or tissue engineering scaffolds made of poly(l-lactic acid) (PLLA) polymer, apatite and apatite/collagen composite coatings were formed on PLLA films within 24 h through accelerated biomimetic processes. In vitro investigation using Saos-2 osteoblast-like cells through cell culture was conducted to assess the biological performance of these biomimetic coatings. The cell morphology on three types of surfaces, viz., PLLA film, PLLA film with the apatite coating, and PLLA film with the apatite/collagen composite coating, was studied using scanning electron microscopy (SEM). Cell viability was estimated using the MTT assay. The differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. The results obtained indicated that the biomimetic apatite and apatite/collagen composite coatings could significantly enhance the proliferation and differentiation of osteoblast-like cells. The apatite/collagen composite coating appears to be promising for the surface modification of PLLA-based devices with much improved interactions with osteoblastic cells.     

Biocompatibility of multi-walled carbon nanotubes grown on titanium and silicon surfaces

Cell adhesion and cell viability of aligned multi-walled carbon nanotube (MWCNT) films were verified using Fibroblast L929 mouse cells. The MWCNTs were produced by a microwave plasma chemical vapor deposition (2.45 GHz) on silicon (Si), with a nickel catalyst, and titanium (Ti), with an iron catalyst. MTT assay and cellular adhesion were used for biocompatibility tests (ISO 10993-5). The results show very high cell viability and many layers of cells adhered on the surface formed by the nanotube tips at films grown on silicon surfaces. The MWCNT grown on Ti surfaces presented lower cell viability and a reduced number of cells on the surface formed by the nanotube tips. The different behavior is most probably related to excess iron contamination present in the case of titanium substrate, while nickel catalyst is probably enclosed by the nanotubes.     

Synthesis and cellular biocompatibility of two nanophase hydroxyapatite with different Ca/P ratio

The cellular biocompatibility of two types of nanophase hydroxyapatites including nanophase standard hydroxyapatite (n-HA) and nanophase calcium deficient hydroxyapatite (n-CDHA) synthesized by a wet chemical method were assessed using primary cultured osteoblasts. Cytotoxicity of both materials was investigated with L929 cell line. The MTT method was used to evaluate the proliferation of osteoblasts on the third day and ALP activity assay was carried out on the fifth day. SEM was used to observe the morphology of the osteoblasts on the third day. Two types of nanophase hydroxyapatite both showed no cytotoxicity. Higher cell proliferation was observed on n-CDHA than n-HA. At the same time, cells spread more actively on the n-CDHA group. The ALP level of n-CDHA was also significantly higher on the former. Our results show that the n-CDHA is more suitable for osteoblasts growth and is also helpful for ALP synthesis.     

Bioglass® coated poly(DL‐lactide) foams for tissue engineering scaffolds

The purpose of this study was to prepare poly(DL‐lactic acid) (PDLLA)/Bioglass® composites of foam‐like structure, to measure the degree of bioactivity of the composites by studying the formation of hydroxyapatite (HA) after immersion in simulated body fluid (SBF) and to test the initial attachment of human osteoblasts within the porous network. It was found that crystalline HA formed on the Bioglass® coated PDLLA foams after 7 days of immersion in SBF. HA formed also on the surfaces of non‐coated PDLLA foams, however the rate and amount of HA formation were much lower than in the composites. The rapid formation of HA on the Bioglass®/PDLLA foam surfaces confirmed the high bioactivity of these materials. Osteoblasts attached within the porous network throughout the depth of the foams. Cell density was found to be higher in the PDLLA/Bioglass® composites compared to the pure PDLLA foams. The composite foams developed here exhibit the required bioactivity to be used as scaffolds for bone tissue engineering.     

