Nanosurface design of dental implant for improved cell
growth and function
Hsu-An Pan1, Yao-Ching Hung2,3, Jin-Chern Chiou4, Shih-Ming Tai1, Hsin-Hung Chen5 and G. Steven Huang1*
1Graduate Program for Nanotechnology, Department of Materials Science and
Engineering, National Chiao Tung University, Hsinchu,300, Taiwan, R.O.C.
2College of Medicine, China Medical University, Taichung, 40402, Taiwan, R.O.C. 3Department of Obstetrics & Gynecology, China Medical University
Hospital,Taichung, 40402, Taiwan, R.O.C.
4Institute of Electrical Control Engineering, National Chiao Tung University,
Hsinchu, 300, Taiwan, R.O.C.
5Fame Dental Clinic, 1F., No.6, Aly. 4, Ln. 27, Sec. 4, Ren-ai Rd., Da-an Dist., Taipei
City 106, Taiwan, R.O.C
E-mail: gstevehuang@mail.nctu.edu.tw
Abstract
A strategy was proposed for the topologic design of dental implants based on the in vitro survey of optimized nanodot structures. An in vitro survey was performed using nanodot arrays with dot diameters ranging from 10 nm to 200 nm. MG63 osteoblasts were seeded on nanodot arrays and cultured for 3 days. Cell number, apoptosis-like, cell adhesion, and cytoskeletal organization were evaluated. Nanodots with a diameter of approximately 50 nm enhanced 44 % cell number, minimized apoptosis-like to 2.7 %, promoted 30 % increase in microfilament bundles, and maximized cell adhesion with a 73 % increase in focal adhesions. Enhancement of ca 50 % in mineralization was observed determined by von Kossa staining and by Alizarin Red S staining. Therefore, we provide a complete range of nanosurface for growing osteoblasts to discriminate nanoscale environment. Nanodot array presents an opportunity to positively and negatively modulate cell behavior and maturation. Our results suggest a topologic approach which is beneficial for the design of dental implants.
1. Introduction
For dental implants, surfaces are moderately roughened to promote osseointegration [1-2]. Studies have shown that osteoblast-like cells favor microstructured surfaces [3-6]. Roughened surfaces enhance the focal adhesion and guide cytoskeletal assembly and membrane receptor organization [7-8]. Moreover, rough implant surfaces have been shown in in vitro experiments to enhance the adsorption of fibronectin and albumin [9-10], which are important extracellular matrix molecules for cell focal adhesion. Methods including acid-etching, plasma-spraying, grit-blasting, vapor deposition, anodization, and other coating technologies have been developed to fabricate micro- and nanostructures. These different modifications, which result in a variety of surface chemistries and topographies, have led to ambiguous responses by osteoblasts [11-13].There are considerable disagreements concerning the optimal physicochemical properties and surface geometries for the endosseous portion of a dental implant.
Identifying the optimal surface for the bio-implant interface is an important task in tissue engineering [14-15]. Many studies indicate that various nanostructured surfaces can influence the in vitro adhesion [16-18], morphology [19-21], proliferation [22], and gene expression [23] of different cell types. The cellular response to a nanostructured substrate depends on the size arrangement of the topographic features and cell type [24-25]. Nanostructures such as nanofibers [26], sharp-tips [16], and nanotubes [27] interact with cells and direct proliferation. The topologic design of implant surfaces is one of the important factors for the fabrication of medical implants [28-29].
Nanoscale modification of the implant surface may alter the surface reactivity of endosseous implants. The surface roughness influences the production of growth
factors, cytokines and mRNAs, suggesting that the substrate modulates the activity of the cells that are adjacent to an implant; this roughness subsequently affects the adjacent skeletal tissue response and implant success [30-31]. Moreover, surface topography affects the amount of bone that is deposited adjacent to the implants and bone, and its formation can be guided by the specific implant topography [32]. Thus, surface topography plays a critical role in the interaction of dental implants with the adjacent tissues [33-34]. Nanoscale topography may provide biomimetic surfaces that support hydroxyapatite mineral formation [35] and the related organic phase guidance of bone mineralization [36].
