Differential regulation of osteoblasts by microstructural features of titanium substrata.
|Abstract:||The osseointegration process of bone implants crucially depends on the interaction between osteoblasts and the topography of the implant surface. Core binding factor alpha 1 (Cbfa1) is an osteoblast specific transcription factor that regulates osteoblast differentiation and expression of genes necessary for the development of a mineralized phenotype. In this study, we investigated the influence of microfabricated topographies on the activation of Cbfa1, in osteoblasts cultured on titanium substrata presenting microgroove gradients. Surface microgrooves with groove and ridge width ranging from 3 to 300 [micro]m, and four different groove depths (4, 6, 10, 14 [micro]m) were patterned on single crystalline silicon wafers using microlithography and deep reactive ion etching (DRIE). Titanium thin films were coated on the microgrooves by radio-frequency magnetron sputtering. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), and profilometry. Immunofluorescence staining of Cbfa1 protein was used to study the differentiation and function of osteoblasts in 3 day (72 h) culture experiments. Microtextured titanium surfaces were shown to promote contact guidance and enhanced Cbfa1 expression and activation, independently of the dimensions of the grooves of the studied microgroove gradients, compared to non micropattered surfaces. In conclusion, implant surface microgeometries may contribute to the regulation of osteoblast differentiation and function by influencing the level of bone-related transcription factors such as Cbfa1 in osteoblastic cells.|
Cell differentiation (Research)
Titanium (Health aspects)
Cellular control mechanisms (Research)
Substrates (Biochemistry) (Properties)
Kokkinos, Petros A.
Kirby, Paul B.
Deligianni, Despina D.
|Publication:||Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 Society for Biomaterials and Artificial Organs ISSN: 0971-1198|
|Issue:||Date: Jan, 2012 Source Volume: 26 Source Issue: 1|
|Topic:||Event Code: 310 Science & research Computer Subject: Company growth|
|Geographic:||Geographic Scope: United Kingdom Geographic Code: 4EUUK United Kingdom|
Upon the placement of an implant into a surgical site, there is a cascade of molecular and cellular processes that provides for differentiation and new bone growth along the biomaterial surface. Previous research has demonstrated that the response of cells and tissues to implant surfaces with micropatterns is unique and reproducible. The advantages of textured implant surfaces over smooth ones have been verified [5, 16, 31]. The goal of a number of current strategies is to provide an enhanced osseous stability through microsurface mediated events . An approach that is being explored for the design of improved cell-surface integration involves the use of microgrooved titanium geometries [1, 9, 11, 16, 20, 22, 24, 26, 28, 29, 31, 32].
Microfabrication techniques have been widely used to produce surface topographies to investigate the processes of cell adhesion, spreading, migration, proliferation, and of gene expression, as they allow precise production of specified topographies . Numerous techniques have been applied to produce micro-textured surfaces. These range from micro-machining and grit blasting to more controlled fabrication methods like photolithography and laser-texturing .
Surface texture of titanium substrates enhances cell responses to biological mediators and differentiation patterns (dioxide-coated polystyrene, titanium alloy and commercially pure titanium wafer) [11, 22, 26]. What still remains unclear are the exact architectural parameters that influence bone cell behavior and the hierarchy of their influence on osteogenesis, a process that extends many microns away from the surface of planar substrates .
Micrometer-sized grooves, either stochastic or organized, on substrates have been shown to influence the behavior of cells including osteoblasts, both in vitro and in vivo. Microgrooves are known to enhance a range of biological processes, such as cellular attachment and protein production , cell proliferation , mineralization of matrix deposition (1) and influence orientation of mineralized ECM and osteogenic cells [22, 32]. Grooved surfaces with a range of groove depths from 3 to 30 [micro]m increased the number of nodules by osteogenic cells . Both microscale pits and grooves stimulated matrix deposition and mineralization, possibly through different mechanisms . Studies have also shown that micro-textured orthopedic implants enhance tissue integration and wound healing [3, 8, 29].