Titanium dioxide (TiO<Subscript>2</Subscript>) nanoparticles filled poly(<Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis> lactid acid) (PDLLA) matrix composites for bone tissue engineering

Titanium dioxide (TiO₂) nanoparticles were investigated for bone tissue engineering applications with regard to bioactivity and particle cytotoxicity. Composite films on the basis of poly(d,l lactid acid) (PDLLA) filled with 0, 5 and 30 wt% TiO₂ nanoparticles were processed by solvent casting. Bioactivity, characterised by formation of hydroxyapatite (HA) on the materials surface, was investigated for both the free TiO₂ nanoparticles and PDLLA/TiO₂ composite films upon immersion in supersaturated simulated body fluid (1.5 SBF) for up to 3 weeks. Non-stoichiometric HA nanocrystals (ns-HA) with an average diameter of 40 nm were formed on the high content (30 wt% TiO₂) composite films after 2 weeks of immersion in 1.5 SBF. For the pure PDLLA film and the low content composite films (5 wt% TiO₂) trace amounts of ns-HA nanocrystals were apparent after 3 weeks. The TiO₂ nanopowder alone showed no bioactivity. The effect of TiO₂ nanoparticles (0.5–10,000 μg/mL) on MG-63 osteoblast-like cell metabolic activity was assessed by the MTT assay. TiO₂ particle concentrations of up to 100 μg/mL had no significant effect on MG-63 cell viability.     

Time-dependent effects of pre-aging polymer films in cell culture medium on cell adhesion and spreading

We have tested the hypothesis that cell adhesion and spreading on polymer films are influenced by the amount of time that the polymer films are pre-aged in cell culture medium. Cell adhesion and spreading were assessed after a 6-h culture on poly(d,l-lactic acid) (PDLLA) films that had been pre-aged in cell culture medium for 30 min, 1, 3 or 7 d. Cell adhesion and spread area were enhanced as the duration of pre-aging PDLLA films in cell culture medium was increased. Materials characterization showed that the hydrophobicity and surface morphology of the PDLLA films changed with increasing length of pre-aging time. These results suggest that cell adhesion and spreading are sensitive to the time-dependent changes in PDLLA hydrophobicity and surface morphology that occur during exposure of the polymer to cell medium for different lengths of time. These results demonstrate that cell response to a degradable, biomedical polymer can change as a function of the amount of time that the polymer is exposed to physiological medium. This article, a contribution of the National Institute of Standards and Technology, is not subject to US copyright.     

Biocompatibility and bioactivity of PDLLA/TiO2 and PDLLA/TiO2/Bioglass® nanocomposites

Biocompatibility and bioactivity of polymer matrix composites containing titanium dioxide (TiO₂) nanoparticles were investigated. The solvent casting method was used to prepare poly (d,l-lactic acid) (PDLLA) films with 0 and 20 wt.% TiO₂ nanoparticles and with 20 wt.% TiO₂ mixed with 5 wt.% micrometre-sized (< 5 μm) Bioglass® particles. The samples were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy Dispersive X-ray (EDX) analyses. A Zygo® light interferometer was used to examine the surface roughness of the samples. The bioactivity and the surface reactivity of the materials were determined by investigating the formation of hydroxyapatite (HA) on the surface of samples upon immersion in simulated body fluid (SBF) for up to 28 days. Heterogeneous distributed HA crystals were found on composite films containing TiO₂ after 21 days exposure to SBF. Cell cytotoxicity and viability were determined by using live/dead and MTS assay on osteoblast-like MG-63 cells. The PDLLA films containing different concentrations of TiO₂ and Bioglass® particulate inclusions showed no effect on cell viability in live/dead assay after incubation period of 7 days. All three groups of samples demonstrated significant increase in relative metabolic activity in MTS assay after 7 days incubation (while a slower proliferation rate was obtained for cells on the PDLLA film containing both TiO₂ and Bioglass® compared to the Thermanox® control). The bioactive behaviour of the nanocomposites may make them attractive materials for fabrication of tissue engineering scaffolds.     

Effects of hydrolysis on dodecyl alcohol-modified bioactive glasses and PDLLA/modified bioactive glass composite films

In this article, mesoporous 58S and 58S bioactive glasses (BGs) were surface modified by dodecyl alcohol through esterification reaction and PDLLA/modified BGs composite films were prepared. The purpose of this study was to investigate the properties of the modified BGs particles and the PDLLA/modified BGs composite films before and after hydrolytic treatment. The modified BGs powders and composite films were treated in boiling water for 20 min to remove the dodecyl chains. After hydrolytic treatment, the modified BGs powders showed increased hydrophilicity and the FTIR analysis revealed that most dodecyl chains were removed. Furthermore, the hydrophilicity of the PDLLA/modified BGs composite films was also greatly improved. The tensile strength of the composite films after hydrolysis decreased slightly, but was still much higher than that of pure PDLLA film. In addition, bone marrow mesenchymal stem cells from dogs on the composite films after hydrolytic treatment showed the highest proliferation rate. The results suggest that hydrolytic treatment is an effective and practicable method to remove alcohol chains from surface-modified BGs and polymers/modified BG composites, which may be used for preparation of bioactive scaffolds for tissue engineering applications.     

