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Poly-lactic 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 Nano Lett., Just Accepted Manuscript • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018
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Poly-lactic
array-driven
osteogenic
2
differentiation of human adipose-derived stem cells determined
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by pillar diameter
4
Shan Zhang1, Baojin Ma1, Feng Liu1, Jiazhi Duan1, Shicai Wang1, Jichuan Qiu1,
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Dong Li2, Yuanhua Sang1, Chao Liu3*, Duo Liu1* and Hong Liu1,4*
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1
7
China
8
2
9
China
State Key Laboratory of Crystal Materials, Shandong University, Jinan, 250100,
Cryomedicine Laboratory, Qilu Hospital, Shandong University, Jinan, 250012,
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3
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Stomatology, Shandong University, Jinan, 250012, China
12
4
Institute for Advanced Interdisciplinary Research, Jinan University, 250022, China
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*
Corresponding Author.
Department of Oral and Maxillofacial surgery, Qilu Hospital, Institute of
Hong Liu,
[email protected];
14
Duo Liu,
[email protected];
15
Chao Liu,
[email protected] 16 17 18 19 20 21 22 1
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Abstract
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Numerous studies have determined that physical cues, especially the nanotopography
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of materials, play key roles in directing stem cell differentiation. However, most
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research on nanoarrays for stem cell fate regulation is based on non-biodegradable
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materials, such as silicon wafers, TiO2, and poly(methyl methacrylate), which are
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rarely used as tissue engineering biomaterials. In this study, we prepared
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biodegradable poly-lactic acid (PLA) nanopillar arrays with different diameters but
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the same center-to-center distance using a series of anodic aluminum oxide nano-well
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arrays as templates. Human adipose-derived stem cells (hADSCs) were selected to
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investigate the effect of the diameter of PLA nanopillar arrays on stem cell
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differentiation. By culturing hADSCs without the assistance of any growth factors or
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osteogenic-induced media, the differentiation tendencies of hADSCs on the nanopillar
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arrays were assessed at the gene and protein levels. The assessment results suggested
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that the osteogenic differentiation of hADSCs can be driven by nanopillar arrays,
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especially by nanopillar arrays with a diameter of 200 nm. Moreover, an in vivo
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animal model of the samples demonstrated that PLA film with the 200 nm pillar array
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exhibits improved ectopic osteogenic ability compared with the planar PLA film after
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4 weeks of ectopic implantation. This study has provided a new variable to investigate
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in the interaction between stem cells and nanoarray structures, which will guide the
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bone regeneration clinical research field. This work paves the way for the utility of
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degradable biopolymer nanoarrays with specific geometrical and mechanical signals 2
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in biomedical applications, such as patches and strips for spine fusion, bone crack
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repair, and restoration of tooth enamel.
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Keywords: poly-lactic acid nanopillar array; nanopillar diameter; adipose-derived
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stem cells; osteogenic differentiation
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Introduction
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Over the years, using physicochemical signals to regulate stem cell fate has become a
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hot research topic in tissue engineering and regenerative medicine.1-3 Biochemical
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factors have been widely used to induce osteogenic differentiation with good
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efficiency. Using bone morphogenetic protein-2 (BMP-2) as an example, after an
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induction period from 7 days to 31 days, BMP-2 (10-100 ng/ml) can effectively
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induce bone nodule formation and calcium deposition compared with a control
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group.4-6 Compared with biochemical growth factors, physical cues that participate in
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stem cell differentiation are also efficient and have almost no potential side effects in
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human bodies.7,8 The most widely used physical cues include light9, magnetism10,
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electricity11 and surface topology of materials12,13. However, the stimulations from
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light, magnetism and electricity are external factors that do not exist in vivo and are
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generally unwanted for surrounding tissue engineering. Therefore, surface topology
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based on nanostructures of materials is the most preferred cue for tissue regeneration
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therapies in the future due to their biological stability and health safety profile. 14,15
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Nanopattern construction has become a major tool for providing specific geometrical
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and mechanical signals on material surfaces, which can potentially elicit a stem cell 3
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response. Many different nanopatterns have been designed and prepared to promote
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stem cell differentiation, especially osteogenic differentiation. For example, the
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effects
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arginine-glycine-aspartic acid (RGD) nanopatterns17,18, and the aspect ratio of
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nanopillar arrays19,20 have been studied. These studies have demonstrated that only
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nanopatterns with special structures can match the requirement for promoting
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osteogenic differentiation. The effects of nanopattern-based physical cues on
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osteogenic differentiation have great importance in biomedical research and tissue
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regeneration. As one of the typical nanoarrays, nanopillar arrays have been intensively
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investigated. Gold-coated silicon nanopillars21 and polycarbonate nanopillar16 can
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enhance the differentiation of stem cells in a designated direction with growth factors
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or differentiation inducing media. Compared with growth factors and small organic
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molecule
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differentiation of stem cells only occurs on the interface between cells and
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nanostructured materials, which can lead to localized differentiation and avoid the
