Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX-XXX
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Cytocompatibility of Titanium Microsphere-Based Surfaces Xiaoxiao Huang,† Lijun Shan,‡ Kui Cheng,† and Wenjian Weng*,† †
School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou 310027, China ‡ Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, University Kebangsaan Malaysia, Bangi, Malaysia ABSTRACT: The topography at the micro/nanoscale level for biomaterial surfaces has been thought to play vital roles in their interactions with cells. However, discovering the interdisciplinary mechanisms underlying how cells respond to micronanostructured topography features still remains a challenge. In this work, ∼37 μm 3D printing used titanium microspheres and their further hierarchical micro-nanostructured spheres through hydrothermal treatment were adopted to construct typical model surface topographies to study the preosteoblastic cell responses (adhesion, proliferation, and differentiation). We here demonstrated that not only the hierarchical micronanostructured surface topography but also their distribution density played critical role on cell cytocompatibility. The microstructured topography feature surface with middle-density distributed titanium microspheres showed significantly enhanced cell responses, which might be attributed to the better cellular interaction due to the cell aggregates. However, the hierarchical micro-nanostructured topography surface, regardless of the distribution density of titanium microspheres, improved the cell−surface interactions because of the enhanced initial protein adsorption, thereby reducing the cell aggregates and consequently their responses. This work, therefore, provides new insights into the fundamental understanding of cell−material interactions and will have a profound impact on further designing micronanostructured topography surfaces to control cell responses. KEYWORDS: ∼37 μm titanium microspheres, micronano structured surface, cytocompatibility, protein adsorption, cell aggregates dimensions of 10−30 μm.30 Microstructured topography could regulate distribution and orientation of cells on surfaces with different sizes.31,32 It has been reported that cells were unable to crawl onto microstructures with a height difference larger than 20 μm.33 And designed microstructures such as micrometer-depth wells will restrict cell distribution and force cell to form aggregates,34,35 which could promote colony-forming efficiency, maximize the cell−cell communication, and have significant influence on cell proliferation and differentiation.36−39 3D printing is an emerging technology that is rapidly becoming an integral method of component manufacture. With rapid and precise prototyping, 3D printing could provide patient personalized implants and exhibit important prospect. Because the surface structure determines cytocompatibility, it is important to investigate the topography of 3D printed implants. As 3D-printed implants were sintered by titanium microspheres with diameters of 10−53 μm, their surface morphology always shows the accumulation structure of microspheres.
1. INTRODUCTION Cells are inherently sensitive to the microenvironment provided by the surface of biomaterials,1−4 which is usually constructed via a well-organized microstructure or even nanostructure. The biomaterial surface composed of microscaled and nanoscaled topography could provide a desirable surface for cell responses by imitating the natural microenvironment.5 Various topographical features, such as ridges,6,7 grooves,8,9 pores,10−12 tubes,13−15 and rods16,17 in micro/ nanoscale, have been reported to have significant influences on cell behaviors. Topography can influence lots of cellular responses such as initial attachment and migration, differentiation and production of new tissues.18−21 It is well-known that nanoscale topography plays important role in cellular response to biomaterial surfaces by regulating protein adsorption.22,23 Proteins adsorbed in different quantities, densities, conformations, and orientations, depending on the local changes in surface properties, including nanoscale topography.24−26 Cell integrin could recognize specific sites of the adsorbed proteins, which is dependent on the orientation and conformation of the adsorbed proteins, which may initiate signaling events and regulate cell adhesion.27−29 The regulation of microscale structures on cells is more intuitive because the dimension is comparable with cellular © XXXX American Chemical Society
