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Influence of topological cues on fibronectin adsorption and contact guidance of fibroblasts on microgrooved titanium Astrid Weidt, Stefan G. Mayr, and Mareike Zink ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00667 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019
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ACS Applied Bio Materials
Influence of topological cues on fibronectin adsorption and contact guidance of fibroblasts on microgrooved titanium Astrid Weidt, a,b Stefan G. Mayr b,c and Mareike Zink *a a
Junior Research Group Biotechnology and Biomedicine, Peter Debye Institute for Soft Matter Physics, Leipzig University, Linnéstr. 5, 04103 Leipzig, Germany. *Email:
[email protected] b Leibniz Institute of Surface Engineering (IOM), Permoserstr. 15, 04318 Leipzig, Germany c
Division of Surface Physics, Felix Bloch Institute for Solid State Physics, Leipzig University, Linnéstr. 5, 04103 Leipzig, Germany
Abstract The choice of suitable nano- and microstructures of biomaterials is crucial for successful implant integration within the human body. In particular, surface characteristics affect the adsorption of various extra cellular matrix proteins. This work illustrates the interaction of protein adsorption and early cell adhesion on bulk microstructured titanium surfaces with parallel grooves of 27 to 35 µm widths and 15 to 19 µm depths, respectively. In contact with low concentrated fibronectin solutions, distinct adsorption patterns are observed on the edges of the ridges. Moreover, NIH/3T3 fibroblasts cultured in serum-free medium for 1 hours, 3 hours and 1 day show enhanced early cell adhesion on fibronectin coated samples compared to uncoated ones. In fact, early adhesion and cell contacts occur mainly on the groove edges where fibronectin adsorption was preferentially detected. Such adsorption patterns support cellular contact guidance on short time scales, since the adsorbed fibronectin proteins acted as a chemical boundary superimposing the topographical cues of the grooved microstructure. In fibronectin-free conditions this chemical boundary is absent after cell seeding and initial cell-surface interaction. Here, cellular fibronectin released by the fibroblasts adsorbs along the grooves after 3 hours and contact guidance occurs delayed. After 1 day, cell adhesion and cell morphology on uncoated and fibronectin coated titanium microgrooves were nearly equilibrated. Thus, surface structures can promote directed adsorption of low concentrated fibronectin which, furthermore, facilitates early cell adhesion. These results give rise to new developments in surface engineering of biomedical implants for improved osseointegration. Keywords: protein adsorption, titanium, microgrooves, contact guidance, early cell adhesion, fibronectin
protein adsorption, cellular requirements on the surface topography and biochemical properties are high. An excellent biocompatibility and corrosion resistance are crucial for biomaterial performance, especially for application as joint replacements or dental implants,17 since ions released due to corrosion influence inflammation reactions.18
Introduction The extracellular matrix (ECM) as a dense network of proteins, proteoglycans and collagen integrates cells to form tissues.1 Nowadays many biomaterials that are inplanted within the human body are coated with ECM molecules that support cell adhesion to the material’s surface.2,3 To this end, cells bind to these material surfaces and sense the physical and chemical properties of their surroundings via different cell-ECM contacts, such as focal adhesions (FAs) and hemidesmosomes.4-6 Integrins link these signals from ligands of the ECM, which contain the binding site RGD (Arg-Gly-Asp)7 to FAs inside the cell, which are further connected to the actin cytoskeleton.8 Thus, for successful integration of foreign materials in the surrounding tissue environment, suitable material properties have to be chosen that encourage the formation of cell-ECM contacts.