Very few reports have been published regarding the mRNA expression levels in osteogenic cells relative to microtopography. Grooved titanium substrata altered fibronectin gene expression, mRNA stability, secretion, and assembly in fibroblasts [4, 22]. It may also be possible that the implant topography may lead to enhanced differentiation of osteoblasts through alterations in transcriptional regulation or gene expression of key osteogenic factors as a result of changes in cell shape due to interaction with the implant surface microtopography . The effects of different implant surface microtopographies on gene expression of key osteogenic factors are not fully understood. The levels of expression of Cbfa1 (Core binding factor alpha 1) and osteocalcin genes were significantly greater on stochastic grooved-textured implant surfaces relative to tissue culture plastic . DNA microarrays have tentatively identified protein activations in osteoblasts that may result from alterations in the features of the substratum topography to which the cell is adhered. It has been previously identified that rat calvarial osteoblasts (RCO) differentiated when cultured on certain topographical cues including microfabricated pits, grooves, and gap-cornerned boxes, in comparison to smooth surfaces. Src, FAK, and ERK 1/2 and Cbfa1 have been found that play key roles in regulating different facets of osteoblast function but their activation by microfabricated substratum topography has not yet been investigated [9, 23].
Although cell response to micrometer topography and in particular surface microgrooves has been reported, studies have often been limited to using stochastically patterned substrata and covering only a small range of microgroove geometrical values and the influence of this interesting microgeometry on the main osteoblast specific transcription factor Cbfa1 has been ignored.
Systematic studies of this microtopography would clearly contribute to our understanding of the osteoblast response. A parallel microgroove gradient surface with continuously changing surface-feature parameters is a powerful tool for such systematic studies. To our knowledge there has not been a report in the literature on the fabrication of microgroove gradients and their application in the study of Cbfa1 expression, activation and osteoblast differentiation. In this work, differentiation of rat calvarial osteoblasts (RCO) has been systematically studied by means of these microgroove gradients. The aim of our study was to evaluate how topographical patterns on titanium surface influence the expression of Cbfa1 which is one of the most important transcription factors responsible for the genetic reprogramming of osteoblasts.
Materials and Methods
The microtextured Ti substrates used in the present study were fabricated at the Nanofabrication Facility of the Microsystems and Nanotechnology Centre (School of Applied Sciences, Cranfield University, United Kingdom). Micro-grooves were first etched in blank silicon wafers (2 inch, Si <100>) by deep reactive ion etching (DRIE) using a STS-Surface Technology Systems, Multiplex ASE-Advanced Silicon Etcher and Shipley S1818 photoresist as a mask. The run time was adjusted for individual wafers to provide a range of microgroove depths. The wafers were then coated with 300 nm thick films of commercially pure Ti (99.99% purity) by RF magnetron sputtering. This microfabrication protocol produced microgroove gradients on titanium substrata, with groove and ridge widths ranging from 3 to 300 [micro]m and four different groove depths (4, 6, 10, and 14 [micro]m). Each of the designed groove width in the produced gradient equaled to its adjacent ridge width. Non patterned "smooth" areas were introduced to each sample as cell growth control areas. All lithographic procedures were conducted in a clean room environment to avoid particle contamination. Microtextured substrates (five substrata per type) were placed in 24-well plates. Substrata were sterilized and the surface activated by oxygen-plasma treatment (Harrick Plasma, Ithaca, USA) for 4 min. Immediately after the plasma treatment, the substrata were covered with media (cc-MEM with 10% fetal bovine serum and 1% antibiotics).
All specimens were examined and characterized by optical microscopy, surface profilometry, scanning electron microscopy (SEM), and atomic force microscopy (AFM) for surface features and uniformity. The microtextured substrates were characterized with a laser profilometer Dektak (3) ST (Surface Profile Measuring System, Veeco Surface Metrology/Veeco Process Metrology, Santa Barbara, CA, USA), and the profiles were measured perpendicular to the long gradient axis. In addition, the three-dimensional geometric patterns were examined in micrographs of SEM (High-Resolution FEI XL30 SFEG analytical SEM). SEM was used to characterize the microstructure and surface topography of the bulk material and the micro-textured features and also for the morphological analysis of osteoblastic cells cultured on these substrates. Samples with attached cells were removed from the media, rinsed with PBS at pH 7.4, fixed in 2.5% glutaraldehyde/paraformaldehyde fixative, dehydrated via a stepwise (30 min each step) alcohol dehydration (30, 50, 70, 80, 90%, and 3 x 100%), dried, gold sputter coated and examined by scanning electron microscopy. For a more detailed characterization of the surfaces in the nanometer regime a Digital Instruments Nanoscope III AFM (Veeco Instruments, Santa Barbara, CA, USA) was used.