Received: August 4, 2017 Accepted: October 2, 2017 Published: October 2, 2017 A
DOI: 10.1021/acsbiomaterials.7b00551 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
glutaraldehyde for 2 h at room temperature. Next, the samples were dehydrated for 10 min at a gradient of ethanol solutions (10, 30, 50, 75, 90, 95, and 100 v/v % in sequence). Afterward, the samples were dried and observed by SEM. After 24 h of culture, the original culture medium was removed and the specimens flushed with PBS for three times, and then 300 μL of 1 mg/mL calcein-AM was added into each well and incubated for 30 min. After gently rinsing three times, the distribution of stained cells were observed under confocal laser scanning microscopy(CLSM). 2.7. Cell Viability. Cell viability was evaluated with CCK-8 (Dojindo Laboratories, Kumamoto, Japan) assay at 1 and 5 days. A 500 μL cell suspension with a regular density of 5 × 104 cells/cm2 (2 × 104 cells/cm2 for low density and 10 × 104 cells/cm2 for high density) was inoculated into a 24-well plate containing the samples. After 1 or 5 days, samples were washed three times with PBS and then transferred to another 24-well plate. After that, samples were incubated for 3 h at 37 °C with a mixed solution of fresh culture media and CCK-8 solution with 10:1 proportion. Finally, at the indicated times, 120 μL of the solution was dispensed into another 96-well plate, and colorimetric measurements of formazan dye were made with the microplate reader at 450 nm. 2.8. Cell Differentiation. Cell differentiation was evaluated by alkaline phosphatase (ALP) activity. MC3T3-E1 cells were inoculated on different samples into a 24-well plate. The culture medium was changed every 3 days. After 7 or 14 days of culture, the samples were transferred to another 24-well plate, cells were lysed and the solution was used for the measurement of ALP activity. ALP activity was determined using an ALP activity assay kit (Wako, Osaka) and then dividing the results by the concentration of cellular proteins determined using a BCA kit, respectively. 2.9. Statistical Analysis. All values are expressed as means ± standard deviation for three independent experiments. Statistical analyses were carried out using one-way analysis of variance (ANOVA) and Scheffe’s post hoc test with the SPSS software for multiple comparison tests (*P < 0.05, **P < 0.01, ***P < 0.001).
In this study, 3D printing used titanium microspheres and their further hierarchical micro-nanostructured spheres through hydrothermal treatment were adopted to construct typical model surface topographies to study their cytocompatibility (adhesion, proliferation and differentiation). MC3T3-E1 cell responses on typical hierarchical micro-nanostructured titanium surface topography were investigated. Also, the mechanism underlying the cell aggregates due to the hierarchical micronanostructured surface topography and their distribution density were discussed.
2. MATERIALS AND METHODS 2.1. Materials. Metal titanium (Erli Titanium Products Co., Ltd.) was used for substrates (10 mm × 10 mm × 0.5 mm). They were ultrasonic cleaned in ethanol (Sinopharm Chemical Reagent Co. Ltd.), and etched by a mixed acid solution with composition of 2.75 M HF (Juhua Chemical Reagent Co. Ltd.) and 3.94 M HNO3 (Sinopharm Chemical Reagent Co. Ltd.). Titanium microspheres for 3D printing (Baoji Lihua Nonferrous Metals Co. Ltd.) were used to construct the microstructure on the substrates. The numbers of titanium microspheres with different size were counted. The diameters of the purchased titanium microspheres ranged from 10 to 53 μm, and the mean diameter of titanium spheres was 37 μm. Sodium hydroxide (NaOH, Sinopharm Chemical Reagent Co. Ltd.) was used to modify the titanium microspheres. 2.2. Preparation. 