Fabrication and Characterization of Three Dimensional Core-Shell

Subscriber access provided by Fudan University

Article

Fabrication and Characterization of Three Dimensional CoreShell Structure Nanofibers Designed for 3D Dynamic Cell Culture Lin Jin, Qinwei Xu, Shreyas Kuddannaya, Cheng Li, Yilei Zhang, and Zhenling Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017

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 24

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

Fabrication and Characterization of Three Dimensional Core-Shell Structure Nanofibers Designed for 3D Dynamic Cell Culture Lin Jin,†,‡* Qinwei Xu, Zhenling Wang†,§* †

‡

Shreyas Kuddannaya,

‡

Cheng Li,

‡

Yilei Zhang, ‡*

The Key Laboratory of Rare Earth Functional Materials and Applications, Zhoukou

Normal University, Zhoukou 466001, P. R. China ‡

School of Mechanical & Aerospace Engineering, Nanyang Technological University,

50 Nanyang Avenue, Singapore 639798, Singapore §

International Joint Research Laboratory for Biomedical Nanomaterials of Henan,

Zhoukou 466001 China KEYWORDS: electrospinning, three dimensional nanofibers, hMSCs, dynamic cell culture, tissue engineering ABSTRACT: Three dimensional elastic nanofibers (3D eNFs) can offer a suitable 3D dynamic microenvironment and sufficient flexibility to regulate cellular behaviour and functional protein expression. In this study, we report a novel approach to prepare 3D nanofibers with excellent mechanical property by solution-assisted electrospinning technology and in-situ polymerization. The obtained 3D eNFs demonstrated excellent biocompatible properties to meet cell culture requires under dynamic environment in vitro. Moreover, these 3D eNFs also promoted human bone marrow mesenchymal stem cells (hMSCs) adhesion and collagen expression under the biomechanical stimulations. The results demonstrated that this dynamic cell culture system could positively impact cellular collagen, but has no significant effect on the proliferation of hMSCs grown in the 3D eNFs. This work may start a new way of constructing 3D cell culture for tissue engineering.

1. INTRODUCTION

ACS Paragon Plus Environment


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 24

3D elastic nanofibers (3D eNFs) are now emerging as one class of important biomaterials. These 3D eNFs can provide cues for monitoring and guidance of cellular development by mechanical simulation,1,

2

can also offer suitable 3D

nanofibrous microenvironment and sufficient flexibility to regulate cellular behaviour and some functional protein expression, such collagen, glycosaminoglycan.3-10 Thus, analysis the relationship between programmed mechanical signals, geometry cues and specific cellular responses in this 3D microenvironment is very important for tissue engineering field. Several approaches including electrospinning, carbonization and self-assembly methods have been used in the fabrication of such nanofibers.11-23 However, these 3D nanofibers, such as PCL fibers,24 PLLA fibers,25 PAN/SiO2 fibers nanofibrous aerogels,26 carbon fiber aerogels,27 carbon tubes, bacterial cellulose fibrils28,29 often failed to be used as 3D eNFs due to their intrinsic unfavourable properties, such as weak mechanical strength and unsuitable interconnected microscopic architectures. Recently, researchers focused on developing novel preparation techniques for 3D eNFs generation, and also exhibited various applications.30-33 Despite their outing potential,

these

3D

nanofibers

still

cannot

accurately

mimic

the

ECM

microenvironment or form organized nanofibrous constructs to regulate cellular behaviour and function expression. Moreover, most of these nanofibers are not completely 3D structured nanofibrous scaffolds but rather 2D nanofiber mesh deposits, which demonstrate poor mechanical strength or unsuitable pore size for cell infiltration and storage. Therefore, it is pivotal to construct homogenous and mechanically viable 3D eNFs capable of forming an interconnected structure with desirable pore size, which will greatly enhance the functionality of the intrinsic nanofibers to be widely applied. In our study, we developed a novel fabrication strategy for 3D eNFs by solution-assisted electrospinning technology and in-situ polymerization (Figure 1b). The resulted 3D eNFs possessed polymer core and polyprrole shell complex structure, and the unique structure not only greatly enhanced the mechanical property and flexibility, but also remained 3D nanofiber architecture. Here, 3D eNFs were used as


