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Letter Cite This: Nano Lett. 2018, 18, 2243−2253

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Polylactic Acid Nanopillar Array-Driven Osteogenic Differentiation of Human Adipose-Derived Stem Cells Determined by Pillar Diameter Shan Zhang,† Baojin Ma,† Feng Liu,† Jiazhi Duan,† Shicai Wang,† Jichuan Qiu,† Dong Li,‡ Yuanhua Sang,† Chao Liu,*,§ Duo Liu,*,† and Hong Liu*,†,∥ †

State Key Laboratory of Crystal Materials, Shandong University, Jinan, 250100, China Cryomedicine Laboratory, Qilu Hospital, Shandong University, Jinan, 250012, China § Department of Oral and Maxillofacial surgery, Qilu Hospital, Institute of Stomatology, Shandong University, Jinan, 250012, China ∥ Institute for Advanced Interdisciplinary Research, Jinan University, Jinan, 250022, China

Nano Lett. 2018.18:2243-2253. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 01/19/19. For personal use only.

‡

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ABSTRACT: Numerous studies have determined that physical cues, especially the nanotopography of materials, play key roles in directing stem cell differentiation. However, most research on nanoarrays for stem cell fate regulation is based on nonbiodegradable materials, such as silicon wafers, TiO2, and poly(methyl methacrylate), which are rarely used as tissue engineering biomaterials. In this study, we prepared biodegradable polylactic acid (PLA) nanopillar arrays with different diameters but the same center-to-center distance using a series of anodic aluminum oxide nanowell arrays as templates. Human adipose-derived stem cells (hADSCs) were selected to investigate the effect of the diameter of PLA nanopillar arrays on stem cell differentiation. By culturing hADSCs without the assistance of any growth factors or osteogenic-induced media, the differentiation tendencies of hADSCs on the nanopillar arrays were assessed at the gene and protein levels. The assessment results suggested that the osteogenic differentiation of hADSCs can be driven by nanopillar arrays, especially by nanopillar arrays with a diameter of 200 nm. Moreover, an in vivo animal model of the samples demonstrated that PLA film with the 200 nm pillar array exhibits an improved ectopic osteogenic ability compared with the planar PLA film after 4 weeks of ectopic implantation. This study has provided a new variable to investigate in the interaction between stem cells and nanoarray structures, which will guide the bone regeneration clinical research field. This work paves the way for the utility of degradable biopolymer nanoarrays with specific geometrical and mechanical signals in biomedical applications, such as patches and strips for spine fusion, bone crack repair, and restoration of tooth enamel. KEYWORDS: Polylactic acid nanopillar array, nanopillar diameter, adipose-derived stem cells, osteogenic differentiation

materials is the most preferred cue for tissue regeneration therapies in the future due to their biological stability and health safety profile.14,15 Nanopattern construction has become a major tool for providing specific geometrical and mechanical signals on material surfaces, which can potentially elicit a stem cell response. Many different nanopatterns have been designed and prepared to promote stem cell differentiation, especially osteogenic differentiation. For example, the effects of the disorder degree of nanopit arrays,16 nanospacing of arginineglycine-aspartic acid (RGD) nanopatterns,17,18 and the aspect ratio of nanopillar arrays19,20 have been studied. These studies have demonstrated that only nanopatterns with special

Over the years, using physicochemical signals to regulate stem cell fate has become a hot research topic in tissue engineering and regenerative medicine.1−3 Biochemical factors have been widely used to induce osteogenic differentiation with a good efficiency. Using bone morphogenetic protein-2 (BMP-2) as an example, after an induction period from 7 days to 31 days, BMP-2 (10−100 ng/mL) can effectively induce bone nodule formation and calcium deposition compared with a control group.4−6 Compared with biochemical growth factors, physical cues that participate in stem cell differentiation are also efficient and have almost no potential side effects in human bodies.7,8 The most widely used physical cues include light,9 magnetism,10 electricity,11 and surface topology of materials.12,13 However, the stimulations from light, magnetism, and electricity are external factors that do not exist in vivo and are generally unwanted for surrounding tissue engineering. Therefore, surface topology based on nanostructures of © 2018 American Chemical Society

Received: November 9, 2017 Revised: March 5, 2018 Published: March 8, 2018 2243

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Scheme 1. Schematic Illustration of the Series of PLA Nanopillar Arrays on the Surface of PLA Film with Different Pillar Diameters but the Same Center-to-Center Distance with Stem Cells Cultured on the Surface

Figure 1. Characterization of the PLA planar film and PLA nanopillar arrays and the cell viability of hADSCs after culturing on different samples for several days. (a) Digital photo of PLA planar film. (d, g, j) SEM images of the AAO template platform with different nanopit diameters of 100, 200, and 300 nm. (b, e, h, k) SEM image of PLA planar film, PLA-100, PLA-200, and PLA-300 platform. (c, f, i, l) SEM images of PLA planar film, PLA100, PLA-200, and PLA-300 oblique view of 45°. (m−x) Live/dead cellular staining of hADSCs after culturing on PLA film, PLA-100, PLA-200, and PLA-300 for 48 h. The live cells were stained green, and the dead cells were stained red. (y) Schematic view of hADSC spreading and growth. (z) Proliferation results of hADSCs cultured on PLA film compared to that on commercial culture plates.