2.2.1. Hydrothermal Modification of Titanium Microspheres. Hydrothermal treatment was adopted to procure porous titanium microspheres. Five molar NaOH was used as hydrothermal solution. A 100 mL Teflon vessel was filled with 80 mL of hydrothermal solution and 5 g of titanium microspheres. Hydrothermal growth was performed under the condition of 80 °C for 24 h in an electric oven. The autoclave was cooled to room temperature and the treated microspheres were removed, ultrasonically cleaned with deionized water and ethanol, and dried in ambient air. 2.2.2. Dispersed Spherical Ti Structure. Cleaned titanium microspheres with designed weight were mixed with 5 mL ethanol to get suspensions with 1, 5, or 20 wt % solid content. The samples were spin-coated with a drop of 20 μL suspension at 1000 rpm for 30 s. During spin-coating, each time before drawing the suspension, suspension needed to be fully mixed by shaking. After spin-coating, all the samples were heat-treated in a muffle furnace at 500 °C for 60 min. 2.3. Surface Characterization. Surface topography was characterized by a field-emission scanning electron microscope (FE-SEM, Hitachi SU-70, working voltage at 3 kV), and the initial cell adhesion on different samples was also observed by SEM. 2.4. Protein Adsorption. Samples were soaked in a 24-well plate with a 500 μL solution of α-MEM containing 10% FBS for 24 h at 37 °C to adsorb protein. The samples were transferred to another 24-well plate and soaked in 500 μL of 1% sodium dodecyl sulfate (SDS, Sinopharm Chemical Reagent Co. Ltd.) solution for 2 h to detach protein. BCA protein assay kit (Beyotime Biotechnology Co. Ltd.) was employed to measure the detached protein content in solution. 2.5. Cell Culture. Mouse calvaria-derived preosteoblastic cells (MC3T3-E1, CRL-2594, and ATCC) were utilized in this study. MC3T3-E1 cells were cultured in alpha-modified Minimum Essential Medium (MEM Alpha, Gibco) supplemented with 10% fetal bovine serum (FBS, PAA, Australia), 1% sodium pyruvate (Gibco), 1% antibiotic solution containing 10 000 units/mL penicillin and 10 mg/ mL streptomycin (Gibco), 1% MEM nonessential amino acids (Gibco). The cells were cultured under a humidified 5.0% CO2 atmospheres at 37 °C. Cultured cells were trypsinized with 0.25% trypsin and 1 mM EDTA (Gibco), centrifuged, and then dispersed in fresh culture medium and subcultured on samples. 2.6. Cell Distribution. After 24 h of culture, the original culture medium was removed and the specimens were flushed with PBS and water each for two times, and then fixed with 500 μL of 2.5%
3. RESULTS 3.1. Topography Features of Various Surfaces. The SEM image of a single titanium microsphere (Figure 1a, b)
Figure 1. SEM images of typical titanium microsphere surface feature before (a, b) and after (c, d) nanostructured treatment.
showed that the surface of the microspheres was quite smooth. After treatment by hydrothermal, the surface became rough, producing nanopores with an average size of 200 nm (Figure 1c, d). However, the diameter of the microspheres was not obviously changed after the hydrothermal treatment (Figure 1c). The microstructured and micro-nanostructured topography surfaces were prepared by spinning the corresponding microB
DOI: 10.1021/acsbiomaterials.7b00551 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 2. SEM images of typical (a−c) microstructured and (d−f) micro-nanostructured topography surface features with different microspheres densities. (a, d) low density; (b, e) middle density; (c, f) high density.
HPT surface, respectively. Each micro-nanostructured surface had higher protein adsorption than microstructured surface at the same density. 3.3. Cell Distribution on Various Surfaces. As shown in Figure 4a, b, 1-day cultured cells were evenly distributed on LT
spheres on the substrate. The dipping suspension concentration had a significantly influence on stacking density of the microspheres; the lower solid content led to a lower density. The amount of the titanium microspheres on the substrate was estimated ∼120 per mm2 for the low-density stacking surface (Figure 2a, d), ∼720 per mm2 for the middle-density stacking surface (Figure 2b, e), and ∼2400 per mm2 for the high-density stacking surface (Figure 2c, f). The surfaces with different density of microspheres were named as LT, MT, or HT, and the surfaces with different density of nanostructured microspheres were named as LPT, MPT, or HPT, respectively. The microspheres had an enough strong adhesion on the substrate since there morphology was almost unchanged after samples soaked in PBS for 7 days. 3.2. Protein Adsorption Behaviors on Various Surfaces. The amount of proteins adsorbed on different samples was measured and the results were shown in Figure 3.