Page 3 of 24

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


cell culture scaffold for mechanical simulation of hMSCs. The results indicated that the prepared 3D eNFs can greatly promote convenient infiltration and adhesion of cells into the nanofibrous scaffold, moreover, under the dynamic culture conditions, hMSCs could secrete collagen in their immediate microenvironments, which noted a great potential of the 3D eNFs in 3D cell culture in vitro. 2. EXPERIMENTAL SECTION 2.1 Fabrication of 3D eNFs We fabricated 3D eNFs by three steps. First, we prepared 3D nanofibers (3D NFs) using electrospinning technique (Figure 1b). A precursor solution was prepared by

dissolving

polyacrylonitrile

(PAN,

MW=100k)

in

the

solvent

of

N,

N-dimethylformamide (DMF) (W: V=10%). The electrospinning was performed using a customized spinning system as previous study.25 The collector for nanofiber deposition in a borosilicate beaker (1000mL) with 800mL of ethanol was shaken every five minutes. Eventually, the nanofiber dispersion was replaced by 800mL DI water three times with rigorous shaking. The fabrication consists of three distinct steps: (1) Collection of nanofibers in solution state; (2) Homogenization of nanofibers; and (3) Freeze-drying assembly. And then, the conductive polymer layer (polypyrrole) was polymerized on the nanofibers as previous study.25 The 3D NFs changed from white to black due to the coating PPy layer on the nanofiber surface. Finally, the 3D eNFs were fabricated by the freeze-drying process. 2.2 Materials Characterization The 3D eNFs were imaged by a SEM system (Hitachi S4800) at an accelerating voltage of 15kV, and the nanofiber diameters were measured using the software (Image J). To clearly show nanofiber network organization with the different pore structure, scaffolds were doped with CeF3:5%Tb nanoparticles in the fabrication process (5mg CeF3:5% Tb nanoparticles were dispersed into 1mL precursor solution). The fluorescence images were performed as previous study.25 2.3 Biocompatibility Analysis



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

The 2D samples were treated for cell culture as our previous study.25 The 3D samples were cut into 24-well TCPs using freezing microtome and freeze-drying method (See details in method Supplementary), then sterilized under UV for 2 h. And then, hMSCs were cultured and incubated onto or into the NFMs, TCPs, 3D NFs, 2D PPy NFs and 3D eNFs. The cell density is 1.2 × 105 cells every well. The cell viability was assessed according to our previous study.25 For detailed cellular morphology analysis on the scaffold, the cells were ﬁxed on the nanofibers through glutaraldehyde (V: V=3%) and changed the solution by DI water 2-3 times, then freezing overnight and dehydrated using freeze dryer for 48h. And then, these cell-nanofiber constructs were imaged by SEM. proliferation and adherence of cells were characterized after 6 h, 3, 7 and 14 days culture based on a DNA analysis method.25 To assess the effect of cells cultured for a long-term period (2 weeks) under dynamic mechanical stimulation, the cell-scaffold constructs were subjected to dynamic loading as per the protocols mentioned in previous works.34, 35 Briefly, each day, the cell-scaffold constructs with growth medium were placed into self-made bioreactor, and then the culture system loaded to 20% strain for 2 h (the frequency is 2Hz). After cyclic loading for cell dynamic culture, the nanofiber-cell constructs were stored at -20°C to test biochemical function express. The obtained samples were treated as previous methods.34 Proliferation and collagen content were quantified at the 14th day using PicoGreen® DNA and orthohydroxyproline assay, respectively. Collagen content of these cells was estimated through measuring the converted hydroxyproline content using a conversion factor of 7.14.36-39 3. RESULTS AND DISCUSSION 3.1 Preparation of 3D NFs As the template, 3D NFs successful fabrication is important for preparing 3D eNFs. To obtain 3D NFs, we developed a water-assisted electrospun nanofiber collection system to achieve the structure reconstruction. As shown in the schematic diagram of the fabrication process (Figure 1b), PAN nanofibers were directly spun into the ethanol solution to make the nanofibers fully extend. With a progressive increase in the electrospinning time, PAN nanofibers were produced and