Primary rat calvarial osteoblasts (RCO) were used in this study. Cells were obtained from rat calvariae and were isolated and sub-cultured as described by Hasegawa et al. and Chehroudi et al. Only RCO of the second (2nd) or third (3rd) passage were used for these experiments. After reaching confluency, cells were detached from the flasks with trypsin-EDTA solution (Sigma-Aldrich), centrifuged at 5000 rpm for 5 min, resuspended in culture media (c DMEM supplemented with 1% antibiotics and 10% fetal bovine serum) and seeded at a concentration of 3500 cells/ [cm.sup.2] on the micro-textured specimens. The concentration of the cells was manually counted with Bright-Line hemacytometer (Sigma). Osteoblasts were incubated for 72 hours (3 days) in short-term experiments at 37[degrees]C in a humidified atmosphere of 93% air and 7% C[O.sub.2].
[FIGURE 1 OMITTED]
Cells were washed with pre-warmed PBS (37[degrees]C) and fixed in a freshly prepared 4% paraformaldehyde solution. After rinsing with PBS, the cells were permeabilized using a 0.5% Triton X-100 solution (Sigma) for 3 min at RT. In order to block non-specific binding sites, the substrata were immersed into a 3% BSA/PBS solution (Sigma) for 30 min at room temperature and incubated with a primary antibody (rabbit polyclonal antibody to Cbfa1, 1:200 dilution in 1% BSA/PBS). Following incubation for 90 min at RT the samples were rinsed with PBS and incubated with the secondary antibody (Alexa Fluor 594 donkey anti-rabbit IgG, 1:200 dilution in 1% BSA/PBS) for 60 min at RT. After a new rinsing step with 1% BSA/PBS, samples were immersed in a 3.7% glutaraldehyde solution for 15 min at RT, rinsed again with PBS and kept therein in order not to dry off. Antibodies to Cbfa1 (Runx2) were from Abcam, Cambridge, MA, USA, and anti-rabbit IgG conjugated to Alexa Fluor 594 from Invitrogen. Before the IF microscopy, samples were rinsed with dd[H.sub.2]O, a small drop of FluoroGuard reagent (Bio-Rad) was added to the seeded micro-textured specimens and coverslips were placed over the specimens. Finally, stained cells were observed and photographed on a fluorescence microscope (Axio IMAGER M1m, Zeiss, Oberkochen, Germany). Images were taken at different positions along the gradient. Five substrata per type were investigated. In addition, the stained cells on the microtextured substrates were examined on a confocal microscope (Microscopy Center, ETH Zurich, Switzerland) (data non shown).
Microtextured titanium substrates
The microgrooved titanium surfaces obtained by sputtering on microfabricated silicon showed well-maintained groove geometry. A nanometer scale surface roughness appears on the microtextured substrates which is negligible in comparison to the groove sizes. There is at least 1000 times difference between the dimensions of the grooves (in microns) and those of the titanium coatings (in nanometers), thus the obtained surfaces showed insignificant surface roughness compared with the groove sizes. The surface roughness can be observed in SEM images (Fig. 1a, 1b, 1c) and AFM images (Fig 3a). A detailed microstructural, visual, and surface metrological characterization has been performed by surface profilometry, SEM, and AFM. The investigated micro textured surfaces consisted of a gradient of parallel microgrooves the width of which ranged from 3 to 300 [micro]m. The titanium coatings were uniform and consistent, continuous and adherent to the substrate even on the side walls of the grooves. In addition, the titanium did not fill up the grooves of the microtexture.
[FIGURE 2 OMITTED]
The highly conductive nature of cpTi resulted in the layer appearing brighter in comparison to the Si substrate (Fig. 1e, 1f). Templates presented an undercut and substrates with 14 [micro]m grooves showed a gradual increase in groove depth with the increase of groove width as can be seen in Figs. 1g, 1h, 1i. Micrographs of the patterned and unpatterned test specimens show that the surfaces are uniform and featureless at the micron level (Fig 1).