Figure 4. SEM images of cells cultured on (a) LT surface and (b−d) LPT surface after 1 day of culture. Circles show the nanostructured microspheres attached by cells.
and LPT surfaces. We counted the number of microspheres in contact with or without cells on LT and LPT surfaces with different diameter on 2 mm2 and the data were shown in Table 1. On LT surface, cells could rarely attach or crawl onto the microspheres. While on LPT surface, the phenomenon of cells crawling to nanostructured microsphere surface can be generally observed. Cells could attach on nanostructured microspheres (Figure 4c) and the filopodia could be obviously observed the nanostructured surface (Figure 4d). It indicated that nanostructure on microspheres could significantly improve cell attachment to the surface. CLSM images showed that cells almost evenly distributed on all the micro-nanostructured surfaces with any density of nanostructured titanium microspheres (Figure 5c−f). Whereas cell aggregates could be observed on LT surfaces on the microstructured surfaces (Figure 5a), they could more frequently be seen on MT surfaces (Figure 5b), and no cell aggregates showed on HT surface.
Figure 3. Protein adsorption behaviors on various surfaces.
The protein adsorption quantity of the flat titanium surface (Control) was 0.224 mg/cm2, and surfaces with microstructures could adsorb much more proteins. According to Figure 3, higher density of the microspheres on substrate led to a higher amount of adsorbed proteins, indicating that titanium microspheres can significantly promote protein adsorption on the surfaces. Moreover, compared with the microspheres, the nanostructured microspheres had higher protein adsorption ability. The protein adsorption quantity was 0.334 mg/cm2 on the LT surface and 0.487 mg/cm2 on the LPT surface, 0.524 mg/cm2 on the MT surface and 0.632 mg/cm2 on the MPT surface, and 0.681 mg/cm2 on the HT surface and 0.829 mg/cm2 on the C
DOI: 10.1021/acsbiomaterials.7b00551 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering Table 1. Microsphere Counts with or without Cells in Different Sizes on 2 mm2 Surface 10−20 μm LT LPT
20−30 μm
30−50 μm
with cells
without cells
percentage (%)
with cells
without cells
percentage
with cells
without cells
percentage (%)
3 17
25 9
10.7 65.4
2 23
42 19
4.5 54.8
0 51
162 109
0 31.9
Figure 5. CLSM images of MC3T3-E1 cells cultured on (a) LT, (b) MT, (c) HT, (d) LPT, (e) MPT, and (f) HPT surface for 1 day. Arrows point to the cell aggregates (scale bar = 200 μm).
Cells were not able to crawl onto microspheres (Figure 6a) and be forced to distribute around the microspheres (Figure
Figure 7. CCK-8 results of MC3T3-E1 cells on different surfaces after 1 day and 5 days of culture.
variation trend between each surface was similar to cell adhesion. 3.4.2. Cell Differentiation. The ALP expression was thought to be an important indicator in early stage of cell differentiation, and the ALP activity of each sample was shown in Figure 8. According to Figure 8, ALP activity of cells cultured on the MT surface was significantly upregulated than that of the other microstructured surfaces and all the micro-nanostructured surfaces both at 7 and 14 days. The difference in cell differentiation for the surfaces showed the similar trend to
Figure 6. CLSM images of MC3T3-E1 cells cultured on (a, b) MT surface and (c, d) MPT surface for 1 day; (a, c) laser focused on substrate; (b, d) laser focused on microspheres. Circles show the microspheres (scale bar = 50 μm).