ate nanopillars16 can enhance the differentiation of stem cells in a designated direction with growth factors or differentiationinducing media. Compared with growth factors and small organic molecule regulation of stem cell differentiation, nanotopography-regulated differentiation of stem cells only occurs on the interface between cells and nanostructured

structures can match the requirement for promoting osteogenic differentiation. The effects of nanopattern-based physical cues on osteogenic differentiation have great importance in biomedical research and tissue regeneration. As one of the typical nanoarrays, nanopillar arrays have been intensively investigated. Gold-coated silicon nanopillars21 and polycarbon2244

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Figure 2. Attachment of hADSCs on PLA nanopillar arrays with different pillar diameters after culturing for 48 h. (a−c, g−i, m−o, s−u) Fluorescence microscopy images of hADSC F-actin, vinculin, and nucleic staining after culturing for 48 h on PLA planar film and PLA nanopillar arrays with different pillar diameters, respectively. (d−f, j−l, p−r, v−x) SEM images of hADSCs after culturing for 48 h on PLA planar film and PLA nanopillar arrays with different pillar diameters. F-actin was stained by Alexa Fluor 568-labeled phalloidin (red); vinculin was stained by an Alexa Fluor 488-labeled antivinculin antibody (green), and cell nuclei were stained by Hoechst 33258 (blue).

materials, which can lead to localized differentiation and avoid the side effects induced by molecule diffusion into the surrounding tissues during tissue regeneration in vivo. In fact, many researchers have demonstrated the enhanced effect of nanopillar arrays on stem cell differentiation, which encouraged more researchers to explore their potential use in practical applications.22−24 Unfortunately, most studies on nanotopography regulation of stem cell fate have not provided evidence for their application in tissue regeneration for the following reasons: (1) Most studies are based on nonbiodegradable materials or degradable polymers that are not well-accepted,25,26 which could not be used as tissue scaffolds. (2) In most cases, the differentiation experiments are conducted with growth factors or differentiation media, making it difficult to judge the effect of nanotopography on differentiation.27,28 (3) Most of the studies are cell experiments in vitro without in vivo experimental data, making it difficult to judge the potential applications in tissue regeneration.29,30 Therefore, it is still unknown whether specific geometrical and mechanical signals can indeed affect bone regeneration in vivo and if they could potentially be applied to clinical applications in the future. Thorough experiments are urgently needed to explore whether the nanotopography of biodegradable materials can regulate stem cell fate without any growth factors or differentiation media in vitro and in vivo to assess the potential application of nanotopography-driven stem cell differentiation. In this study, the effect of nanotopography on stem cell differentiation was demonstrated by growth factor-free stem cell culture on biopolymer film with different nanopillar arrays. Polylactic acid (PLA), a US FDA certified biodegradable biomaterial, is used as a basic material for preparing biodegradable nanostructures. PLA nanopillar arrays with

different diameters but the same distribution density were prepared with a nanoimprint method31−34 using anodic aluminum oxide (AAO) nanowell arrays as templates to investigate the regulation effect of material nanotopological properties on stem cell differentiation. The nanopillar array samples with different diameters were used to assess the effect of nanostructures on stem cell differentiation without growth factors using tissue culture plates and smooth PLA film as controls. In this work, human adipose-derived stem cells (hADSCs), one of the most practical clinically applied autologous stem cells for tissue regeneration,35−37 were chosen as model cells to provide solid support for tissue regeneration application of nanopillar array-driven differentiation. The schematic diagram of the series of PLA nanopillar arrays with hADSCs cultured on the surface is shown in Scheme 1. In addition, an ectopic ossification experiment in nude mice was used to confirm the osteogenic differentiation by nanopillar arrays. Results and Discussion. Characterization of PLA Film and PLA Nanopillar Arrays. The nanopillar arrays with different pillar diameters were prepared on PLA film by a nanoimprint method with AAO nanowell arrays as templates. The surface topographies of the PLA planar film and PLA nanopillar arrays on the film surface were observed by SEM, and the SEM images are shown in Figure 1. Figure 1a shows the digital picture of the PLA planar film, and the surface of the as-prepared PLA film is quite smooth. (The digital picture of the PLA original film with a lower magnification is shown in Figure S1). Figure 1b,c shows the morphology of the PLA film surface, and the surface of the PLA planar film is still smooth even at a high resolution. Figure 1d,g,j shows the topography of a series of AAO templates with different dimensions, and Figure 2245