Page 4 of 24

Page 5 of 24

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


well-dispersed in the ethanol solution (Figure S1). Eventually, 3D nanofibers were obtained by freeze-drying method after the nanofiber dispersion was replaced 3-5 times using deionization (DI) water along with shaking under a strong shear force. By regulating the dispersity, concentration of nanofibers and the freezing conditions, the 3D NFs with desired structured and shape were readily obtained, such as cones, cylindrical and cubes shapes (inset in Figure 1b). 3.2 Structural Characterization of 3D NFs Compared with the traditional NFMs (Figure 2a), the obtained 3D NFs showed a clear 3D architecture (Figure 2d). The nanofibers in 3D NFs formed fibrous networks, and clearly exhibited disperse status, i.e., the 3D NFs have continuous and interconnected pores with a pore size of 10-30µm (Figure 2f). However, the nanofibers in the 2D NFMs (Figure 2b) appeared closely packed together with an average pore size measuring less than 5µm (Figure 2c). Evidence of the pore size and the internal structure of 3D NFs were further confirmed from the fluorescence images (Figure S2). Moreover, the total surface area of 3D NFs (33.08 m2/g) was also much larger than that (11.68 m2/g) of 2D NFMs (Figure S3). The results demonstrated that these obtained 3D NFs could successfully overcome the structural and functional limitations of the 2D fibrous mats. 3.3 Biocompatibility of 3D NFs In order to gain an insight of the cells growth and morphology in the 3D NFs, the cellular responses of 3D NFs were characterized using fluorescent images of cells using a confocal microscope. The cells seeded into the 3D NFs demonstrated cell penetration spread across the whole bulk of 3D nanofibrous scaffold with the cell body firmly attached on the fibrous matrix (Figure 3c). Both F-actin and nuclei staining revealed a well directional spreading along the nanofibers. Further, F-actin staining of the hMSCs showed that the hMSCs were not completely spread out with a flat morphology as often seen in 2D cultures, and are often found hanging on to multiple nanofibers in their immediate vicinity. In contrast, the cells cultured on NFMs (Figure 3b), resembled 2D cell cultures (similar to TCPs) with extended cell area which could be attributed to the cell retention at the NFMs surface due to their



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

inability to penetrate into the bulk of nanofibrous mesh. However, F-actin of cells on the TCPs showed all well-spread morphology with higher cell area due to the isotropic surface of the TCPs which allows hMSCs to spread freely in every direction (Figure 3a) as commonly shown in standard conventional TCP cultures. These observations from the fluorescent images corresponded well with the morphology of the cells (Figure 3d-f) demonstrated that the 3D NFs allow adequate penetration of hMSCs into the interior regions of the nanofibrous system and promote their growth in a 3D microenvironment. For cellular attachment and proliferation, the cell number was normalized by DNA assay at a specific time. Estimation of the relative levels of cellular attachment of the substrates (Figure 3g) shows that the cell attachment after 6 h incubation was 94%, 93% and 98%, respectively. This shows that the cellular attachment of the 3D NFs was slightly higher compared to that on the NFMs and TCPs. From the cell proliferation trend (Figure 3h), it is clearly showed that the number of hMSCs on each sample has rapidly increased after 3 days culture. However, 3D NFs showed much higher cell number compared to the NFMs and TCPs, which may be attributed to the porous 3D architecture and abundance of interconnected pores which provide enough space for adequate cell penetration and proliferation. After 7 days culture, the cell number increased to 238% and 243% on the NFMs and TCPs, respectively. Hence, there is no significant increase in cell number estimated previously on day 3 in culture. Whereas, the cell content of 3D NFs showed a significant increase to 378%, which is much higher compared to that on day 3 in 3D NFs cultured. After14 days in culture, the cell proliferation on the TCPs and NFMs still remained at relatively lower levels. However, the cells grown in the 3D culture environment maintained significantly higher cell viability and proliferation efficiency. These results integrated with cellular morphology and fluorescent images, strongly demonstrated that open, larger pore size and the porous nanofibrous network of the 3D NFs allowed enough space for quick and continuous cellular penetration and growth throughout its structure. These observations collectively indicated that 3D NFs could provide an ideal microenvironment for the cellular penetration, growth and proliferation.