6b). In terms of nanostructured microspheres, cells could attach to the nanostructured microspheres (Figure 6c, d). Therefore, cells could distribute around or on the nanostructured microspheres randomly. 3.4. Cytocompatibility of Various Surfaces. 3.4.1. Cell Viability. The attachment ability of MC3T3-E1 cells to the surfaces was evaluated by the cck-8 assay after 1 day culture. As shown in Figure 7, cells on the microstructured surfaces such as LT, MT and HT exhibited much higher adhesion level than control, a flat titanium substrate. Among them, the MT surface had the best cell adhesion. For the micro-nanostructured surfaces, it was shown that higher density of nano structured microspheres lead to higher cell adhesion. But cell adhesion on HT surface was still lower than MT surface. Furthermore, cellular proliferation on the surfaces was evaluated after 5 days culture. The number of cells on the each surface was increased as compared with those of cells cultured for 1 day, and the
Figure 8. ALP activity of MC3T3-E1 cells on different surfaces after 7 and 14 days of culture. D
DOI: 10.1021/acsbiomaterials.7b00551 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 9. CCK-8 results of different density of MC3T3-E1 cells cultured on different density of microstructured surfaces after (a) 1 and (b) 5 days of culture.
Figure 10. CCK-8 results of different density of MC3T3-E1 cells cultured on nanomicrostructured surfaces after (a) 1 and (b) 5 days of culture.
surface played no positive role in enhancing cytocompatibility as expected. When the cell distribution on these surfaces is concerned, the cells on the micro-nanostructured surfaces almost evenly distributed as shown in Figure 8d−f, whereas the cells on LT microstructured surface tended to aggregate (Figure 9a) and more cell aggregates were observed on MT microstructured surface (Figure 9b). For the microstructured surface, the cells rarely crawled onto a microsphere (Figure 4a and Figure 6a, b), which could attributed that 20 μm is reported as a maximum height for cells to reach. As a consequence, the cells could only distribute on the bottom area around the microspheres. When the density of microspheres on surface increased, the bottom area decreased, the cells tended to form aggregates. And the density is too high, there is no bottom space to accommodate cells, the cells have to adhere onto the microspheres. Hence, only cell aggregates (Figure 5b) obviously appeared on MT microstructured surface due to having suitable bottom area, while little cell aggregates were observed on LT (Figure 5a) or HT (Figure 5c) microstructured surface due to the excessive large or small bottom area. For the micro-nanostructured surface, the cells could crawl or adhere onto the top of the nanostructured microspheres (Figure 4c and Figure 6c, d), this could be attributed that nanostructured microspheres probably gave a strengthened interaction with the cells through a better protein adsorption (Figure 3), as a result, no cell aggregates appeared on micronanostructured surfaces with any microsphere density (Figure 5d−f). When cell seeding density was increased to 10 × 104 cells/ cm2, respectively, the cytocompatibility on all surfaces had no obvious difference (Figure 9a, b and Figure 10a, b), implies that cell density with 10 × 104 cells/cm2 results in cell aggregates on all surfaces due to large amount of cells. While cell seeding density was decreased to 2 × 104 cells/cm2, no obvious
cell adhesion as well as proliferation. Obviously, cells on the MT surfaces had better osteogenic differentiation; it might be ascribed to the initial cell viability. 3.5. Influence of Cell Seeding Density on Cell Viability of the Surfaces. 3.5.1. Surfaces with Different Microstructure. There was no significant difference between cells seeded with low density (2 × 104 cells/cm2) or high density (10 × 104 cells/cm2) in cell adhesion to LT, MT, and HT surfaces after 1 day of cultivation (Figure 9a). However, after cultured for 5 days, cell proliferation with low density seeded cells on MT showed significantly upregulated than that on LT and HT surface. But there was still no significant difference to be observed on high density seeded cells (Figure 9b). It is worth to noting that significant differences were demonstrated between each surface with regular density (5 × 104 cells/ cm2) seeded cells both at 1 day and 5 days of culture. 3.5.2. Surfaces with Nanostructure. As shown in Figure 10, cells seeded with high density had no difference on surfaces with different nanostructure both in cell adhesion or proliferation, whereas cells seeded with low and regular density had significant difference between the MT and MPT surfaces. Furthermore, the proliferation of cells seeded with low density was significantly more improved by MT surface (Figure 10b).