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hADSCs cultured on PLA-200 have an osteoblast-like polygonal morphology, but the cells on all of the other 3 samples maintain a spindle shape; the size of the spindles on PLA-100 is much smaller. The different morphology is also caused by different stretching resistance due to the different space between the neighboring nanopillars. Different mechanical resistances affect the cytoskeletal rearrangement of hADSCs and influence the final morphology, which may further influence gene expression in the nucleus.40 Further, from the results of vinculin staining, we found that vinculin fluorescent intensities are different among the different groups. Vinculin fluorescent intensities on PLA-200 and PLA-300 are higher than on PLA planar film and PLA-100. As reported in previous studies, vinculin plays a key role in stem cell differentiation, and more vinculin may indicate a greater osteogenic differentiation potential.41 Therefore, PLA-200 and PLA-300 may have more positive osteogenic results. To observe the cell adhesion and spread on different substrates more meticulously, SEM images of cells cultured for 48 h were obtained after alcohol gradient dehydration treatment. Due to the smooth surface of PLA planar film, hADSCs can spread naturally with flat pseudopodium and present a typical spindle shape consistent with the cytoskeleton staining (Figure 2d−f). When hADSCs were cultured on PLA100, the spread resistance was greatly increased, and the cell shape became more slender (Figure 2j−l). When hADSCs were cultured on PLA-200, causing less resistance compared with PLA-100, the cell shape changed to a polygon and the pseudopodium became wider, which was also consistent with the cytoskeleton staining (Figure 2p−r). Regarding hADSCs cultured on PLA-300, the resistance was less than on PLA-100 and PLA-200 but still larger than on PLA planar film. Therefore, most of the cells were still spindle shaped but were wider than on PLA planar film, and some of the cells were also polygon shaped (Figure 2v−x). From these results, it is clear that different nanopillar arrays have different effects on cell morphology and pseudopodium spread due to the difference in resistance and hADSC morphology and altered pseudopodium spread. Therefore, we can infer that PLA-200 and PLA-300 may improve the osteogenic differentiation of hADSCs. Considering both the results of cytoskeleton staining and SEM images, these phenomena indicate that the difference in nanopillar diameters can significantly influence the spreading area and morphologies of hADSCs and suggest that nanopillar arrays with different pillar diameters can cause different osteogenic differentiation effects according to the results above. Moreover, cell morphology changes to polygon-shaped cells after culturing with osteoblast-inducing conditional media for at least 1−2 weeks.22,42 However, from the results above, hADSCs cultured on PLA nanopillar arrays changed their morphology after culturing for only 48 h, which is faster than the osteoblast-inducing conditional media or BMP-2. Therefore, nanopillar array-mediated osteogenic differentiation may be more effective in stem cell morphology transformation than osteoblast-inducing conditional media. The results of cytoskeleton staining and the SEM images of hADSCs cultured on TCP are shown in Figure S3 as a blank control. Cell Viability and Proliferation of hADSCs. To investigate the cell viability and proliferation of hADSCs, live/dead staining and a CCK-8 assay were used to observe the ratio of live cells and dead cells and to evaluate cells cultured on the samples qualitatively and quantitatively. As shown in Figure 1m−x, there are almost no dead cells on the PLA planar films

1e−f,h−i,k−l, respectively, shows the nanotopography of PLA nanopillar arrays with pillar diameters of 100, 200, and 300 nm (defined as PLA-100, PLA-200, and PLA-300, respectively) constructed by the AAO template imprinting method. As shown in SEM images, the constructed PLA nanoarrays have nanopillars on the top and pyramids on the bottom. The pyramids of different nanopillar arrays are caused by the hexagonal pyramid morphology of the oxide unit cell of the AAO template, of which the center-to-center distance of the AAO template is 450 nm. The nanopillars on the top have different pillar diameters, which are, respectively, approximately 100, 200, and 300 nm, corresponding to different diameters of nanowells on the AAO templates. The heights of the nanopillars on the top are approximately 100 nm. Therefore, the only difference among the PLA nanopillar arrays is the nanopillar diameter, which provides suitable samples in the perfect model to study the influence of different nanopatterns on the differentiation of hADSCs. The digital photos of the PLA nanopillar array samples are shown in Figure S1. The samples’ size is approximately 2 cm2 × 2 cm2, and the nanoarray construction on the surface did not affect the visible smoothness and transparency of the PLA films. Adhesion and Spreading Morphologies of hADSCs. The hADSCs were cultured on different PLA nanopillar arrays to evaluate the effect of nanopillar diameter on cell adhesion and proliferation. The actin immunofluorescent staining results of hADSCs cultured on PLA nanopillar arrays and PLA planar film after 48 h are shown in Figure 2. The cytoskeleton of hADSCs was stained red by phalloidin-Alexa Fluor 568; vinculin was stained green by an antivinculin antibody (Alexa Fluor 488), and the nuclei were stained blue by Hoechst 33258. The results of the immunofluorescent staining show that the adhesion of hADSCs on PLA films is normal, but the cell spreading areas and morphologies on the different substrates are quite different. Compared with hADSCs on PLA planar films, hADSC spreading on PLA-100 and PLA-200 seems to be limited, and cell spreading areas were generally smaller than on PLA-300. The possible reason could be the different spaces between the neighboring pillars of the different nanopillar arrays.38,39 The space between neighboring pillars on PLA-300 is only 150 nm, which is equivalent and smaller than the pseudopodium of cells. Therefore, cells can easily spread across the spaces between neighboring nanopillars, thereby having little influence on the adhesion and migration of cells. For PLA-100, the diameter of the nanopillars is 100 nm, and the space between the neighboring nanopillars is 350 nm, which is difficult for hADSCs to cross and reach another rod. Therefore, cell migration should be difficult. In addition, the attachment area is limited because the diameter of the pillars on PLA-100 is just 100 nm, leading to the small spreading area of the cells on PLA100. For PLA-200, the diameter of the pillars is 200 nm, and the space between two pillars is 250 nm, which is between PLA300 and PLA-100. Therefore, the spreading area of hADSCs on PLA-200 is between that on PLA-100 and PLA-300. The quantified cell spreading area is shown in Figure S4. Based on the results of previous studies,10 the attachment of the cells not only affects the migration behavior of the stem cells but also affects the differentiation tendency of the stem cells due to the topological signal of the material surface. In addition to spreading areas, the morphologies of hADSCs have apparent differences among each group of samples. As shown in Figure 2a,g,m,s,c,i,o,u, after being cultured for 48 h, 2246