Nanotopographical surface affects cellular behavior in a wide range of cell types including epithelial cells, fibroblasts, myocytes, and osteoblasts [37]. Nanoscale features alter osteoblastic attachment, proliferation, differentiation, and matrix production [38-39]. Up-regulation of osteoblast proliferation is observed on nanoscaled surface of materials such as alumina, titania, and calcium phosphate [40]. An interesting feature of the nanoscale topographic surfaces is the selectivity for cell adhesion. Several investigators have demonstrated the relative diminution of fibroblast adhesion compared to osteoblast adhesion when nano- and micron-structured surfaces were evaluated [41-42]. Nanotopography-induced cellular response has been explored using nanoislands [43]. Osteoblastic cells respond differentially to nanoislands with height varying between 11 and 85 nm. However, until recently, no attempt to utilize systemic nanoscale environment to investigate the an optical range of nanostructure for cell growth.
This study is based on the hypothesis that nanotopography may modulate and control the growth, proliferation, and biological function of osteoblasts. Arrays of nanodots with defined diameters and depths can be fabricated using aluminum
nanopores as a template during the oxidation of tantalum thin films. The dot size and the depth of the dots are well controlled. We previously demonstrated that an integrated nanodevice containing nanodot arrays with dot diameters ranging from 10 nm to 200 nm can be used to evaluate cell behavior. Nanodevices may be used as a detecting platform for the rapid modulation of proliferation, apoptosis, invasive ability, and cytoskeletal reorganization in different cell types [44]. The application of an assembly containing a range of nanostructures should be capable of obtaining parameters that are useful in the design and evaluation of artificial implants for tissue engineering.
2. Materials and methods
2.1 Fabrication of nanodot arrays
The nanodot arrays were fabricated as described [45]. A tantalum nitride (TaN) thin film (200 nm in thickness) was deposited onto a 6-inch silicon wafer; a 400 nm-thick aluminum layer was then deposited onto the TaN layer. Anodization was conducted in 1.8 M sulfuric acid at 5 V or the 10 nm nanodot arrays, in 0.3 M oxalic acid at 25 V for the 50 nm nanodot arrays, in 0.3 M oxalic acid at 100 V for the 100 nm nanodot arrays and in 5 % (w/v) phosphate acid (H3PO4) at 100 V for the 200 nm nanodot arrays. In the first instance, the above aluminum layer oxidized to alumina, accompanied by the outward migration of Al3+ and inward diffusion of O2− driven by the applied electric field, leading to the vertical pore channel growth. The dissolution of alumina at the alumina/electrolyte interface is in equilibrium with the growth of alumina at the Al/Al2O3 interface. As the oxide barrier layer at the pore bottom approaches the TaN/Al interface, the O2− migrating inwards through the alumina barrier layer are continuously injected into the Ta layer and form the tantalum oxide. The underlying tantalum oxide by O2− transported through/from the barrier layer of
the initially formed porous alumina without direct contact of tantalum with the electrolyte. The anodic reaction of TaN results in the formation of tantalum oxide accompanied by formation of hemispherical structures due to volume expansion. Eventually, the aluminum completely transferred into alumina accompanied the end of the all anodic process. The porous alumina was removed by immersion in 5 % (w/v) H3PO4 overnight. A thin layer of platinum (ca. 5nm) was sputtered onto the structure to improve the biocompatibility. The dimensions and homogeneity of the nanodot arrays were measured and calculated from images taken by JEOL JSM-6500 TFE-scanning electron microscopy (SEM). The size of nanodots were counted using ImageJ software and expressed in terms of diameter. We randomly picked 30 nanodots from each substrate field and calculated the diameter of dots. Three random substrate fields were measured per sample and three separate samples were measured for each surface.
2.2 Cell culture
MG63 osteoblast-like cells were originally isolated from a human osteosarcoma. The cell culture experiments were performed with the osteoblastic cell line MG63 (BCRC No. 60279, Bioresources Collection and Research Center, Taiwan). The cells were seeded in substrates and cultured in Eagle’s minimum essential medium with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate. The Eagle’s minimum essential medium was supplemented with 10 % fetal bovine serum (Gibco Invitrogen) at 37 °C in 5 % CO2.
2.3 Scanning electron microscopy
saline (PBS) at 4 ºC for 20 minutes, and then treated in 1 % osmium tetroxide for 30 minutes. Dehydration was performed with a series of ethanol concentrations (5 minute incubations each in 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, and 100 % ethanol) followed by air drying. The specimen was sputter-coated with platinum and examined by JEOL JSM-6500 TFE-SEM at an accelerating voltage of 10 keV.
2.4 Measurement of cell number by cell density
Cells were double stained using 4',6-diamidino-2-phenylindole DAPI and phalloidin. MG63 cells were harvested and fixed using 4% paraformaldehyde diluted in PBS for 30 min, followed by 3 washes in PBS. Cell membranes were permeabilised during 10 min incubation in 0.1 % Triton X-100, followed by 3 PBS washes. MG63 cells were incubated with phalloidin and nuclei counterstained with DAPI for 15 min at room temperature. Samples were mounted and imaged using a Leica TCS SP2 confocal microscope. Cell number was counted using ImageJ software and expressed in terms of cell density. Six different substrate fields were measured per sample and three separate samples were measured for each surface.