4. DISCUSSION Titanium microspheres and nanostructured titanium microspheres at low, middle, and high density were deposited on substrate to construct microstructured surfaces and micronanostructured surfaces (Figures 1 and 2). When preosteoblast cells in a routine density of 5 × 104 cells/cm2 were seeded, the microstructured surface with middle-density had much better cell viability (Figure 7) and differentiation (Figure 8) than other surfaces, and surprisingly, the incorporation of nanopore structure onto the microspheres for the micro-nanostructured E
DOI: 10.1021/acsbiomaterials.7b00551 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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(4) Lim, J. Y.; Donahue, H. J. Cell sensing and response to microand nanostructured surfaces produced by chemical and topographic patterning. Tissue Eng. 2007, 13 (8), 1879−1891. (5) Muhammad, R.; Lim, S. H.; Goh, S. H.; Law, J. B. K.; Saifullah, M. S. M.; Ho, G. W.; Yim, E. K. F. Sub-100 nm patterning of TiO2 film for the regulation of endothelial and smooth muscle cell functions. Biomater. Sci. 2014, 2 (12), 1740−1749. (6) Watari, S.; Hayashi, K.; Wood, J. A.; Russell, P.; Nealey, P. F.; Murphy, C. J.; Genetos, D. C. Modulation of osteogenic differentiation in hMSCs cells by submicron topographically-patterned ridges and grooves. Biomaterials 2012, 33 (1), 128−136. (7) Wang, P.-Y.; Wu, T.-H.; Chao, P.-H. G.; Kuo, W.-H.; Wang, M.J.; Hsu, C.-C.; Tsai, W.-B. Modulation of cell attachment and collagen production of anterior cruciate ligament cells via submicron grooves/ ridges structures with different cell affinity. Biotechnol. Bioeng. 2013, 110 (1), 327−337. (8) Andersson, A. S.; Olsson, P.; Lidberg, U.; Sutherland, D. The effects of continuous and discontinuous groove edges on cell shape and alignment. Exp. Cell Res. 2003, 288 (1), 177−188. (9) Sprague, E. A.; Tio, F.; Ahmed, S. H.; Granada, J. F.; Bailey, S. R. Impact of Parallel Micro-Engineered Stent Grooves on Endothelial Cell Migration, Proliferation, and Function An In Vivo Correlation Study of the Healing Response in the Coronary Swine Model. Circ.: Cardiovasc. Interventions 2012, 5 (4), 499−507. (10) Janshoff, A.; Lorenz, B.; Pietuch, A.; Fine, T.; Tarantola, M.; Steinem, C.; Wegener, J. Cell Adhesion to Ordered Pores: Consequences for Cellular Elasticity. J. Adhes. Sci. Technol. 2010, 24 (13−14), 2287−2300. (11) Zhang, J. C.; Wu, L. B.; Jing, D. Y.; Ding, J. D. A comparative study of porous scaffolds with cubic and spherical macropores. Polymer 2005, 46 (13), 4979−4985. (12) Liu, P.; Zhang, H.; Liu, H.; Wang, Y.; Yao, X.; Zhu, G.; Zhang, S.; Zhao, H. A Facile Vapor-Phase Hydrothermal Method for Direct Growth of Titanate Nanotubes on a Titanium Substrate via a Distinctive Nanosheet Roll-Up Mechanism. J. Am. Chem. Soc. 2011, 133 (47), 19032−19035. (13) Kroustalli, A. A.; Kourkouli, S. N.; Deligianni, D. D. Cellular Function and Adhesion Mechanisms of Human Bone Marrow Mesenchymal Stem Cells on Multi-walled Carbon Nanotubes. Ann. Biomed. Eng. 2013, 41 (12), 2655−2665. (14) Cao, X.; Yu, W.-q.; Qiu, J.; Zhao, Y.-f.; Zhang, Y.-l.; Zhang, F.-q. RGD peptide immobilized on TiO2 nanotubes for increased bone marrow stromal cells adhesion and osteogenic gene expression. J. Mater. Sci.: Mater. Med. 2012, 23 (2), 527−536. (15) Zhang, L.; Hemraz, U. D.; Fenniri, H.; Webster, T. J. Tuning cell adhesion on titanium with osteogenic rosette nanotubes. J. Biomed. Mater. Res., Part A 2010, 95A (2), 550−563. (16) Park, E. J.; Peixoto, A.; Imai, Y.; Goodarzi, A.; Cheng, G.; Carman, C. V.; von Andrian, U. H.; Shimaoka, M. Distinct roles for LFA-1 affinity regulation during T-cell adhesion, diapedesis, and interstitial migration in lymph nodes. Blood 2010, 115 (8), 1572− 1581. (17) Matsutani, T.; Tanaka, T.; Tohya, K.; Otani, K.; Jang, M. H.; Umemoto, E.; Taniguchi, K.; Hayasaka, H.; Ueda, K.; Miyasaka, M. Plasmacytoid dendritic cells employ multiple cell adhesion molecules sequentially to interact with high endothelial venule cells - molecular basis of their trafficking to lymph nodes. Int. Immunol. 