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side effects induced by molecule diffusion into the surrounding tissues during tissue
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regeneration in vivo. In fact, many researchers have demonstrated the enhanced effect
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of nanopillar arrays on stem cell differentiation, which encouraged more researchers
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to explore their potential use in practical applications.22-24 Unfortunately, most studies
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on nanotopography regulation of stem cell fate have not provided evidence for their
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application in tissue regeneration for the following reasons: 1. Most studies are based
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on non-biodegradable materials or degradable polymers that are not well-accepted25,26,
of
the
disorder
regulation
of
degree
stem
cell
of
nanopit
arrays16,
differentiation,
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nanospacing
of
nanotopography-regulated
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which could not be used as tissue scaffolds; 2. In most cases, the differentiation
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experiments are conducted with growth factors or differentiation media, making it
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difficult to judge the effect of nanotopography on differentiation;27,28 3. Most of the
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studies are cell experiments in vitro without in vivo experimental data, making it
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difficult to judge the potential applications in tissue regeneration.29,30 Therefore, it is
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still unknown whether specific geometrical and mechanical signals can indeed affect
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bone regeneration in vivo and if they could potentially be applied to clinical
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applications in the future. Thorough experiments are urgently needed to explore
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whether the nanotopography of biodegradable materials can regulate stem cell fate
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without any growth factors or differentiation media in vitro and in vivo to assess the
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potential application of nanotopography-driven stem cell differentiation.
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In this study, the effect of nanotopography on stem cell differentiation was
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demonstrated by growth factor-free stem cell culture on biopolymer film with
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different nanopillar arrays. Polylactic acid (PLA), a US FDA certified biodegradable
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biomaterial, is used as a basic material for preparing biodegradable nanostructures.
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PLA nanopillar arrays with different diameters but the same distribution density were
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prepared with a nanoimprint method31-34 using anodic aluminum oxide (AAO)
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nano-well arrays as templates to investigate the regulation effect of material
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nanotopological properties on stem cell differentiation. The nanopillar array samples
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with different diameters were used to assess the effect of nanostructures on stem cell
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differentiation without growth factors using tissue culture plates and smooth PLA film
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as controls. In this work, human adipose-derived stem cells (hADSCs), one of the 5
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most practical clinically applied autologous stem cells for tissue regeneration35-37,
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were chosen as model cells to provide solid support for tissue regeneration application
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of nanopillar array-driven differentiation. The schematic diagram of the series of PLA
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nanopillar arrays with hADSCs cultured on the surface is shown in Scheme 1. In
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addition, an ectopic ossification experiment in nude mice was used to confirm the
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osteogenic differentiation by nanopillar arrays.
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Scheme 1. Schematic illustration of the series of PLA nanopillar arrays on the surface
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of PLA film with different pillar diameters but the same center-to-center distance with
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stem cells cultured on the surface.
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Results and discussion
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Characterization of PLA film and PLA nanopillar arrays
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The nanopillar arrays with different pillar diameters were prepared on PLA film by a
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nanoimprint method with AAO nano-well arrays as templates. The surface
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topographies of the PLA planar film and PLA nanopillar arrays on the film surface
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were observed by SEM and the SEM images are shown in Figure 1. Figure 1a shows 6
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the digital picture of the PLA planar film, and the surface of as-prepared PLA film is
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quite smooth (the digital picture of the PLA original film with a lower magnification
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is shown in Figure S1). Figure 1b and c show the morphology of the PLA film surface,
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and the surface of the PLA planar film is still smooth even at a high resolution. Figure
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1d, g and j show the topography of a series of AAO templates with different
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dimensions, and Figure 1e-f, 1h-i and 1k-l, respectively, show the nanotopography of
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PLA nanopillar arrays with pillar diameters of 100 nm, 200 nm and 300 nm (defined
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as PLA-100, PLA-200 and PLA-300, respectively) constructed by the AAO template
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imprinting method. As shown in SEM images, the constructed PLA nanoarrays have
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nanopillars on the top and pyramids on the bottom. The pyramids of different
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nanopillar arrays are caused by the hexagonal pyramid morphology of the oxide unit
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cell of the AAO template, of which the center-to-center distance of the AAO template
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is 450 nm. The nanopillars on the top have different pillar diameters, which are,
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respectively, approximately 100 nm, 200 nm, and 300 nm, corresponding to different
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diameters of nano-wells on the AAO templates. The heights of the nanopillars on the
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top are approximately 100 nm. Therefore, the only difference among the PLA
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nanopillar arrays is the nanopillar diameter, which provides suitable samples in the
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perfect model to study the influence of different nanopatterns on the differentiation of
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hADSCs. The digital photos of the PLA nanopillar array samples are shown in Figure
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S1. The samples’ size is approximately 2×2 cm2, and the nanoarray construction on
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the surface did not affect the visible smoothness and transparency of the PLA films.