Titanium as the most used implant material17 offers excellent inert material characteristics; it is researched, tested and utilized for in vivo applications for almost 35 years.19-24 Nevertheless, titanium and its oxides lack sufficient protein adsorption properties and especially the adsorption of proteins on unstructured surfaces is barely supported.25-27 Thus, biocompatibility and bioactivity of titanium and titanium dioxide can still be improved, for instance by tuning surface properties in terms of surface roughness13,28 or by using efficient biofunctional coatings that bind to the surface. While e.g. FN coatings support specific cell adhesion, other polymers such as poly-L-lysine enhance cell adhesion via unspecific, viz. electrostatic interactions.8,29,30 Most often, the concentration of proteins in coating solutions is chosen reasonably high to achieve protein layers and protein matrix formation on the substrates.25,26,31 In addition, defined cell adhesion can be initiated by distinct fibronectin patterns on specific areas, such as pillars.32,33
In general, implant materials have initial contact to body fluids such as blood which contain among other proteins also fibronectin (FN) – a major component of the ECM with the integrin binding site RGD.9 Adsorption of these proteins to the implant surface takes place within seconds to minutes.10 However, the amount of adsorbed proteins as well as the adhesion time scale are influenced by surface characteristics such as roughness, wettability and chemical composition of the surface.11-13 Depending on the surface density of the adsorbed proteins, the adhesion strength of cells varies.14 Moreover, the interaction of adsorbed proteins to the implant must exceed the forces exerted by the cells during spreading and migration, otherwise those may peel off and no longer support cell adhesion.15 Thus, specific protein adsorption characteristics, such as the activation of particular binding sites, are beneficial for enhanced cellular mechanotransduction.16 Nevertheless, besides good
However, especially in in vitro experiments distinct adsorption patterns on structured and unstructured metal surfaces resulting from adsorption of lowconcentrated protein solutions, have hardly been considered for years. As shown by de Luca et al. in 2015, osteoblast cells cultured on microgrooved stainless steel surfaces align along the grooved and perform contact guidance.34 Such behavior is not surprising. However, as further reported in this study, fibronectin from serum included in culture medium only adsorbs on the edges of the microgrooves which is considered an important factor for contact guidance besides spatial cues and structures of the size of adherent cells. However, here only the effect of protein adsorption on microgrooved surfaces on long-time cell behavior was investigated and the influence of protein
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adsorption on initial cell attachment to structured metals is still unclear. Thus, protein adsorption on structured surfaces in low concentrated fibronectin solutions still bears a high potential for investigations and – in turn – improved surface bioactivity.10
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Furthermore, adsorption of FN present in complete growth medium of NIH/3T3 fibroblasts was analyzed. Therefore, 800 µl of Dulbecco's Modified Eagle's Medium (DMEM) (41966029 Thermo Scientific™), supplemented with 10 % calf serum (12133C Sigma-Aldrich Co. LLC.) and 1 % penicillin/streptomycin antibiotic solution (PS) (PAA P11-010 10000 U/ml penicillin, 10 mg/ml streptomycin) were pipetted onto the cleaned sample and were incubated at room temperature for 45 min. Subsequently, adsorbed FN on the titanium surfaces was marked using an indirect immunostaining with primary polyclonal Anti-FN antibody produced in rabbit (F3648 Sigma, München) and secondary polyclonal Alexa Fluor® 488-conjugated Goat Anti-Rabbit IgG (H+L) antibody (AP132JA4 EMD Millipore, Darmstadt), both in a dilution of 1:500. The antibodies were directly applied to the incubated samples after washing with phosphate buffered saline (PBS). Before imaging with a Carl Zeiss Axio Scope.A1 (Carl Zeiss Microscopy GmbH) equipped with a Zeiss AxioCam ICm1 Rev.1 camera with 10× magnification, samples were washed briefly with Millipore ultrapure water to prevent salt crystal formation after drying.
Protein adsorption is also often hardly considered when studying contact guidance and how cells sense their environment. Even though cells respond to the predominant nano- or microstructure, the influence of anisotropic cues and various surface topographies on cell behavior cannot be predicted without often extensive cell experiments.35-38 Nevertheless, these nano- and microtopographies are highly relevant for cell adhesion and proliferation and, thus, the research efforts are fully justified. Easy to reproduce and well investigated structures such as grooves, pillars and pits are preferable for simpler comparison of the adhesion effects of cell types as well as material characteristics.30 They can further be employed for improved prosthesis performance in vivo.39 In this study we investigate the effect of titanium microgrooves on protein adsorption and its influence on early cell adhesion. In detail we focus on the question whether the underlying microgrooved structure of titanium surfaces or FN adsorption is the origin for early contact guidance, viz. do cells perform contact guidance only because of spatial cues or if protein adsorption prior to cell adhesion determines cell shape and orientation during the adhesion process. Physical characteristics of the employed surface topographies, as roughness and wettability, as well as chemical composition of the substrates are illustrated and distinct adsorption patterns of FN depending on the surface characteristics are observed. The early cell adhesion and cell morphology of NIH/3T3 fibroblasts within 1 hour, 3 hours and 1 day after plating cells on the substrates are examined and reveal that early cell adhesion is influenced by the surface characteristics and the adsorbed FN patterns. In fact, early cell adhesion and contact guidance occur as a two-step process in which first FN adsorbs to the edges of the microgrooves and subsequently cells bind to these proteins during early adhesion.