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Figure 3. Osteogenic differentiation of hADSCs cultured on a culture plate, PLA planar film, and PLA nanopillar arrays with different pillar diameters. (a) ALP activity of hADSCs on day 7, 14, and 21, normalized to total protein concentration. (b−d) Q-PCR analysis of the expression of osteoblast-specific genes (b) Runx-2, (c) OPN, and (d) OCN on day 7, 14, and 21, normalized to actin expression. (e) Calcium nodules stained by Alizarin Red S show extracellular calcium deposits by hADSC-derived osteoblasts on day 21. All data represent the mean ± standard deviation (n = 3). 0.01 < *p < 0.05, **p < 0.01.

in surface resistance derived from nanoarrays with different nanopillar diameters. The cell spreading and growth schematic image is shown in Figure 1y. Cell proliferation is therefore hampered by different degrees of surface resistance. From the cell viability result on day 5, we observed that cell proliferation is hampered by PLA nanopillar arrays at the 100 nm diameter due to the greatest resistance on the PLA-100 surface, and the cell number increases by only less than 5% of that on day 3, which is consistent with the results of hADSC spread mentioned above. Previous studies have shown that the decrease of cell proliferation is generally related to cell apoptosis and cell differentiation. Therefore, the hampered proliferation of hADSCs on PLA nanopillar arrays may also suggest improved osteogenic differentiation.43,44

and less than 5% of dead cells on PLA nanopillar array samples with different pillar diameters after culturing for 48 h, indicating the cytocompatibility of PLA films before and after the construction of superficial nanopatterns. The results of live/ dead staining of hADSCs cultured on TCP are shown in Figure S3 as a blank control. From the CCK-8 assay results shown in Figure 1z, the hADSCs cultured on these samples have a proliferation trend from day 1 to day 3, and the cell number on day 3 is more than twice that on day 1, indicating good biocompatibility of both PLA planar and nanopillar array samples. On day 5, hADSCs on TCP and planar PLA film continue to proliferate. Inversely, hADSCs on PLA nanopillar array samples have almost stopped proliferating. This result should be attributed to the difference 2247

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Figure 4. Immunofluorescence staining of osteogenic markers. (a−j) OPN of hADSCs on commercial culture plates, PLA planar film, and PLA nanopillar arrays with different diameters after 21 days of culture. (k−t) OCN of hADSCs on commercial culture plates, PLA planar film, and PLA nanopillar arrays with different diameters after 21 days of culture. Cell nuclei were stained blue by Hoechst 33258, and F-actin was stained red by phalloidin-Alexa Fluor 568. OPN and OCN were stained green.

activity in hADSCs on PLA-200 corresponds to the result of the most significant cell morphology transformation in this group (shown in Figure 2m−o). To further investigate the osteogenic differentiation degree, quantitative polymerase chain reaction (q-PCR) was used to assess the differentiation at the genetic level by measuring the relative RNA content in hADSCs cultured on different PLA substrates. Runx2, which regulates a number of other genes associated with osteogenesis, is a specific transcription factor expressed during osteogenic differentiation. Osteopontin (OPN) is a type of glycoprotein and plays a key role in the mineralization process of bone matrix during bone metabolism. Osteocalcin (OCN) is another important protein during osteogenic differentiation and is considered a marker in the mineralization period. Therefore, the expressions of Runx2, OPN, and OCN are commonly used as markers for osteogenesis characterization.48,49 The three osteogenic marker genes of hADSCs were examined on days 7, 14, and 21 by qPCR. As shown in Figure 3b, the expression level of Runx2 in hADSCs cultured on PLA-200 is the most up-regulated. After culturing for 21 days, the Rux2 expression level is almost 2.5fold compared to hADSCs cultured on a culture plate for 7 days. Similarly, as shown in Figure 3c, the OPN expression level of hADSCs cultured on PLA-200 is the most up-regulated on day 7 and 14 and is almost 3-fold compared to hADSCs cultured on a culture plate for 7 days, but on day 21, the expression level of cells on PLA-200 is surpassed by cells cultured on PLA-100. The increased expression of Runx2 and OPN is lost on day 21, potentially because the genes are relatively early markers during osteogenic differentiation. The expression of OCN in hADSCs cultured on PLA-200 is upregulated, and after culturing for 21 days, the expression level