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Figure 1. Characterization of the PLA planar film and PLA nanopillar arrays, and the
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cell viability of hADSCs after culturing on different samples for several days. a.
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Digital photo of PLA planar film. d, g, j. SEM images of the AAO template platform
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with different nanopit diameters of 100, 200 and 300 nm. b, e, h, k. SEM image of
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PLA planar film, PLA-100, PLA-200 and PLA-300 platform. c, f, i, l. SEM images of
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PLA planar film, PLA-100, PLA-200 and PLA-300 oblique view of 45°. m-x.
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Live/dead cellular staining of hADSCs after culturing on PLA film, PLA-100,
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PLA-200, and PLA-300 for 48 hours. The live cells were stained green, and dead cells
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were stained red. y. Schematic of hADSC spreading and growth. z. Proliferation
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results of hADSCs cultured on PLA film compared to that on commercial culture
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plates.
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Adhesion and spreading morphologies of hADSCs 8
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hADSCs were cultured on different PLA nanopillar arrays to evaluate the effect of
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nanopillar diameter on cell adhesion and proliferation. Actin immunofluorescent
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staining results of hADSCs cultured on PLA nanopillar arrays and PLA planar film
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after 48 hours are shown in Figure 2. The cytoskeleton of hADSCs was stained red by
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phalloidin-Alexa Fluor 568, vinculin was stained green by an anti-vinculin antibody
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(Alexa Fluor® 488) and the nuclei were stained blue by Hoechst 33258. The results of
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the immunofluorescent staining show that the adhesion of hADSCs on PLA films is
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normal, but the cell spreading areas and morphologies on the different substrates are
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quite different.
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Compared with hADSCs on PLA planar films, hADSC spreading on PLA-100 and
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PLA-200 seems to be limited, and cell spreading areas were generally smaller than on
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PLA-300. The possible reason could be the different spaces between the neighboring
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pillars of the different nanopillar arrays.38,39 The space between neighboring pillars on
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PLA-300 is only 150 nm, which is equivalent and smaller than the pseudopodium of
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cells. Therefore, cells can easily spread across the spaces between neighboring
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nanopillars, thereby having little influence on the adhesion and migration of cells. For
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PLA-100, the diameter of the nanopillars is 100 nm, and the space between the
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neighboring nanopillars is 350 nm, which is difficult for hADSCs to cross and reach
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another rod. Therefore, cell migration should be difficult. In addition, the attachment
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area is limited because the diameter of the pillars on PLA-100 is just 100 nm, leading
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to the small spreading area of the cells on PLA-100. For PLA-200, the diameter of the
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pillars is 200 nm, and the space between two pillars is 250 nm, which is between 9
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PLA-300 and PLA-100. Therefore, the spreading area of hADSCs on PLA-200 is
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between that on PLA-100 and PLA-300. The quantified cell spreading area is shown
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in Figure S4. Based on the results of previous studies,
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not only affects the migration behavior of the stem cells but also affects the
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differentiation tendency of the stem cells due to the topological signal of the material
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surface.
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In addition to spreading areas, the morphologies of hADSCs have apparent
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differences among each group of samples. As shown in Fig. 2a, g, m, s, and c, i, o, u,
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after being cultured for 48 hours, hADSCs cultured on PLA-200 have an
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osteoblast-like polygonal morphology, but the cells on all of the other 3 samples
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maintain a spindle shape, and the size of the spindles on PLA-100 is much smaller.