2.5 Immunostaining of vinculin and the microfilament bundless
The cells were harvested and fixed with 4 % paraformaldehyde in PBS for 15 minutes and then washed three times in PBS. The membranes were permeabilized by an incubation in 0.1 % Triton X-100 for 10 minutes, washed three times in PBS, blocked with 1 % bovine serum albumin (BSA) in PBS for 1 hour, and then washed three times in PBS. The samples were incubated with an anti-vinculin antibody (properly diluted in 0.5 % BSA) and phalloidin for 1 hour, incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody for 1 hour, and then washed three times in PBS. Immunostaining with anti-vinculin antibody and phalloidin were performed. For
each experimental condition, the number of vinculin plaques and microfilament bundles per cell were counted and compared to that for cells that were cultured on a flat surface. The diameter of actin filament is ~8 nm which is beyond the resolution of our microscope. The fibrous structure that we observed is apparently microfilament bundles. Actin filaments are assembled in two general types of structures: bundles and networks. These structures are regulated by many other classes of actin-binding proteins. With confocal microscopy, an estimation for the number of microfilament bundles can be obtained by building a 3-d cell superimposed image. Since the length and exact diameter of microfilament bundles are difficult to quantify the measurement of microfilament number is meant to be a semi-quantification to estimate the cytoskeleton organization of cultured osteoblasts. Twelve cells were measured per sample and three separate samples were measured for each surface. The plots were fitted using Origin software (Northampton, USA).
2.6 von Kossa staining
MG63 cells were harvested and fixed with 95 % ethanol for 1 hour and then washed three times with DI water. The samples were treated with a 5 % silver nitrate solution, exposed to UV light for 20 minutes, and then washed three times with DI water. The samples were treated with a 5 % thiosulphate solution for 5 minutes and then washed three times with DI-water [46-47]. The phosphate ion precipitation was demonstrated following dark brown colored nodular staining confirming the formation of minerals in the osteoblast cultures. The mineralized nodules were counted under microscope. Three random substrate fields were calculated per sample and three separate samples were measured for each surface.
The MG63 cells on the substrates were washed with PBS and fixed with 4 % paraformaldehyde for 10 min. The fixed cells were soaked in 0.5 % Alizarin Red S in PBS for 10 minutes at room temperature and then washed with water to remove the remaining stain [48-49]. The extent of mineralized nodule formation based on number of nodules was determined by Alizarin Red S staining at 7 day. The mineralized nodules were counted under microscope. Three random substrate fields were calculated per sample and three separate samples were measured for each surface.
2.8 Statistics
The experimental Data were expressed as the mean±standard deviation. One-way analysis of variance followed by Tukey-post test was used for statistical analysis (SPSS 13.0 software, Chicago, USA), and the level of significance was set at *P<0.05.
3 Results
3.1 Nanotopology of dot arrays
Nanodot arrays were fabricated on tantalum-coated wafers by anodic aluminum oxide (AAO) processing. Tantalum oxide nanodot arrays with dot diameters of 10 nm, 50 nm, 100 nm, and 200 nm were constructed on silicon wafers. To provide a biocompatible and unique interacting surface, platinum of ca. 5nm thickness was sputter-coated onto the top of the nanodots. SEM images showed diameters of 10 ± 3 nm, 52 ± 6 nm, 102 ± 9 nm, and 212 ± 19 nm for the 10 nm, 50 nm, 100 nm, and 200 nm dot arrays, respectively (Figure 1); the dot-to-dot distances were 22.8 ± 4.6 nm, 61.3 ± 6.4 nm, 108.1 ± 2.3 nm, 194.2 ± 15.1 nm, and the average heights were 11.3 ± 3 nm, 51.3 ± 6 nm, 101.1 ± 10 nm, 154.2 ± 28 nm, respectively. The dimensions of the nanodots were well-controlled and highly defined.
3.2 The topology controlled the cell number, apoptosis-like, and adhesion of MG63 osteoblasts
MG63 osteoblasts were cultured on fabricated nanodot arrays at densities of 1,000 cells per square centimeter. The cells were harvested 3 days after seeding. SEM was performed to examine the cell number and apoptosis-like morphology of the cells (Figure 2). The number of focal adhesions is the hallmark for cell attachment and can be evaluated by immunostaining against vinculin. The organization of the cytoskeleton was visualized by immunostaining for the microfilament bundles (Figure 3). To evaluate the size effect of the nanodot arrays, the percent cell number, the percent apoptosis-like, the number of focal adhesions, and the number of microfilament bundles were drawn against the dot diameters (Figure 4).