Summarizing all of the results of the cytoskeleton, cell viability, and proliferation, hADSCs cultured on these samples have good cell viabilities and proliferation in general. However, the differences in adhesion and spreading morphologies are obvious. According to previous reports, differences in cell adhesion and morphology may be closely associated with stem cell differentiation.45,46 Therefore, we hypothesize that PLA nanopillar arrays with different diameters may have different effects on hADSC osteogenic differentiation, among which PLA-200 may have the most positive effect according to the morphology transformation results. Osteogenic Differentiation of hADSCs. To investigate the effect of different PLA nanopillar arrays with different diameters on osteogenic differentiation of hADSCs, cells were cultured on a series of different PLA films with different surface nanopatterns (PLA film, PLA-100, PLA-200, and PLA-300) in culture medium without osteogenic supplements for different time periods. In addition, culture plates were used as a blank control group. To evaluate the osteogenesis of hADSCs, typical osteogenic markers at protein, gene, and functional levels were characterized. Alkaline phosphatase (ALP) is an important early phenotypic marker for the osteogenic differentiation of stem cells.47 Therefore, detection of ALP activity is essential to evaluate the osteogenic differentiation degree of hADSCs. Figure 3a shows the relative ALP protein activity, normalized to total protein content of hADSCs cultured on different PLA substrates. On day 14, the ALP activity of hADSCs on all samples increased dramatically to approximately twice that of cells on day 7, among which hADSCs cultured on PLA-200 showed the highest ALP activity. However, on day 21, ALP activity of hADSCs on all substrates decreased because ALP is an early marker of osteogenic differentiation. The greatest ALP 2248

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Figure 5. ALP immunohistochemical staining and H&E staining of an ectopic osteogenesis tissue slice. (a) ALP immunohistochemical staining result of tissue slices above a PLA planar film scaffold. (b) ALP immunohistochemical staining result of tissue slices above a PLA-200 scaffold. (c) H&E staining result of tissue slices above a PLA planar film scaffold. (d) H&E staining result of tissue slices above a PLA-200 scaffold. M represents muscle tissue. F represents fibrocollagenous connective tissue. C represents calcification area. The red dashed line represents the interface between new tissue and the PLA film. The green circles with dashed lines represent the location of blood vessels.

increased to approximately 3.5-fold compared to hADSCs cultured on a culture plate for 7 days (Figure 3d). These results indicate that hADSCs cultured on PLA-200 have higher osteogenesis-related gene expression than those on other PLA substrates and culture plates, demonstrating that PLA200 can further promote the osteogenic differentiation of hADSCs. Further, based on the expression levels of Runx-2, OPN, and OCN, hADSCs cultured on PLA-100 and PLA-300 also exhibit osteogenic differentiation to some degree, albeit weaker than on PLA-200. Alizarin Red S staining is another commonly used functional indicator of osteoblasts.50,51 Calcium nodules caused by cell calcium secretion can be specifically bound with Alizarin Red S and turn red, a widely used visualization marker of osteogenesis. After culturing for 21 days, the mineralized nodules of hADSCs on these substrates were stained by Alizarin Red S to evaluate the degree of calcium deposit. As shown in Figure 3e, there are significantly more mineralized calcium nodules on PLA-200 than the other groups. The percentage of positive Alizarin Red S staining area was quantified by ImageJ, and the results are shown in Figure S5. The calcification area % of PLA-200 is the largest at 44.52%. This result further indicates that PLA-200 promotes the most effective osteogenic differentiation. The above results have demonstrated that PLA-200 can promote the osteogenic differentiation of hADSCs to the greatest degree, and PLA-100 and PLA-300 can also promote osteogenic differentiation to some degree according to the results of q-PCR. This result required further investigation via protein expression analysis. Therefore, immunofluorescence staining of typical bone-specific proteins OPN and OCN was used to detect the protein expression of hADSCs cultured on different substrates after 21 days of culture. As shown in Figure 4, the OPN immunofluorescence intensity of hADSCs cultured on PLA-200 is much stronger than cells cultured on other PLA substrates or culture plates, indicating that the hADSCs on PLA-200 have a higher OPN protein expression. Similarly, the

