Inducing Temporal and Reversible Autophagy by Nanotopography for

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Inducing temporal and reversible autophagy by nanotopography for potential control of cell differentiation Wen Song, Mengqi Shi, Mingdong Dong, and Yumei Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11699 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Inducing temporal and reversible autophagy by nanotopography for potential control of cell differentiation Wen Song1, 2, Mengqi Shi1, Mingdong Dong2*, and Yumei Zhang1*

1

The State Key Laboratory of Military Stomatology & National Clinical Research Center for

Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University, Xi’an 710032, China 2

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus 8000, Denmark

KEYWORDS: tissue engineering; cell differentiation; autophagy; nanotopography; cell membrane

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

ABSTRACT

Tuning autophagy has become a new strategy to control cell differentiation in tissue engineering. The nanosized surface is well-known for its ability to interfere intracellular procedures while its role in autophagy regulation is unclear. In this study, we found that the nanotubes (NTs) structure was able to induce enhanced mTOR-independent autophagy in osteoblasts compared to the flat surface. Further analysis revealed that the autophagy was just temporally promoted by NTs in the initial day contact and it was also reversible by exchanging the substrate nanotopographies. The actin filaments were significantly dispersed and there were numerous filopodia on the leading edge of cells grown on the NTs surface. The intracellular Ca2+ was significantly increased on NTs surface. Moreover, the phenomenon was also found on different nanotopographies as well as in different cell lines. These indicated that the cell membrane stretch might be the central regulation factory. Finally, we found that the NTs surface exhibited enhanced autophagy-dependent osteogenic differentiation efficacy. In addition, the enhancement on NTs surface could be remembered. In conclusion, the nanotopographic surface is able to induce temporal, reversible and memorable autophagy via the cell membrane stretch, which may be used as a versatile method to control cell differentiation.

INTRODUCTION Control of cell differentiation is the central idea in tissue engineering.1 There are numerous methods to manipulate cell differentiation by either the chemical drugs stimulation2 or physical structures inducing.3 Autophagy is a conserved intracellular self-digestion system featured by


2

Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


double-membraned autophagosomes formation.4 It plays an important role in cell homeostasis maintenance in response to the environment stimuli. Recently, it has attracted more and more attention because a rising evidence suggest that the autophagy is closely related to various cellular processes including tumorigenicity,5 neural degradative diseases,6 bacteria invasion,7 cell differentiation8 and et al. In the osteogenic differentiation, for example, autophagy is activated during the osteogenic induction and depletion of it will cause osteopenia.9-10 Similarly, during myoblast differentiation, autophagy is required and protects against apoptosis.11-12 Consequently, the autophagy has become a new target for cell differentiation manipulation in tissue engineering. A variety of nanoparticles such as silica nanoparticles,13 gold nanoparticles,14 silver nanoparticles,15 graphene quantum dots,16 carbon nanotubes,17 lipoplex and polyplex,18 has been confirmed of triggering autophagy. Importantly, the autophagy induced by nanoparticles is also closely related to their biological outcomes. For example, the silica nanoparticles triggered autophagy is beneficial for osteoblast differentiation.13 During gene delivery, autophagy plays as a barrier against exogenous DNA18 and the transfection efficiency can be modulated by smallmolecule regulators of autophagy.19 Considering the coupled machinery of autophagy and endocytosis,20 it is reasonable to understand the interaction between the uptake of nanoparticles and autophagy activation, which may make nanoparticles be the most popular nanomaterials for autophagy stimulation. In fact, tuning autophagy is also proven feasible through the surface modification of nanoparticles, such as different chemical groups17, 21 and peptide coating.22 However, most of the nanoparticles are non-degradable in cells so that the internalized nanoparticles will stay inside the cell permanently, which may cause unpredictable potential cytotoxicity.23 In addition, since the endocytosis of nanoparticles is irreversible, the autophagy induction process cannot be interrupted or terminated once the nanoparticles are administrated.