The cell growth was closely associated with the surface topology. The number of MG63 cells initially increased when the diameter of the nanodots increased. The cell number reached a maximum at a nanodot diameter of approximately 50 nm but dropped dramatically for the 100 nm and 200nm nanodot arrays. The cell number reached a maximum of +143.9 % at a dot diameter of 48.79 nm (Figure 4A, Table 1). A significant decrease of the cell number was observed for the cells grown on the 200 nm nanodots (65 % cell number).
Decreased cell number occurred with MG63 cells seeded on larger nanodots. The decrease of cell number is very likely due to programmed cell death. Cells that underwent apoptosis exhibited an abnormal morphology that was identified in the SEM images. The percentages of apoptotic-like cells versus dot diameters were plotted for the MG63 cells. Minimal apoptosis-like occurred when the dot diameter approached 50 nm (Figure 4B). The cells started to show thickening and mounting when the dot size was larger than 100 nm; considerable thickening and mounting
were observed when the dot size was 200 nm. On the contrary, cells grown on a flat surface and on 10nm and 50 nm nanodot arrays were flat and extended. Cells grown on 50 nm nanodots exhibited the most extended morphology.
The abnormality of apoptosis-like event was observed by apoptosis-like cell morphology of SEM images. Nanotopography-induced apoptosis shares some common features with anoikis, the apoptosis induced by the loss of cell adhesion. Both events were initiated at the bio-nano interface. The loss of focal adhesions and lamellipodia collapse were key features of both phenomena. However, anoikis is triggered by forcing epithelial cells to grow in suspension, and signaling is detectable in minutes to hours. Nanotopography-induced apoptosis-like events became evident only after days of incubation.
The organization of the cytoskeleton is an important index for cell growth. Although there is no quantitative measurement for cytoskeletal organization, the number of cytoskeletal fibers is a well-recognized estimation. Cells grown on a flat surface and on 10 nm, 50 nm, and 100 nm nanodots exhibited well-defined microfilament bundles in the cytoplasm. However, there was a visible loss of microfilament bundles in cells grown on the 200 nm nanodots (Figure 2, Figure 4C).
The formation of focal adhesions is a hallmark for the proper attachment of cells and can be estimated by the degree of vinculin staining. The formation of focal adhesions versus the dot diameter exhibited a trend that was similar to that for the cell number versus the dot diameter. There was an initial increase of focal adhesion formation that gradually decreased when the nanodot diameter exceeded 50 nm (Figure 4D). The maximum number of focal adhesions occurred with a dot diameter of 59.4 nm; at this diameter, there was a 73.2 % increase in the number of focal adhesions compared to those formed by cells cultured on a flat surface (Table 1).
3.3 The mineralization of MG63 cells was associated with the nanotopology
The mineralization process is a hallmark for the function of osteoblasts. To investigate the modulation of the mineralization process, MG63 cells were cultured onthe integrated nanodot array device for 7 days. Mineralization in cell culture monolayers has been determined using quantitative methods with von Kossa and Alizarin Red S staining. The phosphate ion precipitation was visualized as dark crystals following von Kossa staining (Figure 5). The calcium deposition was stained bright red following Alizarin Red S staining (Figure 6). By von Kossa staining, a high density of nodular phosphate ion precipitation was identified in cells grown on the 50nm nanodots (Figure 7). A 43.7 % increase in phosphate ion precipitation occurred in cells cultured on 45.1 nm nanodots compared to cells grown on a flat surface (Table 1). By Alizarin Red S staining, a high density of nodular calcium deposition was identified in cells grown on 10 nm and 50nm nanodots (Figure 7). The quantification of mineralization by Alizarin Red S staining indicated a 54.8 % increase in mineral content in cells cultured on 45.9 nm nanodots (Table 1). The nanotopography should provide biomimetic surfaces that support mineral formation and guide bone mineralization [37].