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The different morphology is also caused by different stretching resistance due to the
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different space between the neighboring nanopillars. Different mechanical resistances
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affect the cytoskeletal rearrangement of hADSCs and influence the final morphology,
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which may further influence gene expression in the nucleus.
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results of vinculin staining, we found that vinculin fluorescent intensities are different
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among the different groups. Vinculin fluorescent intensities on PLA-200 and
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PLA-300 are higher than on PLA planar film and PLA-100. As reported in previous
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studies, vinculin plays a key role in stem cell differentiation, and more vinculin may
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indicate greater osteogenic differentiation potential.41 Therefore, PLA-200 and
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PLA-300 may have more positive osteogenic results.
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the attachment of the cells
40
Further, from the
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Figure 2. Attachment of hADSCs on PLA nanopillar arrays with different pillar
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diameters after culturing for 48 hours. a-c, g-i, m-o, s-u. Fluorescence microscopy
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images of hADSC F-actin, vinculin and nucleic staining after culturing for 48 hours
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on PLA planar film and PLA nanopillar arrays with different pillar diameters,
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respectively. d-f, j-l, p-r, v-x. SEM images of hADSCs after culturing for 48 hours on
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PLA planar film and PLA nanopillar arrays with different pillar diameters. F-actin
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was stained by Alexa Fluor 568-labeled phalloidin (red), vinculin was stained by an
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Alexa Fluor 488-labeled anti-vinculin antibody (green), and cell nuclei were stained
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by Hoechst 33258 (blue).
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To observe the cell adhesion and spread on different substrates more meticulously,
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SEM images of cells cultured for 48 hours were obtained after alcohol gradient
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dehydration treatment. Due to the smooth surface of PLA planar film, hADSCs can 11
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spread naturally with flat pseudopodium and present a typical spindle shape consistent
2
with the cytoskeleton staining (Figure 2d-f). When hADSCs were cultured on
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PLA-100, the spread resistance was greatly increased, and the cell shape became more
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slender (Figure 2j-l). When hADSCs were cultured on PLA-200, causing less
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resistance compared with PLA-100, the cell shape changed to a polygon and the
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pseudopodium became wider, which was also consistent with the cytoskeleton
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staining (Figure 2p-r). Regarding hADSCs cultured on PLA-300, the resistance was
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less than on PLA-100 and PLA-200 but still larger than on PLA planar film. Therefore,
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most of the cells were still spindle shaped but were wider than on PLA planar film,
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and some of the cells were also polygon shaped (Figure 2v-x). From these results, it is
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clear that different nanopillar arrays have different effects on cell morphology and
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pseudopodium spread due to the difference in resistance and hADSC morphology and
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altered pseudopodium spread. Therefore, we can infer that PLA-200 and PLA-300
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may improve the osteogenic differentiation of hADSCs.
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Considering both the results of cytoskeleton staining and SEM images, these
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phenomena indicate that the difference in nanopillar diameters can significantly
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influence the spreading area and morphologies of hADSCs and suggest that nanopillar
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arrays with different pillar diameters can cause different osteogenic differentiation
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effects according to the results above. Moreover, cell morphology changes to
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polygon-shaped after culturing with osteoblast-inducing conditional media for at least
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1-2 weeks.22,42 However, from the results above, hADSCs cultured on PLA nanopillar
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arrays changed their morphology after culturing for only 48 hours, which is faster 12
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than the osteoblast-inducing conditional media or BMP-2. Therefore, nanopillar
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array-mediated osteogenic differentiation may be more effective in stem cell
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morphology transformation than osteoblast-inducing conditional media. The results of
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cytoskeleton staining and the SEM images of hADSCs cultured on TCP are shown in
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Figure S3 as a blank control.
6 7
Cell viability and proliferation of hADSCs
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To investigate the cell viability and proliferation of hADSCs, live/dead staining and a
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CCK-8 assay were used to observe the ratio of live cells and dead cells and to
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evaluate cells cultured on the samples qualitatively and quantitatively. As shown in
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Figure 1m-x, there are almost no dead cells on the PLA planar films and less than 5%
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of dead cells on PLA nanopillar array samples with different pillar diameters after
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culturing for 48 hours, indicating the cytocompatibility of PLA films before and after
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the construction of superficial nanopatterns. The results of live/dead staining of
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hADSCs cultured on TCP are shown in Figure S3 as a blank control.