In vitro behavior of layer-by-layer deposited molecular oligoelectrolyte films on Ti–6Al–4V surfaces

Layer-by-layer self-assembled films of molecular oligoelectrolytes were used to modify Ti–6Al–4V surfaces in order to test their ability as potential drug delivery system. With regard to medical application the in vitro behavior of the modified material was investigated. The Ti–6Al–4V (6% aluminium, 4% vanadium) material was treated in a layer-by-layer (LbL) process with 2, 4, 6 and 8 layers of molecular oligoelectrolytes 1 and 2 and thereby doped with a fluorescent reporter molecule 2. Human osteoblasts were cultured for a period up to 5 days on the modified material. Ti–6Al–4V surfaces without modification were used as control. In order to investigate the in vitro behavior of the coating as well as the influence of components of the coating on osteoblastic cells, respectively, cell proliferation, differentiation and attachment of hFOB cells were observed by means of cell number, osteoblastic gene expression and fluorescence microscopy. Degradation behavior of the OEM (oligoelectrolyte multilayer film) was examined using optical spectroscopy. Measurement data imply that the layer-by-layer coating was successfully assembled on the Ti surface and endures steam sterilization. The fluorescence signal in cell culture medium increased strictly linear with increasing pre-assembled number of layers on the surface. Proliferation rates of the cells in experimental groups did not differ significantly from each other (P ≥ 0.783). Differentiation pattern was not significantly changed by the coating. The fluorescent reporter component of the film was absorbed by osteoblastic cells and was detected by fluorescence microscopy.     

The use of rat, rabbit or human bone marrow derived cells for cytocompatibility evaluation of metallic elements

Rat, rabbit and human bone marrow cells were cultured according to the method previously reported for cells of rat origin [1] and were exposed, or not (control), to corrosion products of a Co–Cr orthopaedic alloy as well as to metal salts containing Co2+, Cr3+ and Cr6+. Cells were cultured for 21 days and analysed for the following biochemical parameters: intracellular MTT reduction (i.e. cell viability/proliferation), alkaline phosphatase (ALP) activity and protein production. Morphological observations included both histochemistry (detection of ALP-positive cells, calcium and phosphate deposits) and scanning electron microscopy (SEM). Control cultures of rat and rabbit cells showed higher proliferation rates than human cells at the start of culture, but they all reached similar values on day 21. Protein production was parallel to cell proliferation. In contrast, ALP activity of rat cultures was much stronger than rabbit or human cultures. All cell types were able to develop the osteogenic phenotype in vitro.Co–Cr extract caused inhibitory effects on cell viability, on ALP activity and, to a lower extent, on protein production of all rat, rabbit and human cell cultures. Compared to rat and rabbit cultures, human cultures were the most sensitive to metal ions exposure.     

Surface modification of poly-L-lactic acid films by electrostatic self-assembly to promote vascular smooth muscle cells growth

The intention of this study was to surface modify the poly-L-lactic acid (PLLA) film and evaluate the effects of the surfaces on the growth of vascular smooth muscle cells (VSMCs) in vitro. Collagen and hyaluronic acid (HA) were utilized as polycation and polyanion in this study. Layer-by-layer (LBL) self-assembly technique was used to lead to the formation of multilayer moleculer on the poly-L-lactic acid (PLLA) film surfaces. Collagen/HA layers was overcasted coating on the PLLA surface after the activation layers by poly-(ethyleneimine) (PEI). The structure and morphology of the multilayer molecular were examined by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectrophotometer and atomic force microscope (AFM), respectively. The ATR-FTIR analysis illuminated the presence of collagen on the PLLA surface. The AFM results showed the multilayer appeared on PLLA surface. The VSMCs were adopted to evaluate the cyto-compatibility of the modified PLLA films. It was found that the viability of VSMCs on the modified PLLA films were greater than that on original PLLA films and tissue culture plastic after ten days culture (p < 0.05). Scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) data also confirmed the homogeneous results. These data suggest that collagen/HA coat can be successfully adopted in the surface modification of PLLA film through LBL technique, and also can enhance its cell compatibility.     