2007, 19 (9), 1031−1037. (18) Biggs, M. J. P.; Richards, R. G.; Gadegaard, N.; Wilkinson, C. D. W.; Dalby, M. J. The effects of nanoscale pits on primary human osteoblast adhesion formation and cellular spreading. J. Mater. Sci.: Mater. Med. 2007, 18 (2), 399−404. (19) Roach, P.; Farrar, D.; Perry, C. C. Surface tailoring for controlled protein adsorption: Effect of topography at the nanometer scale and chemistry. J. Am. Chem. Soc. 2006, 128 (12), 3939−3945. (20) Bettinger, C. J.; Orrick, B.; Misra, A.; Langer, R.; Borenstein, J. T. Micro fabrication of poly (glycerol-sebacate) for contact guidance applications. Biomaterials 2006, 27 (12), 2558−2565.
difference in cell adhesion (Figures 9a and 10a) but significant difference in cell proliferation (Figure 9b and Figure 10b), implies that cell density with 2 × 104 cells/cm2 leads to no cell aggregates at early stage of cell culture because of the small amount of cells, whereas cell aggregates form with an increase in cell amount after 5 days of culture. The cell aggregates on a surface are reported to favor cytocompatibility35 because cell proximity supports regulation of paracrine and autocrine signals36 and promotes cell−cell communication at early stage.37−39 Hence, we suggest that cell aggregates are responsible for the present microstructured surface with middle-density to have best cytocompatibility at a routine cell density of 5 × 104 cells/cm2.
5. CONCLUSIONS In summary, we demonstrated that not only the micronanostructured surface topography, but also their distribution density played critical role on cytocompatibility. These results might be further attributed to the better cellular interaction due to cell aggregates by suitable synergistic design of hierarchical micro-nanostructured surface topography and distribution density of 3D printing used Ti microspheres. It was also suggested that nanostructured surface feature might indeed enhance the initial protein adsorption as well as cell−surface interactions, but it might be not conducive to cell−cell interactions because of the homogeneous cell distributions that suppressed cell aggregates formation to some extent. This work therefore demonstrates the central role of cell aggregates in mediating cell responses on micro-nanostructured topography surface and implicates this interface as helping in optimizing osteointegration of Ti-based orthopedic and dental implants.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kui Cheng: 0000-0003-4828-6450 Funding
National Natural Science Foundation of China (51472216, 51772273, 51372217, 31570962, 51502262), Zhejiang Provincial Natural Science Foundation (LY15E020004), the 111 Project under Grant B16042, the Fundamental Research Funds for the Central Universities (2017XZZX008-05) and State Key Lab of Silicon Materials Foundation (SKL2017-13). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Dong Lingqing for helping revise the language. REFERENCES
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DOI: 10.1021/acsbiomaterials.7b00551 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsbiomaterials.7b00551 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