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From the CCK-8 assay results shown in Figure 1z, the hADSCs cultured on these
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samples have a proliferation trend from day 1 to day 3, and cell number on day 3 is
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more than twice that on day 1, indicating good biocompatibility of both PLA planar
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and nanopillar array samples. On day 5, hADSCs on TCP and planar PLA film
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continue to proliferate. Inversely, hADSCs on PLA nanopillar array samples have
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almost stopped proliferating. This result should be attributed to the difference in
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surface resistance derived from nanoarrays with different nanopillar diameters. The 13
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cell spreading and growth schematic is shown in Figure 1y. Cell proliferation is
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therefore hampered by different degrees of surface resistance. From the cell viability
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result on day 5, we observed that cell proliferation is hampered by PLA nanopillar
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arrays at the 100 nm diameter due to the greatest resistance on the PLA-100 surface,
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and the cell number increases by only less than 5% of that on day 3, which is
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consistent with the results of hADSC spread mentioned above. Previous studies have
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shown that the decrease of cell proliferation is generally related to cell apoptosis and
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cell differentiation. Therefore, the hampered proliferation of hADSCs on PLA
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nanopillar arrays may also suggest improved osteogenic differentiation.43,44
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Summarizing all of the results of the cytoskeleton, cell viability and proliferation,
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hADSCs cultured on these samples have good cell viabilities and proliferation in
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general. However, the differences in adhesion and spreading morphologies are
13
obvious. According to previous reports, differences in cell adhesion and morphology
14
may be closely associated with stem cell differentiation.45,46 Therefore, we
15
hypothesize that PLA nanopillar arrays with different diameters may have different
16
effects on hADSC osteogenic differentiation, among which PLA-200 may have the
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most positive effect according to the morphology transformation results.
18 19
Osteogenic differentiation of hADSCs
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To investigate the effect of different PLA nanopillar arrays with different diameters on
21
osteogenic differentiation of hADSCs, cells were cultured on a series of different PLA
22
films with different surface nanopatterns (PLA film, PLA-100, PLA-200 and 14
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PLA-300) in culture medium without osteogenic supplements for different time
2
periods. In addition, culture plates were used as a blank control group. To evaluate the
3
osteogenesis of hADSCs, typical osteogenic markers at protein, gene, and functional
4
levels were characterized.
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Alkaline phosphatase (ALP) is an important early phenotypic marker for the
6
osteogenic differentiation of stem cells.47 Therefore, detection of ALP activity is
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essential to evaluate the osteogenic differentiation degree of hADSCs. Figure 3a
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shows the relative ALP protein activity, normalized to total protein content of
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hADSCs cultured on different PLA substrates. On day 14, the ALP activity of
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hADSCs on all samples increased dramatically to approximately twice that of cells on
11
day 7, among which hADSCs cultured on PLA-200 showed the highest ALP activity.
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However, on day 21, ALP activity of hADSCs on all substrates decreased because
13
ALP is an early marker of osteogenic differentiation. The greatest ALP activity in
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hADSCs on PLA-200 corresponds to the result of the most significant cell
15
morphology transformation in this group (shown in Figure 2m-o).
16
To further investigate the osteogenic differentiation degree, quantitative polymerase
17
chain reaction (q-PCR) was used to assess the differentiation at the genetic level by
18
measuring the relative RNA content in hADSCs cultured on different PLA substrates.
19
Runx2, which regulates a number of other genes associated with osteogenesis, is a
20
specific transcription factor expressed during osteogenic differentiation. Osteopontin
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(OPN) is a type of glycoprotein and plays a key role in the mineralization process of
22
bone matrix during bone metabolism. Osteocalcin (OCN) is another important protein 15
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during osteogenic differentiation and is considered a marker in the mineralization
2
period. Therefore, the expressions of Runx2, OPN and OCN are commonly used as
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markers for osteogenesis characterization.48,49 The three osteogenic marker genes of
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hADSCs were examined on days 7, 14, and 21 by q-PCR.
5 6
Figure 3. Osteogenic differentiation of hADSCs cultured on a culture plate, PLA
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planar film, and PLA nanopillar arrays with different pillar diameters. a. ALP activity
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of hADSCs on day 7, 14 and 21, normalized to total protein concentration. b–d.
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Q-PCR analysis of the expression of osteoblast-specific genes (b) Runx-2, (c) OPN, 16
ACS Paragon Plus Environment
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and (d) OCN on day 7, 14 and 21, normalized to actin expression. e. Calcium nodules
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stained by Alizarin Red S show extracellular calcium deposits by hADSC-derived
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osteoblasts on day 21. All data represent the mean ± standard deviation (n = 3).
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0.01
