Cell adhesion on substrates is accompanied by significant changes in shape and cytoskeleton organization, which affect subsequent cellular and tissue responses, determining the long-term success of an implant. Alterations in osteoblast stiffness upon adhesion on orthopaedic implants with different surface chemical composition and topography are, thus, of central interest in the field of bone implant research. This work aimed to study the mechanical response of osteoblasts upon adhesion on chitosan-coated glass surfaces and to investigate possible correlations with the level of adhesion, spreading, and cytoskeleton reorganization. Using the micropipette aspiration technique, the osteoblast elastic modulus was found higher on chitosan-coated than on uncoated control substrates, and it was found to increase in the course of spreading for both substrates. The cell-surface contact area was measured throughout several time points of adhesion to quantify cell spreading kinetics. Significant differences were found between chitosan and control surfaces regarding the response of cell spreading, while both groups displayed a sigmoidal kinetical behavior with an initially elevated spreading rate which stabilizes in the second hour of attachment. Actin filament structural changes were confirmed after observation with confocal microscope. Biomaterial surface modification can enhance osteoblast mechanical response and induce favorable structural organization for the implant integration.
Cell adhesion to surfaces is a key regulator of prominent biological processes. The adherent cell undergoes significant shape changes, including the initial membrane deformation as well as the extensive cytoskeleton reorganization [
In bone tissue engineering research, the design of chemically modified surfaces allows the development of functional biomaterials, since it has been referred to the fact that biological tissues interact mainly with the outermost atomic layers being generally about 0.1–1 nm. Osteoblasts that have to integrate the orthopaedic implant are in direct contact with its surface, and the long-term acceptance of the material mostly relies on the initial adhesion phase and the subsequent mechanical response upon adhesion [
Correlations between extracellular matrix (ECM) characteristics and cell mechanical properties have been of great research interest aiming at understanding how these physical and mechanical alterations affect cellular behavior. The force-deformation responses provide insight into similarities and differences in cytoskeletal microstructure between cells adhered to different substrates and in different time points of the adhesion process. The elastic moduli, viscosity, and other mechanical and rheological parameters are measures which may determine changes of the cell mechanical and rheological properties under adhesion stress and may reflect their dependence on the ECM properties [
A variety of methods have been developed to quantify cellular mechanical properties, such as viscoelasticity and deformability, mainly at the single cell level of analysis. These approaches include micropipette aspiration [
During the last years, the biological phenomena occurring after the first minutes of cell to surface contact were subjected to thorough examination and much effort was done to apply biophysical or biomechanical approaches to the analysis of the cell-substrate interface and to interrogate emerging binding events. There are limited studies through examining osteoblast mechanical properties on different ECM substrates or through examining osteoblast response to mechanical stimuli when adhered to different substrates.
Chitosan is a high molecular weight, linear polycationic heteropolysaccharide, consisting of N-acetyl-glucosamine and D-glucosamine. It is produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans, giving chitosans with varying degree of deacetylation (DD). Due to its biocompatibility, biodegradability, and nontoxic properties, it has become a promising biomaterial for a wide range of biomedical applications. It has been accepted in the biomaterials field as a structural analogue of glycosaminoglycans (GAGs). Thus, chitosan may be able to mimic certain biological activities of GAGs, including binding with growth factors and adhesion proteins [
While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. In this study, the mechanical properties of osteoblasts were determined; during the adhesion procedure on chitosan and alterations of the mechanical measures with the cell spreading rate was correlated. The purpose of the work was to better understand the way in which chitosan affects these cellular responses so as to shed light onto the design and development of implants that would create such a microenvironment to provoke coordinated molecular and mechanical activities.