OCN immunofluorescence images of hADSCs cultured on different substrates show the same result as those of OPN. OCN expression is mostly promoted by PLA-200 and is secondarily promoted by PLA-300. Both the results of OPN and OCN expression in cells after culturing for 21 days further suggest that PLA-200 can promote osteogenic differentiation of hADSCs to the greatest degree. In addition, the morphology transformation of hADSCs on PLA-200 is also quite obvious compared to other groups after 21 days of culture. hADSCs on PLA-200 are more polygon in shape, while cells cultured on culture plates, PLA planar film, and PLA-100 are still fibrous. hADSCs cultured on PLA-300 also display a morphological transformation, which was not as obvious as on PLA-200. This phenomenon corresponds to the morphology transformation after 48 h caused by a different surface resistance (shown in Figure 2), and we conclude that PLA-200 can promote the osteogenic differentiation of hADSCs in vitro to the greatest degree. To assess the in vivo ability of the PLA nanoarray with a reasonable nanopillar diameter to promote osteogenesis, we used PLA-200 as the experimental group and PLA planar film as a control group to serve as ectopic osteogenic scaffolds. After 4 weeks of subcutaneous implantation, PLA scaffolds were removed from the implantation sites. After being frozen and sectioned, the tissue slices were immunohistochemically stained for CD3 (for T cells) and CD11c (for macrophages) to assess the inflammatory response, and we found that there was almost no inflammation (Figure S6). The osteogenesis in the tissue slices were assessed by ALP (brown color) immunohistochemical staining and H&E staining.52−55 In Figure 5, no inflammation on the interface of the PLA and the tissue was observed. A membrane structure formed on the surface of the PLA film scaffold, which permeated the interspace between the PLA and the tissue. The membrane creates a close relationship between the PLA film scaffold and the subcutaneous tissue, which helps stabilize the osteogenic environment. Moreover, 2249

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X-100, bovine serum albumin, goat serum, and Alizarin Red were obtained from Sigma. Alexa Fluor 568, Hochest 33258, and Trizol Reagent were obtained from Invitrogen. OPN and OCN primary and secondary antibodies were obtained from Abcam. Runx2, OPN, and OCN primers for q-PCR were obtained from Sangon Biotech Co., Ltd. (Shanghai). α-MEM, fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco. Construction of PLA Nanopillar Arrays. One gram of PLA powder was dissolved in 10 mL of dichloromethane with mechanical stirring, and the mixture was poured into a 90 mm glass culture dish. After standing overnight, the PLA film was peeled off gently. After cutting into 2 × 2 cm2 pieces, the PLA film piece was overlapped by an AAO template on the right side and uniaxially pressed at 20 MPa by a desktop powder presser. Then, the PLA film was peeled off from the AAO template, and the nanostructured film nanopillar arrays on surface of one side were achieved. The nanopatterned PLA films were cut into 1 × 1 cm2 pieces to be used in further experiments. The AAO templates and constructed PLA nanopillar arrays were characterized by a HITACHI S-4800 scanning electron microscope. The nanowell diameters of a series of AAO nanowell templates are, respectively, 100, 200, and 300 nm with the same center-to-center distance of 450 nm. Therefore, the PLA nanopillar arrays have pillar diameters of approximately 100, 200, and 300 nm with the same center-to-center distance of approximately 450 nm. In this study, we defined the series of PLA nanopillar arrays as PLA-100, PLA-200, and PLA-300 according to the different diameters. Tissue culture plates and smooth PLA films were used as controls. Human Adipose-Derived Stem Cells (hADSCs) Isolation, Characterization, and Cultivation. hADSC was a gift from the Qilu Hospital of Shandong University in Jinan. The use of hADSCs was approved by the Ethics Committee of Shandong University, Qilu Hospital, and written informed consent was obtained from all participants. Cells were cultured in a primary medium containing α-MEM supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO2 in air at 37 °C. Third-passage hADSCs were used for all of the experiments. hADSCs Viability, Proliferation, and Spreading on PLA Nanopillar Arrays and PLA Planar Film. The PLA nanopillar arrays and PLA planar film samples were first sterilized in 75% ethanol overnight and then washed three times using sterile Hanks’ Balanced Salt Solution (HBSS). Then, 0.1 mL of the cell suspension containing 2 × 104 cells was seeded on the samples in 24-well plates, and 1 mL of the complete medium was added. To investigate the cell attachment to the PLA nanopillar arrays and PLA planar film, after culturing for 48 h, the hADSCs were imaged by immunofluorescence measurements of F-actin, vinculin, and their nuclei. After washing with PBS three times, hADSCs were fixed with a 4% paraformaldehyde solution for 5 min and washed with PBS three times. Then, after permeation with 0.1% Triton X-100 for 3 min and blocking with 1% bovine serum albumin for 30 min, cells were stained with an antivinculin antibody labeled by Alexa Fluor 488 at a 1:100 dilution for 3 h and phalloidin-conjugated Alexa Fluor 568 at a 1:200 dilution for 30 min and incubated with Hoechst 33258 for 5 min to stain the nuclei. Finally, after washing with PBS three times, hADSCs were examined under an inverted fluorescence microscope (Olympus IX71). To investigate the survival of hADSCs on PLA films, live/ dead staining was used to visually evaluate the survival status of