Top-and side-view micrographs were taken of the surfaces in order to measure groove dimensions, examine the effects of the processing parameters on groove geometry, and to study observable physical characteristics. The micro-grooved surfaces were found to exhibit relatively uniform surface morphologies. A slight variation in microgroove depth and width was observed with groove spacing. Specifically, the micro-grooves became less steeply sloped as groove spacing increased. Consequently, a slight increase in groove depth and a slight decrease in groove width were observed as groove spacing increased. The titanium coating on the smooth test specimens is uniform, continuous, and adherent to the substrate. The SEM evaluation of the coated and uncoated microtextured test specimens revealed that the microtexturing is uniform. Additionally, the titanium was successfully and uniformly sputter-coated on the microtextured test specimens. The side walls of the grooves were successfully coated with titanium. AFM images were taken of grooved samples without attached cells (Fig 3 bi). Figure 3bii shows scans of the grooved substrates.
[FIGURE 3 OMITTED]
Primary rat calvarial osteoblasts (RCO) were cultured for 72 h (3 days) on the micropatterned titanium substrates in order to analyze the effects of the tested surface topographies on osteoblastic differentiation and function. An overview of osteoblasts on the gradients is shown in Figure 2. The images of RCO cells on all studied titanium surfaces indicated groove guidance effects independently from microgroove depth (2, 6, 10 and 14 [micro]m) and width (ranging from 3 to 300 [micro]m). The cells' morphology and growth features were clearly revealed by SEM micrographs following culture for 72 h on Ti. Figure 2 (a-f) shows typical osteoblast morphology on "flat" unpatterned and microgrooved Ti substrata. It has been observed that the guidance effect became gradually weaker with increasing the groove width but it did not totally disappear even as the width approached values compared to the dimensions of the osteoblasts. Cells cultured on unpatterned control areas of the substrates were randomly oriented and well dispersed, mostly flattened, presenting numerous cytoplasmic extensions and lamellipodia, spreading in all directions and lacking any particular orientation. Cells on the "flat" control areas showed a rounder footprint with a few small filamentous extensions at the edge of the cell membrane. In contrast, cells attached on the microgrooved areas were directionally oriented in parallel with the microgrooves and appeared to align in the direction of the grooves, presenting a highly elongated spindle shape. These cellular morphologies aligned along the grooved surface can be seen in SEM and AFM images (Figures 2, 3). The larger groove sizes displayed cell growth within the grooves and on ridge surfaces with minimal impact on cell guidance. The smaller grooves only supported cell growth on the ridges however appeared to influence cell contact guidance a great deal more. It can be seen that cellular elongation becomes less pronounced as groove spacing increases. Interestingly, cells were elongated along the grooves wall and bottom in microgrooves of higher width compared with that of the osteoblastic cells (Figs. 2e, 2f, 3bii). With decreasing of the microgroove width, cellular morphology changed with cells presenting some long filamentous extensions. By increasing the microgroove width, cells were more spread with filamentous extensions. The substrates with depth of grooves of 4 [micro]m showed the greatest degree of contact guidance. Cells cultured on the 4 [micro]m surface extend on the ridge surface. Cell behavior on the 10 and 14 [micro]m samples were quite similar with a greater cell extension compared to the less deep grooves.
[FIGURE 4 OMITTED]
RCO cells were cultured onto the microgrooved surfaces, and following 72 h were fixed and immunofluorescently labeled with antibodies against Cbfa1. Cellular differentiation was illustrated by the visualization of Cbfa1 in osteoblasts attached on the microtextured surfaces. Figure 4 shows representative images of the stained RCO cells, cultured on substrates bearing groove gradients of 2 [micro]m (a i-iii), 6 [micro]m (b i-iii), 10 [micro]m (c i-iii), and 14 [micro]m (d i-iii) depth, showing the immunocytochemical localization of Cbfa1 transcription factor. Images display an overview of different areas of the gradients. Images are taken from the same sample. A low level and randomly distributed staining was observed on smooth surfaces, while a more intense and evident staining was seen on the microgroove gradient. Cbfa1 was significantly upregulated on micropatterned surfaces, in comparison with the nonpatterned surfaces.