For the purpose of this study, human bone marrow stromal cells were obtained by aspiration from the femoral diaphysis of patients aged 50–70 years old who underwent total or elective hip replacement. All donors have been preoperatively controlled for systematic and local infection and malignancy; specimens taken intraoperatively were sent for histology and cultures for bacteria. Approval was obtained from the Ethical Committee of the University of Patras and the study was carried out in accordance with the Helsinki Declaration. From each donor, a “single cell” suspension was prepared by repeatedly aspirating the cells successively through 19- and 21-gauge needles. The cell suspension was cultured, as described elsewhere [
Cover glasses were cleaned in piranha solution (30% (v/v) of hydrogen peroxide and 70% (v/v) of concentrated sulfuric acid) for 1 h, rinsed thoroughly with 18.2 MΩ water, dried in air, and heated in vacuum oven at 120°C for 1 h. Then the cleaned substrates were dipped into 1% (v/v) APTES solution in ethanol/water (95% : 5% by volume) for 10 min and washed with pure ethanol three times. After drying in a stream of nitrogen, the silanized surfaces were heated in vacuum oven at 120°C for 1 h in order to cross-link the silanized layer on the glass surface. The silanized glasses were immersed in 1 wt% glutaraldehyde (GA) solution for 3 h at room temperature, followed by rinsing with excess 18.2 MΩ water for 24 h in order to remove free GA. The glasses were incubated in 0.4 mg/mL chitosan solution (pH 5) for 24 h at room temperature. The specific chitosan used in the study was
For morphological observation, osteoblasts were seeded on the control and modified surfaces at a density of 4 × 104 cells/cm2 and examined with scanning electron microscopy (SEM). The cells attached to the surfaces were gently washed with PBS to remove nonadherent or loosely adherent cells and then fixed for 20 min with 2.5% GA in PBS. After thorough washing with PBS, the cells were dehydrated through a series of ethanol-water solutions for 20 min each, using increasing concentrations of ethanol up to 100%. The samples were then gold sputtered in vacuum and examined using a JEOL-JSM 6300 SEM microscope (accelerating voltage between 100 V and 30 kV, we worked with 20 kV, about 103 Pa specimen chamber pressure, and current about 1.5 nA). Experiments were conducted three times for each spreading time (2, 7.5, 15, 30, 45, 60, and 75 min). Five samples for each surface (glass, chitosan) were prepared in each experiment. SEM micrographs were captured digitally on three representative fields of each sample. The average cell area (
To quantify osteoblast spreading, cell-substrate contact area (subsequently referred to as “cell area”) was determined by tracing the outline of the cell at different time points using ImageJ (NIH). This method was the inverse of the one reported in recent study of deadhesion dynamics [
For researching the mechanical and rheological properties of the osteoblasts the elastic shear modulus,
Schematic illustration of the micropipette experimental setup.
Micropipettes were prepared by borosilicate glass tubes as described in our previous work [
The membrane elastic shear modulus,
Representative images of the aspirated osteoblasts in three sequenced time points of adhesion are shown in Figure
Micropipette aspiration of 3 osteoblasts at different phases of cell adhesion (30 min, 60 min, and 90 min seeding time). The radius of the pipette is 2.2–2.4
Cells were seeded onto the control and the chitosan surfaces at a density of 4 × 104 cells/cm2 and were allowed attaching for 30 or 60 min. The samples were stained for F-actin with dye Alexa Fluor 488 phalloidin (A12379, Invitrogen) and for nucleus with DAPI (90229, Millipore) and observed with an inverted confocal microscope (ECLIPSE TE-2000U, Nikon) and 60x magnification.
Statistical analysis was performed using SPSS statistical software (version 15.0, SPSS Inc.). Overall differences between groups were assessed by analysis of variance (ANOVA) and individual paired comparisons were analyzed post hoc with Scheffe’s test. Results were considered to be statistically significant when
Quantitative measurement of the area of the attached cells using Image Pro software showed that the osteoblast-substrate contact area was higher when the cells were attached on the chitosan than on the control surface (Figure
Mean ± SD values of cell area of osteoblasts attached on control and chitosan-coated surfaces after different seeding times;
To quantify spreading kinetics, we plotted the normalized cell area as a function of time and fitted the experimental data with the Boltzmann equation to obtain the time constants
Quantification of cell shape changes during adhesion.