Page 6 of 24

Page 7 of 24

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


3.4 Fabrication and Characterization of 3D eNFs To further enhance the mechanical property, conductive polymer (PPy) shell layer was polymerized on the nanofiber surface using in-situ polymerization. Pyrrole is used as polymeric monomer and FeCl3 is used as the oxidant, respectively. Compared to the original 3D nanofibrous templates, the obtained 3D eNFs retained the porous structure, interwoven architecture (Figure 4a) and the pore sizes of the native 3D NFs. The coated PPy layer could be seen clearly by TEM, high magnification SEM image (Figure 4b, Figure S4-6), and the FTIR spectrum（Figure S7) also indicated that PPy layer successfully formed on the nanofibers. More importantly, the coating layer of the nanofibers greatly improved the mechanical toughness and elastic property of 3D NFs. The curves of compression and photos of obtained 3D eNFs were displayed (Figure 4c and d) in air and DI water, respectively. The 3D eNFs demonstrated linear elasticity behaviour when they were in a low strain (< 10%) in air. After this point, a great mechanical strength increase was observed for nanofibers to adapt to changes structure deformation and relevant energy absorption. The obtained 3D eNFs were released after 40% compressive strain 40%, during which the stress is 4.02 KPa (in air) and 1.2 KPa (in DI water), respectively. Despite the relative decrease of compression stress in water compared to that in air, the 3D eNFs could be well-restored to its original state as it in the air. After ten cycles, the 3D eNFs still recovered well, close to its original state (Figure 5), these results may be due to the comprehensive effects of this unique “core-shell” structure and PPy layer along the nanofibers which could have played a combined role in enhancing the nanofibers strength. On the other hand, this synergistic effect also makes 3D eNFs adjust to absorb of the external force. These results demonstrated that the 3D eNFs not only give enough space for easy penetration, attachment and proliferation of cells, but can also provide mechanically viable 3D nanofibrous micro-environment with sufficient flexibility to regulate cellular behaviour and functional protein expression under dynamic culture condition. The electrochemical performance of 3D eNFs was characterized in the 0.5M Na2SO4 salt solution as previous method.25 3D eNFs (21.2mg) were immersed in



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

Na2SO4 solution. The CV spectras (Figure S8) indicated 3D PPy nanofibers exhibited high charge carrying capacity when they were under a given voltage and much better electrochemical property and conductivity. Combining excellent mechanical property with porous architecture mark, we believe that the 3D eNFs can further expand their biomedical application as bio-scaffold, such as cell cultures with electrical stimulation, neuronal tissue engineering, dynamic cell culture, etc. 3.5 Celluar Response under Dynamic Culture in 3D eNFs To explore the application of the 3D NFs for in vitro dynamic cell culture with mechanical stimulation, hMSCs with a density of 1.2×105/well were seeded into the 3D eNFs. The cellular responses of dynamic tensile loading on hMSCs-seeded into the 3D eNFs were evaluated by fluorescence and SEM images of cells, proliferation and the collagen expression. Cells were cultured for long-term period (2 weeks) under dynamic mechanical stimulation protocols described in previous works (Figure 6).32, 33

Figure 7b1, b2 shows confocal images of F actin stained hMSCs under the

unstimulated state (without mechanical load application), show uniformly spread cell distribution on all levels along the randomly aligned nanofibers. Furthermore, a closer look on cell morphology indicated that nanofibers could provide ample surface and space to promote cell adhesion and growth (Figure 7b3). However, while cells are grown under dynamic culture conditions, the F-actin and nuclei staining reveal the alignment of cells along the nanofibers (Figure 7c1, c2), with in distinct directions, which may be attributed to the continuous mechanical stimulations promoted most cells to attach and grow on specialized nanofibers while adapting themselves to dynamic environment. The cell morphology after two weeks dynamic cultured also indicated that cells could firmly attach and grow along the nanofibers, while also encapsulating a few nanofibers at the attachment sites (Figure 7c3). In stark contrast, cells grown on the 2D PPy-NFs gathered together, the morphology of these cells showed a contracted state, and the cells were loosely connected to each other. The results are usually not desirable for hMSCs under a long time survival, growth or functional protein expression (Figure 7a1-3).34 From the above results, we propose that the dynamic state cell culture condition could make cells grow and survive along the