4 Discussion
Nanotopography affects cell growth and function; however, the control of cell growth or function is still not well defined. The topology of titanium oxide nanopores affects human mesenchymal stem cell (hMSC) adhesion and differentiation. Larger nanopores (70 nm to 100nm in diameter) induce elongation and differentiation into osteoblast-like cells [50]. The analysis of hMSC culture on nano-patterned polystyrene and polydimethylsiloxane [51] indicated that the nanotopography may modulate cell behavior by altering the expression profiles of integrins and the
assembly of focal adhesions, which can lead to changes in the cytoskeletal organization and the mechanical properties of the cell. However, nanostructures do not always promote cell growth and function. In our previous studies, we have shown the differential growth of NIH-3T3 cells on nanodot arrays with dot diameters ranging from 10 nm to 200 nm [52]. Cells grew normally on the 10 nm arrays and on flat surfaces. However, the 100 nm and 200 nm nanodot arrays induced apoptosis-like events. The occurrence of apoptosis is due to the loss of focal adhesions. The nanotopographic surface, similar to other substrates including polystyrene and silicon, enhances focal adhesion formation, proliferation and the spreading of various adherent cell types [53-54]. In vitro evidence indicates that the topologic design may have additional benefits in addition to the conventional implant design [44, 55].
It should be noted that the description of the 3-dimentional topology is complicated. Our system used the dot diameter as a variable owing to the monotonous variation and the homogeneity of the structure and thus should be treated as a simplified version of topology. When plotting the cell number versus the dot diameter, maximal growth is identified with nanodots approximately 50 nm in diameter and decayed for diameters larger than 100 nm. It has been proved that the distance between the microfilaments adhering focal adhesions is approximately 50 nm to 70 nm [56-57]. The 50 nm dot-to-dot distance may have provided anchoring points that were the most suitable for the assembly of focal adhesions in migrating cells [58]. Consequently, 100 nm and 200 nm nanodots, which have dot-to-dot distances that are much longer than 50 nm, do not support the formation of properly distanced focal adhesions. Flat surface and 10 nm nanodots surface should provide anchoring points for osteoblasts. Our results showed that minor increase of growth and function for 10 nm nanodots compared to flat surface. The enhancement maximized at 50 nm dot
surface. Apparently, topologic effects other than anchoring distance, such as physical stress caused by different topologies, might play roles in the current study. This finding suggests that carefully designed nanostructures should provide a better environment for cell growth.
Our structure is one of the rare topographies that promote growth and induce cell death within the same structure by varying the dimensions. This method will assist the design and fabrication of nanostructures on artificial implants that may perform contrasting functions on different surfaces. The molecular mechanism that underlies the optimal dot size that stimulates maximum growth and mineralization for osteoblasts is not clear. The range between 50-80 nm is a universal length scale for integrin clustering and activation in cell adhesion [59]. The 50 nm dot surfaces coincidently provide a biocompatible environment for the formation of focal adhesion and the subsequent intracellular organization of the microfilaments. The results indicate that nanotopography may direct the cell behavior via pathways not completely overlapped with surface coating. The hydroxyapatite or β-tricalcium phosphate coating with 50 nm nanodot structure may further improve the surface biocompatibility and promote cell maturation for dental implant.
5 Conclusion
In this study, we evaluated the topologic effects on the growth and function of osteoblastic cell line MG63. MG63 cells were grown on nanodot arrays ranging in diameter from 10nm to 200nm. The cell number, morphology, adhesion, cytoskeleton, and mineralization were evaluated. Nanodot diameters ranging from 46 nm to 60nm provided the optimized condition for the cell number, cell adhesion, and mineralization of MG63 cells. Nanodots lager than 100 nm retarded the growth and suppressed the functional expression of the cells. Here, we propose a strategy for the
topologic design of dental implants based on an in vitro survey of optimized nanodot structures. The nanostructure is capable of modulating the in vitro growth and function of osteoblasts and is optimal with a nanodot size of approximately 50 nm in diameter. These results will contribute to designing functional surfaces that control cell behavior and promote cell maturation for dental implants.
The heights and distance between two dots were used to define topology in the current study. However, heights are important parameters needed to be considered for topology. Because the surface tension applied on the cells is normally based on how the surfaces elongate the cells by two adhesion sites. Due to the limitation of the current fabrication, it requires additional study to explore whether the distance between two dots is more critical than the diameter of the dots (or the heights). However, 50 nm nanodots provided the optimized growth environment for osteoblast, both in grow and function. Application of this topology in the dental implants is expected.
Acknowledgements
This study was supported in parts by the "Aim for the Top University Plan" of the National Chiao Tung University and Ministry of Education, Taiwan, R.O.C. and the National Science Counsel grant 100-2923-B-009-001-MY3. The authors also acknowledge funding support from the Air Force Office of Scientific Research (AFOSR, FA2386-11-1-4094).