Surface characterization and biological response of carbon-coated oxygen-diffused titanium having different topographical surfaces

The materials (C-ODTi) with different topographical surfaces that possess interstitial oxygen atoms into the host titanium lattice and an upper nanometric surface layer of anatase-TiO₂ covered by a carbon thin layer were fabricated in this study. The carbon thin layer on the surface of C-ODTi was composed of amorphous carbon and nano-graphite crystals. In vitro tests, using human bone marrow-derived mesenchymal cells (hBMCs), were performed to check cytotoxicity, examining in particular cell morphology, cell proliferation, cell differentiation, and mineralization capability. After 10 days of culture a higher degree of cell viability was observed on the surface of C-ODTi with an abraded surface. We also observed that hBMCs cultured in direct contact with C-ODTi maintained their capability to express alkaline phosphatase activity (ALP) and formed mineralized nodules similar to the control cultures. Our results demonstrate that the carbon layer coating on the surface of C-ODTi possess better biological response than commercially pure titanium (cp Ti), which was evidenced by the higher proliferation rates of osteoblasts, higher osteo-differentiation and a higher mineralization capability.     

PolyNaSS grafting on titanium surfaces enhances osteoblast differentiation and inhibits <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> adhesion

Titanium surface modifications to simultaneously prevent bacterial adhesion but promote bone-cell functions could be highly beneficial for improving implant osseointegration. In the present in vitro study, the effect of sulfonate groups on titanium surfaces was investigated with respect to both S. aureus adhesion and osteoblast functions pertinent to new bone formation. Commercial pure titanium (cpTi) squares were oxydized (Tiox), grafted with poly(sodium styrene sulfonate) groups (Tigraft) by covalent bonding using radical polymerization, and were characterized by infrared spectroscopy (HATR-FTIR) and colorimetry. Bacterial adhesion study showed that Tigraft exhibited high inhibition of S. aureus adhesion S at levels >90 %, when compared to cpTi (P < 0.05). In contrast osteoblasts adhesion was similar on all three titanium surfaces. While the kinetics of cell proliferation were similar on the three titanium surfaces, Alkaline phosphatase-specific activity of osteoblasts cultured on Tigraft surfaces was twofold higher than that observed on either on Tiox or cpTi surfaces (P < 0.01). More importantly, the amount and the distribution of calcium-containing nodules was different. The total area covered by calcium-containing nodules was 2.2-fold higher on the Tigraft as compared to either Tiox or cpTi surfaces (P < 0.01). These results provide evidence that poly(sodium styrene sulfonate) groups grafting on cpTi simultaneously inhibits bacteria adhesion but promote osteoblast function pertinent to new bone formation. Such modified titanium surfaces offer a promising strategy for preventing biofilm-related infections and enhancing osteointegration of implants in orthopaedic and dental applications.     

Effect of novel chitosan-fluoroaluminosilicate resin modified glass ionomer cement supplemented with translationally controlled tumor protein on pulp cells

Dental materials that can promote cell proliferation and function is required for regenerative pulp therapy. Resin modified glass ionomer cement (RMGIC), a broadly used liner or restorative material, can cause apoptosis to pulp cells mainly due to HEMA (2-hydroxyethyl methacrylate), the released residual monomer. Recent studies found that chitosan and albumin could promote release of protein in GIC while translationally controlled tumor protein (TCTP) has an anti-apoptotic activity against HEMA. The aim of this study was to examine the effect of chitosan and albumin modified RMGIC (Exp-RMGIC) supplemented with TCTP on pulp cell viability and mineralization. Exp-RMGIC+TCTP was composed of RMGIC powder incorporated with 15 % of chitosan, 5 % albumin and supplemented with TCTP mixed with the same liquid components of RMGIC. The effect of each specimen on pulp cells was examined using the Transwell plate. From the MTT assay, Exp-RMGIC+TCTP had the highest percentages of viable cells (P < 0.05) at both 24 and 74 h. Flow cytometry revealed that, after 24 h, Exp-RMGIC+TCTP gave the lowest percentages of apoptotic cells compared to other groups. There was no difference in alkaline phosphatase (ALP) activity among different formula of the specimens, while cells cultured in media with TCTP had higher ALP activity. Von Kossa staining revealed that RMGIC+TCTP, and Exp-RMGIC+TCTP had higher percentages of calcium deposit area compared to those without TCTP. It was concluded that Exp-RMGIC supplemented with TCTP had less cytotoxicity than RMGIC and can protect cells from apoptosis better than RMGIC supplemented with TCTP.     