Page 8 of 24

Page 9 of 24

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


specialized directions on the nanofibers. After 2 weeks of cyclic conditioning, we not observed obvious difference of cellular DNA content between the loaded simulation microenvironment and the nonloaded state (Figure 7d, the value was calculated by OD of cells cultured under static state), but both of these groups show significantly much higher cell survival compared to that on the TCPs. However, after 2 weeks dynamically loaded culture, the hMSC cultured 3D eNFs showed relatively higher collagen content compared to the unstimulated controls (Figure 7e). The results suggested that cyclic loaded mechanical conditioning has no significant effect on the proliferation of hMSCs grown in the 3D eNFs, but could positively impact collagen expression. Previous studies had found that MSCs cultured under the cyclic mechanical strain simulation could be induced endogenous synthesis of potent growth factors, which is able to influence regulat lineage specification.32, 33 For instance, the cyclic tensile could increase osteogenic growth factor response and upregulate the transcription of RUNX2.32, 33 As nanofibers in our 3D eNFs scaffolds would stretch when a uniaxial compression was applied, we hypothesized that the osteogenic lineage happened in our experiment. Anti-RUNX2 antibody, a common osteogenic biomarker, was utilized for immunocytochemistry staining. After staining cells on Day 14, however, there was no visible RUNX2 expression in neither cells cultured in 3D eNFs with dynamic loading nor cells cultured in static 3D eNFs as shown in Figure 8. The positive control groups, no matter they were cultured with dynamic loading or not, showed RUNX2 expression obviously. There is no evidence that the osteogenic differentiation happened in our experiment. The difference of collagen expression and morphology in dynamic and static 3D eNFs was not related to osteogenesis. The cellular response of hMSCs under dynamic culture indicated that the obtained 3D eNFs could be used to build 3D cell culture system and provide appropriate mechanical stimulation to exert effect on the collagen expression, morphology and cellular activity. These results also indicated that our strategy could provide a comprehensive effect on cellular behaviour combined mechanical



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 10 of 24

stimulation with 3D microenvironment. Thus, the 3D eNFs could supply more parameters and possibility for cell functional expression,40 which has obvious advantages than the single geometry cues. 4. CONCLUSION In conclusion, we demonstrated core-shell structure 3D eNFs could be fabricated by solution-assisted electrospinning technology and in-situ polymerization. The obtained 3D eNFs exhibited outstanding recoverable mechanical property, stable 3D porous structure and excellent biocompatibility. Cell culture (in vitro) results showed that 3D eNFs could be favour to promote cellular proliferation; moreover, the collagen expression of hMSCs under mechanical stimulation after 2 weeks culture was much higher compared to that without mechanical stimulation. Our results demonstrated that the obtained 3D eNFs could provide 3D cell culture microenvironment and mechanical stimulation to regulate cellular growth behaviour. we believe that the 3D eNFs will have broad application in biomedical field, such as cell culture with electrical and mechanical stimulation, neuronal tissue engineering, controllable drug release, etc. Supporting Information Supporting Information is available free of charge

via the Internet at

http://pubs.acs.org. The graph of fabrication process 3 DNFs (Figure S1), confocal images and BET surface area of the 3D NFs (Figure S2-3),, TEM image and high magnification SEM image of single PPy core-shell nanofiber (Figure S4), SEM image of PPy tubes in the 3D eNFs (Figure S5-6), FTIR spectrum of 3D eNFs (Figure S7) and CV curves of 3D eNFs (Figure S8). ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China (21404124 and 51572303), A*STAR AOP project (1223600005), the Tier-1 Academic Research Funds from the Singapore Ministry of Education (RGT 30/13),


Page 11 of 24

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


L. J. acknowledges the program of Innovative Talent (in Science and Technology) in University of Henan Province (17HASTIT007), and Z. W. acknowledges Program for Innovative Research Team (in Science and Technology) in University of Henan Province (14IRTSTHN009). AUTHOR INFORMATION

*E-mail for: Jin Lin : [email protected] *

E-mail for Z.L.W: [email protected].