It can be noted the presence of Cbfa1 mainly out of the nuclei at 72 h on all microfabricated topographies. Immunostaining revealed that Cbfa1 signal was not localized in the nucleus and remained cytoplasmic at the tested time period. Cbfa 1 appeared in a diffuse form around the perinuclear region. Substratum surface topographies seem not to stimulate the nuclear translocation at 72 h. On smooth surfaces, Cbfa1 activation was evident mainly throughout the cytoplasm of the cells. Cbfa1 phosphorylation remained high only on microgrooved controls, with a significant reduction evident on all nonpatterned surfaces.
Orthopedic biomaterials have often been modified to include elements of microscale topography, with the aim of increasing osteogenesis and promoting greater tissue integration. (12) Microgrooves on various biomaterial surfaces have been implicated in altering in vitro cell behaviours. Titanium surface microgrooves have been verified to display positive effects on osteoblast behaviour, whereas effects of various microgroove dimensions, including widths, on osteoblast activity remain to be demonstrated .
Amongst the myriad observations on the influence of substratum surface topography on cell behavior, intracellular signal transduction has received relatively little attention in comparison with adhesion, morphology, alignment and cytoskeletal organization . As alterations in microfabricated substratum topography have been shown to influence osteoblast differentiation, we hypothesized that substratum topography composed of a gradient of parallel microgrooves could differentially regulate intracellular signaling cascades related to osteoblast differentiation, subsequently leading to the activation of the osteoblast specific transcription factor Cbfa1.
Microgroove gradients produced in the present study cover most of the microgroove values typically found in osteoblastic cell studies on a single surface. The advantage of a microgroove gradient surface is clearly that cell experiments can be performed over a wide range of microgroove values under identical experimental conditions. Only the response of osteoblasts to roughness by means of surface topography gradients has been studied [14, 15]. The application of microgroove gradients for the investigation of osteoblastic differentiation by the activation of Cbfa1 transcription factor has not been reported previously.
The cells' morphology and growth features were clearly revealed by SEM micrographs (Fig. 2). Surfaces observation indicated the groove guidance effects despite different microgroove depths and widths. The groove depth was a weak controlling parameter when groove width is much larger than the cell size. A suitable groove width can bracket a cell body. If the groove fails to do so, cells may bridge over the groove without contacting the bottom surface of the groove. This may partially explain why the effects of groove depth on cell orientation vary with groove width . Observations of the current study were in accordance to literature data [3, 19].
The texturing of a substrate surface might be an important tool to establish a three-dimensional environment for bone cells. Hereby the cells are stimulated towards a behavior, which is more in accordance with natural bone tissue . This is in accordance with the Cbfa1 pattern of expression on the microgrooved substrate areas compared to the unpatterned control regions. The established 3D microenvironment seems to be important for Cbfa1 activation and osteoblastic differentiation. The mechanical stabilization produced by surface topography could play a role in the observed different behavior.
Recent studies have demonstrated that the implant surface microtopography itself can affect committed osteoblast gene expression. Few studies, however, have analyzed the effect implant microtopographies have on differentiation of cells into osteoblasts . The reports regarding the analysis of mRNA expression of osteogenic cells on micro- or micromachined grooved surfaces are very few [2, 4, 17, 18, 22].
Cbfa1 is a "master" regulator of osteoblast differentiation from mesenchymal precursors and bone formation and can directly stimulate transcription of osteoblast-related genes such as those encoding osteocalcin, type I collagen, osteopontin, and collagenase 3 [6, 13]. The purpose of our study was to elucidate how a precise microgrooved titanium surface topography alters the expression of Cbfa1, an indispensable transcription factor for osteoblast differentiation and expression.