after the differentiation of hADSCs, an enriched vascular network was observed (green circles with broken line), which can provide sufficient nutrients for osteogenesis. Compared with the ALP immunohistochemical staining result of the PLA planar film, PLA-200 supported tissue slices exhibit a higher concentration of ALP, indicating improved osteogenesis of the tissue on the PLA-200 scaffold. Although there is no osseous structure observed by H&E staining, some bone-like microstructures appear above PLA-200. In contrast, almost no bonelike structure was observed on the PLA planar film. Therefore, from the results of ectopic osteogenesis in vivo, we conclude that compared with the PLA planar film, PLA-200 has an improved ability to promote osteogenesis. To the best of our knowledge, this is the first investigation of the effect of nanopillar array structure with specific geometrical and mechanical signals on osteogenesis in vivo. This result provides powerful guidance for osteogenesis and bone regeneration in tissue engineering and regenerative medicine. Conclusion. In conclusion, we have demonstrated that the fate of hADSCs cultured on PLA nanopillar arrays can be determined by the nanopillar diameter without any differentiation growth factors. The osteogenic differentiation of hADSCs can be driven by nanopillar arrays on the surface of PLA film after culturing for 7, 14, and 21 days, which is as efficient as BMP-2. Compared with PLA-100 and PLA-300, cells cultured on PLA-200 are more likely to change their shape to a polygon morphology even within 48 h. The assessment of gene expression and protein characterization further confirmed that PLA-200 can mostly drive osteogenic differentiation of hADSCs without extra osteogenic growth factors. PLA-100 and PLA-300 can also promote osteogenesis of hADSCs to some degree, although the effects are weaker than that of PLA-200. Moreover, an in vivo animal model using the samples demonstrated that PLA film with a 200 nm pillar array exhibits an improved ectopic osteogenesis ability compared with planar PLA film after 4 weeks of ectopic implantation. The reliable evidence for nanopillar array-driven osteogenic differentiation of stem cells in vitro and in vivo confirmed that nanotopography can regulate the fate of stem cells and fine-tune the bone regeneration process. This work demonstrated that the construction of well-designed nanostructures on biopolymer material surfaces can be used as tissue engineering scaffolds, leading to safe and rapid tissue restoration, which will have widespread applications in bone tissue engineering and regenerative medicine. Most importantly, this work is a powerful guide for clinical research fields of bone regeneration. PLA film with nanopillar arrays on the surface is suitable for the regeneration of bone and large areas periodontal erosion due to the observed bone regeneration effect. In addition, the other side of PLA film with no nanoarrays on the surface has a low osteogenic efficiency, which prevents other tissues from sticking to the newly formed bone. Therefore, this work paves the way for the application of degradable biopolymer nanoarrays with specific geometrical and mechanical signals in clinical bone regeneration. Methods. In this work, we aimed to fabricate a series of PLA nanopillar arrays on the surface of PLA film with different pillar diameters and the same center-to-center distance. Materials. AAO templates in pieces of 2 × 2 cm2 were obtained from Shenzhen Topmembranes Technology Co., Ltd. (China). PLA with a molecular weight of 500 000 was obtained from Jinan Daigang Biomaterial Co., Ltd. Dichloromethane was obtained from Sinopharm Chemical Reagent Co., Ltd. Triton 2250

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culture plate were fixed in 4% paraformaldehyde for 30 min and then stained with 2% Alizarin Red at pH 4.2 for 10 min. After rinsing with distilled water three times, the Alizarin Red S stained samples were viewed by a digital camera Immunofluorescence Staining. After culturing for 21 days, the hADSCs on different samples were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. Then, they were permeabilized using 0.1% Triton X-100 for 10 min and blocked with 10% goat serum solution for 30 min at room temperature. After blocking, cells were incubated overnight at 4 °C with primary antibodies at 1:500 dilution against osteocalcin (mouse monoclonal anti-OCN, Abcam) and at 1:1000 dilution against osteopontin (rabbit polyclonal antiOPN, Abcam). Goat antirabbit and goat antimouse secondary antibodies labeled by Alexa Fluor 488 at 1:200 dilutions in 5% goat serum solution were used for staining OPN and OCN for 1 h at room temperature, respectively. After rinsing off the second antibody with PBS, the cells were further stained by phalloidin-conjugated Alexa Fluor 568 for 30 min and Hochest 33258 for 5 min to stain the F-actin and nuclei, respectively. Images of the stained samples were observed with an Olympus IX71 inverted microscope. Ectopic Implantation of hADSC-Seeded PLA Scaffolds. Animal studies were implemented according to the guidelines of the Institutional Animal Care and Use Committee of Shandong University. A subcutaneous ectopic model was prepared using 6-week-old SCID mice (Nara Biotech, Seoul, Korea). A total of 10 male mice were divided into two groups (n = 5 per group): (1) PLA planar film scaffold (control) and (2) PLA-200 scaffold. Prior to sample implantation, hADSCs (cell number: 2 × 104) were seeded onto individual PLA scaffolds and precultured for 3 days. The samples were subcutaneously implanted into the back of the mice after anesthesia with chloral hydrate (1 mL dose per mice); animals were sacrificed via overdose of anesthesia after 4 weeks of implantation. Histological Analysis. Harvested samples were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin wax, and cut in 5 mm thick sections. The thin sections were then subjected to ALP immunohistochemical staining and hematoxylin/eosin (H&E) staining according to the instructions. The osteogenic potential of the implanted PLA film scaffolds was evaluated using ImageJ (NIH) to measure the area of mineralized matrix from the von Kossa images (×200 magnification). Statistical Analysis. Statistical analysis was performed using one-way ANOVA in GraphPad Instant software (GraphPad Software). Data were reported as the mean standard deviations, and statistical significance was accepted at P < 0.05.