Fibronectin Coating To investigate the effect of fibronectin (FN) adsorption on early cell adhesion, uncoated and FN coated microgrooved titanium substrates were used for cell culture. For coated surfaces HiLyte Fluor™ 488 labeled FN (FNR02-A Cytoskeleton, Inc.) was employed to the titanium samples in a concentration of 0.5 µg/cm2 in Millipore ultrapure water with subsequent drying at room temperature in sterile conditions. Cell Culture To test the cellular adhesion on the microstructured titanium samples NIH/3T3 embryonic mouse fibroblasts (ATCC, CRL-1658, Manassas, VA) were employed. Prior to cell seeding onto titanium samples, NIH/3T3 were cultured in complete growth medium composed of Dulbecco's Modified Eagle's Medium (DMEM) (41966029 Thermo Scientific™), supplemented with 10 % calf serum (12133C Sigma-Aldrich Co. LLC.) and 1 % penicillin/streptomycin antibiotic solution (PS) (PAA P11-010 10000 U/ml penicillin, 10 mg/ml streptomycin). Culture medium was renewed every two days and cells were cultured in an atmosphere of 5 % CO2 at 37 °C. Cells were passaged before reaching 80 % confluence. In detail, cells were incubated with 0.25 % trypsin-ethylenediaminetetraacetic acid (EDTA) solution (PAA Laboratories GmbH, L11-004) at 37 °C until detachment.
Experimental Titanium Samples Six titanium samples were processed by photolithography and acid etching to obtain grooved surface topographies with parallel aligned microgrooves as described in detail in Supporting Information. Surface structure and chemistry were studied in detail by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), 3D confocal microscopy (µsurf) and contact angle measurements (see Supporting Information). Three polished samples were left for control, in the following labeled as smooth samples.
The investigation of cellular behavior in terms of morphology and adhesion was obtained by seeding NIH/3T3 fibroblasts onto clean titanium samples in serumfree culture conditions. In detail, coated and uncoated titanium samples were taken under the sterile laminar flow hood, placed into a Petri dish with a droplet of 750 µl to 800 µl DMEM in the center of the sample. Cell adhesion and cell morphology after 30 min, one hour and three hours were tested by seeding cells at a concentration of 10 000 cells/cm2 in DMEM, while for cell inspections one day after seeding, the cell density was decreased to 4000 cells/cm2 due to possible cell division.
Protein Adsorption For qualitative studies of protein adsorption, HiLyte Fluor™ 488 labeled fibronectin (FN, FNR02-A Cytoskeleton, Inc.) was applied to the titanium samples at room temperature in a concentration of 0.5 µg/cm2, resulting in a FN concentration in Millipore ultrapure water solution of 2.5 µg/ml. The concentrations correspond to the amount of FN present in complete growth medium of NIH/3T3 fibroblasts. The protein adsorption from FN solution was observed and pictures were captured in intervals of 10 min until drying at 10× and 40× magnification using a Carl Zeiss Axio Scope.A1 (Carl Zeiss Microscopy GmbH).
Fluorescent Staining To analyze the morphology of cells and cellular adhesion, actin filaments were stained using TRITC-conjugated phalloidin (P1951 Sigma-Aldrich Co. LLC.) in a dilution of 1:600. Focal adhesions (FAs) were localized by staining vinculin, which was detected using the primary monoclonal Anti-Vinculin antibody, clone VIIF9 (7F9) (MAB3574 Merck Millipore) in a dilution of 1:400 and the secondary polyclonal Alexa Fluor® 633-conjugated Goat anti-Mouse IgG (H+L) antibody (A21050 Invitrogen) in a dilution of 1:100.
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ACS Applied Bio Materials
The indirect immunostaining of FA and the actin-cytoskeleton was carried out at room temperature. Cells were fixed with 4 % paraformaldehyde for 15 min to 20 min. Subsequently, cells were washed twice with wash buffer containing 0.05 % Tween-20 in 1×PBS. Cell membranes were permeabilized with 0.1 % Triton X-100 in PBS. Cells were washed twice with wash buffer. Subsequently, blocking solution composed of 1 % bovine serum albumin (BSA) in 1×PBS was applied. After 30 min the primary antibody was diluted in blocking solution and added to the sample for 60 min. Cells were washed with wash buffer using three longer washing steps of 5 min to 10 min each. Then the secondary antibodies and TRITC-conjugated phalloidin were diluted in PBS and added for 60 min. After washing the cells three times for 5 min to 10 min with wash buffer, cells were covered with PBS to prevent drying. The samples were stored, if necessary, at 4 °C in the refrigerator. Steps involving fluorescent dyes were performed in the dark.