等离子体处理偶联胶原提高聚乳酸材料的细胞相容性

本研究通过结合氨气等离子体处理以及胶原改性的方法来改善聚乳酸材料的细胞相容性。水接触角分析表明氨气等离子体处理可以改善聚乳酸材料的亲水性,促进细胞的粘附。光学显微镜以及扫描电镜的分析结果表明,氨气等离子体处理后通过胶原改性可以进一步促进细胞在聚乳酸材料表面的生长。并且,与涂覆胶原相比,偶联胶原可以更好地使胶原蛋白固定在聚乳酸材料表面,从而表现出更好的细胞相容性。     

Evaluation of the attachment,proliferation, and differentiation of osteoblast on a calcium carbonate coating on titanium surface

Titanium has been reported to have some limitations in dental and orthopaedic clinical application. This study described a coating process using a simple chemical method to prepare calcium carbonate coatings on smooth titanium (STi) and sandblasted and acid-etched titanium (SATi), and evaluated the biological response of the materials in vitro. The surfaces of STi, SATi, calcium carbonate coated STi (CC-STi) and calcium carbonate coated SATi (CC-SATi) were characterized for surface roughness, contact angles, surface morphology and surface chemistry. The morphology of MG63 cells cultured on the surfaces was observed by SEM and Immuno-fluorescence staining. Cell attachment/proliferation was assessed by MTT assay, and cell differentiation was evaluated by alkaline phosphatase (ALP) activity. MG63 was found to attach favorably to calcium carbonate crystals with longer cytoplasmic extensions on CC-STi and CC-SATi, resulting in lower cell proliferation but higher ALP activity when compared to STi and SATi respectively. Moreover, CC-SATi is more favorable than CC-STi in terms of biological response. In conclusion, the calcium carbonate coatings on titanium were supposed to improve the osteointegration process and stimulate osteoblast differentiation, especially in early stage. And this method could possibly be a feasible alternative option for future clinical application.     

Biomaterial surface properties modulate in vitro rat calvaria osteoblasts response: Roughness and or chemistry?

The aim of this study was a better understanding of the regulation mechanisms of in vitro osteoblast activity on biomaterials. Rat osteoblast behaviour on different surfaces was studied. Surfaces with different roughness (and a similar surface chemistry) or with different surface chemistry (and a similar roughness) were compared. Cellular morphology was observed by scanning electron microscopy and cell adhesion was quantified using an image analysis system. Osteoblast proliferation was quantified by a MTT test and total protein content and alkaline phosphatase (ALP) activity were evaluated by spectrophotometry. Data were compared by statistical analysis.

Results showed that NiTi surface roughness did not influence osteoblasts morphology, adhesion, total protein content and ALP activity whereas it modulated cell proliferation. Roughness was shown to stimulate cell proliferation. For smooth surfaces exhibiting two different chemical compositions, adhesion rate was found to be higher on Thermanox® than on NiTi whereas proliferation was shown to be smaller. ALP activity was also modulated by surface chemistry. Thus, cell adhesion and ALP activity were found to be more governed by surface chemistry than by roughness whereas cell proliferation was shown to be modulated by roughness (this effect increasing during cell culture) and by chemistry (this effect remaining stable in time) together. Total protein content and cell morphology were found to be independent of both parameters (roughness and chemistry). Effects of surface chemistry were discussed in terms of wettability and electron acceptor/donor properties of the surfaces of interest. Immunofluorescence images of adhesion proteins could not demonstrate differences between the three surfaces.