Examining the changes in the mechanical properties of osteoblasts during spreading, the results showed that the elastic modulus,
Mean ± SD values of elastic modulus of osteoblasts attached on control and chitosan- coated surfaces after different seeding times;
Differences were observed in the elastic modulus of osteoblasts attached on chitosan as compared to the cells attached on the control surface throughout all culture times (Figure
The imaging of the cell spreading after 30 and 90 minutes is showed in Figure
Fluorescent staining of the F-actin cytoskeleton (red) and DNA (blue), showing cell attachment and spreading after 30 and 90 minutes of cell seeding on chitosan-coated and control glass surfaces.
The cytoskeletal organization is determined by actin labeling with phalloidin. This staining demonstrates the presence of stress fibers in the cells cultured on the samples. Osteoblasts on chitosan were significantly different in appearance, being profoundly spread and demonstrating a morphology with a well-organized actin cytoskeleton. Filopodia and lamellipodia cytoplasmic digitations were noticeably more evident on the chitosan samples for both time points tested.
Alterations in osteoblast mechanical properties upon adhesion are likely to be an important factor in the modulation of bone cell functions and in the cell response to biomaterials. During the adhesion process, prominent cell shape changes take place which affect many cellular functions, like growth, proliferation, differentiation, and motility, all detrimental for the implant success [
Chitosan, a structural analogue of GAGs, which may be able to mimic certain biological activities, such as binding with growth factors and adhesion proteins, has been used to modify the surface properties for enhancing the attachment of osteoblasts [
In this work we used the micropipette aspiration technique to measure the elastic modulus of osteoblasts on chitosan-coated and control substrates in different time points of cell adhesion. The aim was to quantify alterations in the mechanical properties during the initial phase of the cell-substrate interaction and to investigate possible correlations with the progress of adhesion and the cytoskeleton reorganization.
Our results demonstrate higher values of the elastic modulus of osteoblasts attached on chitosan-coated glass compared to the uncoated control surface, indicating that the surface characteristics indeed have an effect on cell mechanical properties. This finding is generally consistent with several works in the literature. Takai et al. [
The lengthened and most flattened cells are believed to be strongly adhered to the surface, unlike the more compact and higher ones. In an attempt to establish a relationship between the local mechanical properties and the cytomorphological aspects of the cell, Simon et al. using AFM showed that the values of elastic moduli for strongly adherent cells were between 8 and 400 kPa, while for the weakly adherent cells the values varied between 0.6 and 60 kPa [
In our study, using a phalloidin labeling method, we demonstrated that remarkable differences appeared in the apparent morphology of the osteoblasts on the two substrates during the spreading process. The structural changes of the filaments have occurred as early as 30 minutes of cell attachment, confirming the early process of cell spreading on the chitosan nanostructured surface, compared to the glass flat control surface. This early upregulation of filopodia might be in direct correlation with the adhesive bonds formed, which would also further support our results of increased adhesion strength [
Several properties of chitosan have been reported to strongly influence cell attachment and bioactivity
Subtle differences in actin filament spatial reorganization have been suggested to have a substantial effect on cellular mechanical behavior. Jaasma et al. [
Our results are in agreement with the above findings. Interestingly, there is a significant increase in filopodia projections on the control surface between 30 and 90 minutes of adhesion (Figure
It would be of special interest to further correlate these results with our previous findings on adhesion strength of osteoblasts on chitosan [
The results of the present study reveal that chitosan substrate stimulates fast osteoblast response, displaying rapid cell spreading and cytoskeleton reorganization. Cell adhesion has taken place in three distinct phases, as determined by the spreading kinetics. The osteoblast mechanical properties were significantly changed, with the cell exhibiting higher stiffness when there was a depletion of the cell excess membrane reservoir.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors wish to thank Anthoula Kroustalli, Ph.D. candidate at University of Patras, for her valuable contribution regarding the confocal microscopy images, as well as Pantelis Patsanis, graduate engineer of the Department of Mechanical Engineering and Aeronautics, University of Patras, for his help with the analysis of SEM imaging.