cells. After incubation for 48 h in 24-well plates, the culture medium was replaced by 200 μL of serum-free α-MEM medium containing 0.5 μM calcein AM and 3 μM propidium iodide (PI), respectively. After incubation for 15 min at 37 °C, the cells were washed three times with PBS and examined under an inverted fluorescence microscope. To further observe the attachment of the hADSCs on these PLA samples, after 48 h of incubation, the MSCs were washed with PBS for three times and then fixed with 2.5% glutaraldehyde overnight. Then cells were dehydrated using a series of alcohol solutions (30%, 50%, 70%, 80%, 90%, 95%, 98%, and 100%) and lyophilized at −60 °C for 6 h. Finally, the cells were observed under a HITACHI S-4800 scanning electron microscope after 50 s of Au spraying at a current of 20 uA. A Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technology) was used to quantitatively evaluate the cell proliferation on PLA nanopillar arrays, PLA planar films, and culture plates after cultivation for 1, 3, and 5 days in 96-well plates. First, the culture medium was replaced with 100 μL of serum-free α-MEM medium plus 10% CCK-8 solution per well. After 1.5 h of incubation at 37 °C, the production of watersoluble formazan dye was assayed at a wavelength of 450 nm by a microplate reader (Multiscan MK3; Thermo Fisher Scientific, Inc.). Three parallel replicates for each group were performed. Osteogenic Differentiation of ADSCs. To investigate the osteogenic differentiation of hADSCs on the PLA nanopillar arrays, PLA planar films, and culture plates, the cells were seeded on the samples and incubated for 7, 14, and 21 days in a primary complete culture medium. The culture medium was changed every 2 days during the experiments. Total Intracellular Protein Content and Alkaline Phosphatase (ALP) Activity. After culturing for 7, 14, and 21 days, the hADSCs on different samples were digested from the samples and lysed by cell lysis buffer for total intracellular protein content and ALP activity assays. The total intracellular protein content was assayed using a micro BCA protein assay kit (Thermo Scientific) by measuring the absorbance of the reaction solution at 570 nm. The ALP activity was analyzed using a Lab Assay ALP activity assay kit (Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s instructions. Briefly, p-nitrophenyl phosphate (pNPP) was used as a substrate for ALP, to be hydrolyzed into p-nitrophenol and phosphoric acid in the carbonate buffer (pH 9.8) at 37 °C. The staining reaction was terminated by the addition of 0.2 M NaOH, and the released p-nitrophenol was a yellow color optically measured at a 405 nm wavelength. The relative ALP activity was normalized to the protein content of cells cultured on different samples (n = 3 for each group). Quantitative PCR (q-PCR). After culturing for 7, 14, and 21 days, the hADSCs on different samples were treated with Trizol Reagent (Invitrogen) to extract the total RNA. The total RNA concentration and purity were determined using a Q-5000 spectrophotometer (Quawell) at 260/280 nm. The q-PCR analysis was performed using the 7500 Real Time PCR system (Applied Biosystems) for one housekeeping gene, β-actin, and the three genes of interest, Runx2, osteocalcin (OCN), and osteopontin (OPN) (Table S1 for primer sequence, Supporting Information). The relative transcript levels of the target gene were normalized to β-actin and expressed as the mean ± SD (n = 3 for each group). Alizarin Red S Staining. After 21 days of cultivation, the hADSCs on the PLA nanopillar arrays, PLA planar films, and

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b04747. Additional information and figures, including the photo of PLA planar films, surface roughness of PLA samples, hADSCs viability on TCP, quantification results of spreading areas and Alizarin Red staining, inflammation immunohistochemical staining of tissue slices, and oligonucleotide sequences of q-PCR primers (PDF) 2251

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Tel: +86-531-88362807. ORCID

Yuanhua Sang: 0000-0001-7989-7014 Duo Liu: 0000-0002-5503-2566 Hong Liu: 0000-0003-1640-9620 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant nos. 51372142 and 51402063), the Fundamental Research Funds of Shandong University (2014QY003-09, 2014JC019), and the Program of Introducing Talents of Discipline to Universities in China (111 Program no. b06015), Shandong University-Karolinska Institute Collaborative Laboratory for Stem Cell Research. Many thanks to Doctor Dong Li from Qilu Hospital for the supply of hADSC.

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