water to remove salt deposits on the surface. In general, a humidity of 50 % to 70 % was adjusted. For enhancing the resolution, the humidity was reduced to 30 % in some cases. Image Processing and Data Analysis of Cellular Adhesion and Cell Morphology To analyze images obtained by fluorescence microscopy and CLSM for cell morphology, a self-written Fiji macro39 was used to evaluate cell areas and cell perimeters and cell orientations. A brightness threshold was set manually and an edge detection routine was applied. Thereby, only objects in the size range from 200 µm2 to 400 µm2 were analyzed to exclude cell coagulates and small non-cellular particles. Holes in the cell objects were closed automatically. Objects touching the edge of the image were excluded from analysis. Objects were additionally filtered for their dimensionless shape factor σ (circularity) with 𝐴
𝜎 = 4𝜋 , where A corresponds to the cell area and p to the cell perimeter. Only 𝑝
To visualize possible cellular FN production and its localization, cells were cultured in serum-free conditions. After 30 min, 1 h and 3 h after seeding, an indirect immunostaining with primary polyclonal Anti-FN antibody produced in rabbit (F3648 Sigma-Aldrich Co. LLC.) and a secondary polyclonal Alexa Fluor® 488-conjugated Goat Anti-Rabbit IgG (H+L) antibody (AP132JA4 Merck Millipore) was carried out, both in a dilution of 1:500 with the same immunostaining procedure described above for the FA. Additionally, F-actin was stained using TRITC-conjugated phalloidin (P1951 Sigma-Aldrich Co. LLC.) in a dilution of 1:300.
objects having a shape factor in the range of 0.03 to 1.00 were analyzed. The value 1 corresponds to a perfectly circular object, whereas a value approaching 0 depicts a completely irregular object. Cell orientations were evaluated by fitting the best ellipse to the individually analyzed cell and detecting the orientation of the major axis of the ellipse. On average, 2390 individual cells per sample are analyzed on FN coated substrates. On uncoated substrates, on average 2880 individual cells are analyzed per sample. To obtain the number of cells on relative positions on small grooved microstructured samples (either inside the grooves, on the ridges or on both, respectively), cells were counted using the Fiji Cell Counter. At least 1250 cells per sample are detected.
Fluorescence Microscopy To analyze protein adsorption and cell morphology after the above described staining procedures, a Carl Zeiss Axio Scope.A1 (Carl Zeiss Microscopy GmbH) equipped with a Zeiss AxioCam ICm1 Rev.1 camera was used to acquire images in 5×, 10× and 20× magnification. Images and movies were recorded using ZEN lite software (Carl Zeiss Microscopy GmbH).
For qualitative analysis, image stacks gained from spinning disk confocal microscopy were merged to obtain a 3D image of cellular arrangement within the microgrooves of the titanium surface using ZEN Software (Carl Zeiss Microscopy GmbH).
Additionally, images used to evaluate the cell morphology were captured with a Leica TCS SP2 confocal laser scanning microscope (CLSM) (Leica Microsystems GmbH) combined with a 10× objective. Cell adhesion and protein adsorption were studied in detail using an inverted Zeiss Axio Observer.Z1 research microscope (Carl Zeiss Microscopy GmbH) combined with a Yokogawa CSU-X1A 5000 spinning disk confocal scanning unit (Yokogawa Electric Corporation) (SpiDi). A Hamamatsu Orca Flash 4.0 camera (Hamamatsu Photonics K.K.) connected to a 1.2 × EMCCD camera adapter captured images observed with either a LCI Plan-Neofluar 63× /1,3 Imm Korr Ph3 M27 for (D=0.15mm to 0.19mm) (420881 9970 000, Carl Zeiss Microscopy GmbH) for samples with FN coating or a C-Apochromat 40× /1,20 W Korr for (D=0.14mm to 0.19mm) (441757 9970 000, Carl Zeiss Microscopy GmbH) for samples without FN coating. Solid-state lasers with wavelengths of 488 nm, 561 nm and 638 nm were used to excite the fluorophores. Images were recorded using ZEN software (Carl Zeiss Microscopy GmbH).
Sample Cleaning Procedure After cell adhesion experiments, cells were trypsinized with 1 ml 0.25 % trypsinEDTA for 10 min to 15 min at 37 °C. Subsequently, samples were washed twice with phosphate buffered saline (PBS). Proteins were dissolved by adding 1.5 ml Laemmli solution with 17.5 mmol dithiothreitol (DTT) per sample, heating it up to the boiling point 3 times in a microwave. Subsequently, titanium samples were sonicated in acetone, 80 % ethanol and distilled water for 5 min each. In between the sonication steps, the samples were washed gently using a conventional soft cellulose sponge and distilled water to remove cell residues. The samples were dried in nitrogen stream and stored in petri dishes sealed with Parafilm. Statistical significance Independent two-sample t-tests for unequal sample size were used to evaluate statistical significance. Values differing by p