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 30

Consequently, it might be more compatible and flexible if the autophagy can be stimulated via non-invasive way from the interface between substrate topography and cell membrane. The nanosized surface is able to induce different cell behaviors through the interface between cells and materials. It is well known that the titanium implant surface nanotopography is closely related to its osteogenic differentiation inducing capability.24-25 Although the behind mechanism has not been fully elucidated, it is actually an ideal example of tuning cell fate through the noninvasive extracellular environment without entering into intracellular compartments. In addition, since numerous researchers have confirmed the interaction between osteogenic differentiation and autophagy,26 it strongly suggests that the autophagy can also be modulated via the substrate topography. In this study, the nanotubes (NTs) surface was fabricated on etched titanium implant surface and the mirror polished surface (denoted as Flat) was taken as control. We confirmed that the NTs could induce both temporal and reversible autophagy within osteoblast MC3T3-E1 cells, which was closely related to the interface reaction between nanotopography and cell membrane. This phenomenon was the also existed on different nanotopographies as well as in different cell lines. In addition, the nanotopography induced autophagy was required for osteogenic differentiation and could be remembered after removal of the substrate, which might be used as a scaffold-free technique in tissue engineering. RESULTS AND DISCUSSION Mild autophagy is activated by NTs. With conventional anodization, the regular vertical aligned nanotubular surface (NTs) with the diameter of ~100 nm was fabricated on titanium surface (Figure 1A). The MC3T3-E1 cells were grown directly on the NTs or polished titanium


4

Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


surface (Flat). After 24 hours culture, the monodansylcadaverine (MDC) staining was performed to display autophagic vacuoles27 and examined by confocal laser scanning microscope (CLSM). It was apparent that the number of intracellular autophagic vacuoles was increased significantly on NTs surface (Figure 1B). To directly observe the vacuoles, cells were subjected to transmission electron microscopy (TEM) observation. In agreement with the CLSM, an increased autophagic vacuoles number was detected within the cells that grown on NTs surface (Figure 1C, lower magnification). Since there was no exogenous substances invasion, the contents of autophagosomes were mainly the intracellular damaged organelles (Figure 1C, higher magnification). On the contrary, autophagy induction by nanoparticles may be an attempt to degrade the perceived foreign or aberrant stuff so that it can often see the accumulation of nanoparticles in autophagosomes.28 In the meanwhile, the excess accumulation of nanoparticles in the lysosomal system may be harmful to the normal degradation process and result in toxicity.29-30 To further confirm the existence of autophagy, the total protein was isolated from cells that grown on different surfaces, in the absence or presence of vacuole acidification inhibitor ammonium chloride (AC). Without AC, the LC3-II expression on NTs surface was ~3fold higher than that on the Flat surface (Figure 1D). When in the presence of AC, the LC3-II expression was upregulated on both surfaces and it was still significantly higher on NTs surface (Figure 1D). The LC3-II origins from the lipidation of LC3-I in the cytoplasm, which is called LC3 conversion.31 During autophagy, the LC3-II is recruited to the membrane of autophagosomes so that it is the most frequently used indicator as autophagosomes number. Meanwhile, autophagy is a dynamic process and the LC3-II will be consequently degraded in autolysosomes, which can be interrupted by lysosomes acidification inhibitors. Consequently, the LC3-II level will be increased if the lysosome degradation is inhibited, which is named


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 30

autophagy flux.32 Both the LC3 conversion and autophagy flux have been observed on the NTs surface, which can be assured that the autophagy is activated by contacting with the nanotopography. It is notable that excess autophagy caused by the environment is harmful to cell homeostasis and may accelerate cell death.33 In our observation, the NTs surface triggered autophagy is mild and may be beneficial for cell functions. In fact, the NTs surface is a wellknown structure that can improve osteoblast activities without affecting the viability.34 There are two activation pathways of autophagy, e.g. the mammalian target of rapamycin (mTOR)-dependent and –independent pathways, which are classified based on whether the mTOR complex-1 (mTORC1) expression is changed.35 In the next, the mTOR level was examined by western blot. There were no significant differences in p-mTOR expression between NTs and Flat (Figure 1E), indicating that the nanotopography induced autophagy was mTORindependent. In the conventional nutrient starvation caused autophagy, the mTORC1 is inhibited and cooperates with protein phosphatase 2A to initiate the autophagy response.36 Since there are no cell metabolism differences between NTs and Flat surface, the mTORC1 expression may not be influenced in this situation.


6

Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. The titanium nanotubes (NTs) topography induced more autophagic vacuoles formation without affecting mTOR activity compared to the flat titanium surface. MC3T3-E1 cells were seeded onto NTs surface and Flat surface (A) to allow for 24 hours contacting. (B) The MDC staining was performed to exhibit the autophagic vacuoles formation and imaged by CLSM. (C) TEM images of the cells. Arrows showed the autophagic vacuoles. The right panel is the higher magnification of the indicated part. (D) MC3T3-E1 cells were grown on NTs and Flat surfaces for 24 hours. The ammonium chloride (10 mM) was added 6 hours before the total protein extraction. LC3-II activity was analyzed by western blot and normalized to GAPDH. *P

Inducing Temporal and Reversible Autophagy by Nanotopography for

Recommend Documents