In the present study, a low level and randomly distributed immunofluorescent staining for Cbfa1 was observed on smooth surfaces, while a more intense and evident staining was seen on the microgroove gradient. Immunostaining revealed that Cbfa1 signal was not localized to the nucleus and remained cytoplasmic at the tested time period. Substratum surface topographies seem not to stimulate the nuclear translocation of Cbfa1 at 72 h. Microgrooves have been found to encourage mitosis in the direction parallel to the microgrooves and discourage mitosis in the direction perpendicular to the microgrooves. (7) Interestingly, some of the immunocytochemical staining images have captured osteoblastic cells under division with two obvious nuclei present along the microgroove. Changing the pattern of the cell shape may affect the ability of the cell to differentiate into an osteoblast. HEPM cells demonstrated the ability to differentiate from a preosteoblast cell to an osteoblast over a 3-week time course .
Real-time PCR showed significant increases in Cbfa1 and osteocalcin gene expression in cells cultured on rough and grooved implant microtopographies . Cbfa1 gene expression was significantly increased on roughened titanium substrates . Substratum topography composed of 30 [micro]m deep grooves, 10 [micro]m deep gap cornered boxes and 30 [micro]m deep tapered pits, was found that stimulates significant increases in tyrosine phosphorylation in rat calvarial osteoblats, and regulates the phosphorylation of Src, FAK, and ERK V, as well as, the nuclear translocation of ERK V and the osteocalcin transcription factor, Cbfa1 .
Immunocytochemistry revealed that ERK V also localized to the nuclear compartment at all timepoints greater than 1 week on the microfabricated topographies. The nuclear translocation of ERK V correlated closely with the timing of translocation of the Cbfa1 . The short culture period of 72 h used in our study may be the reason of the lack of Cbfa1 nuclear translocation and of the observed perinuclear immunolocalization. Moreover, the different architecture of the substrates and the important difference of the cell culture period limit the comparison of the present study with the one of Hamilton and Brunette .
Previous studies on the effect of microtopography on osteoblast activity have produced conflicting results, demonstrating that, although some surfaces promote osteblast differentiation, others have a negative effect . It is generally difficult to make true comparisons of these types of studies. The surfaces used as cell culturing substrates are manufactured using different techniques and are often characterized only in terms of simple surface roughness parameters. Moreover, the culturing time is quite different in the relative studies, thus introducing more complexity when trying to compare the experimental data. Differences in gene expression on implant surfaces may be attributed to variations in rates of cell number. Sometimes, oppositional behaviors are due to use of osteoblasts at different maturation states. Some studies have also used transformed cells from osteosarcoma, and there is a concern that these cells may not respond to surface morphologies in a manner typical of normal osteoblasts. A further point to which attention has to be drawn is the level of confluence. It is known that cells behave differently once they reach confluence [14, 15].
The key finding of the present investigation was that Cbfa1 was activated and upregulated in osteoblasts cultured on gradient microgrooved topographies, thus supporting previously reported data regarding osteoblastic responses due to microgroove geometries.
Microlithographic and coating techniques were an effective means of fabricating the titanium substrates used in this study, which were composed of parallel microgroove gradients and were used to study the effects of microtopography on osteoblastic differentiation. The Cbfa1 expression and immunolocalization in osteoblastic cells did not show any preference for a particular groove size under the experimental conditions of the study, while the microgrooved surfaces demonstrated again the contact guidance effect of the grooves in comparison to unpatterned control surfaces. Observations presented in our study demonstrate that implant micro-architectural surface properties may contribute to the regulation of osteoblast differentiation by influencing the level of expression of bone-associated regulatory transcription factors such as Cbfa1.
Special acknowledgements to Dr T. Kunzler (LSST, Dept. of Materials, ETH Zurich, Switzerland) for assistance with immunofluorescent staining. DRIE of the substrates was performed with the help of Dr. Christopher Shaw (Microsystems and Nanotechnology Centre, Cranfield University, UK).
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Petros A. Kokkinos, (1) Robert Wright, (2) Paul B. Kirby, (2) and Despina D. Deligianni (1)
(1) Laboratory of Biomechanics and Biomedical Engineering, Department of Mechanical Engineering and Aeronautics, University of Patras, GR 26500 Rion, Patras, Greece; (2) Microsystems and Nanotechnology Centre, Materials Department, School of Applied Sciences, Cranfield University, Bedfordshire MK43 0AL, United Kingdom
Corresponding author: Despina Deligianni, email@example.com
Received 30 August 2011; Accepted 9 October 2011; Available online 8 February 2012
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