*

E-mail for Y.L.Z.: [email protected]. Fax: 65-67942035; Tel: 65-67905952

REFERENCES (1)

Kliment, C. R.; Englert, J. M.; Crum, L. P.; Oury, T. D. A Novel Method for Accurate Collagen and Biochemical Assessment of Pulmonary Tissue Utilizing One Animal. Int. J. Clin. Exp. Pathol. 2011, 4, 349-355.

(2)

Henrionnet, C.; Wang, Y.; Roeder, E.; Gambier, N.; Galois, L.; Mainard, D.; Bensoussan, D.; Gillet, P.; Pinzano, A. Effect of Dynamic Loading on MSCs Chondrogenic Differentiation in 3-D Alginate Culture. Biomed. Mater. Eng. 2012, 22 (4), 209-218.

(3)

Dvir, T.; Timko, B. P.; Kohane, D. S.; Langer, R. Nanotechnological Strategies for Engineering Complex Tissues. Nat. Nanotechnol. 2011, 6, 13-22.

(4)

Krogman, K. C.; Lowery, J. L.; Zacharia, N. S.; Rutledge, G. C.; Hammond, P. T. Spraying Asymmetry into Functional Membranes Layer-by-Layer. Nat. Mater. 2009, 8, 512-518.

(5)

Jain, A.; Betancur, M.; Patel, G. D.; Valmikinathan, C. M.; Mukhatyar, V. J.; Vakharia, A.; Pai, S. B.; Brahma, B.; MacDonald, T. J.; Bellamkonda, R. V. Guiding Intracortical Brain Tumour Cells to an Extracortical Cytotoxic Hydrogel Using Aligned Polymeric Nanofibres. Nat. Mater. 2014, 13, 308-316.

(6)

Onoe, H.; Okitsu, T.; Itou, A.; Kato-Negishi, M.; Gojo, R.; Kiriya, D.; Sato, K.; Miura, S.; Iwanaga, S.; Kuribayashi-Shigetomi, K.; Matsunaga, Y. T.; Shimoyama, Y.; Takeuchi, S. Metre-Long Cell-Laden Microfibres Exhibit Tissue Morphologies and Functions. Nat. Mater. 2013, 12, 584-590.



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

(7)

Page 12 of 24

Lutolf, M. P.; Gilbert, P. M.; Blau, H. M. Designing Materials to Direct Stem-Cell Fate. Nature 2009, 462, 433-441.

(8)

Lee, S.; Leach, M. K.; Redmond, S. A.; Chong, S. Y. C.; Mellon, S. H.; Tuck, S. J.; Feng, Z. Q.; Corey, J. M.; Chan, J. R. A Culture System to Study Oligodendrocyte Myelination Processes Using Engineered Nanofibers. Nat. Methods 2012, 9, 917-922.

(9)

Jin, L.; Wu, D. C.; Kudannaya, S.; Zhang, Y. L.; Wang, Z. L. Fabrication, Characterization, and Biocompatibility of Polymer Cored Reduced Graphene Oxide Nanofibers．ACS Appl. Mater. Interface 2016, 8, 5170-5177.

(10)

Miller, J. S.; Stevens, K. R.; Yang, M. T.; Baker, B. M.; Nguyen, D. H. T.; Cohen, D. M.; Toro, E.; Chen, A. A.; Galie, P. A.; Yu, X.; Chaturvedi, R.; Bhatia, S. N.; Chen, C. S. Rapid Casting of Patterned Vascular Networks for Perfusable Engineered Three-Dimensional Tissues. Nat. Mater. 2012, 11, 768-774.

(11)

Murphy, W. L.; McDevitt, T. C.; Engler, A. J. Materials as Stem Cell Regulators. Nat. Mater. 2014, 13, 547-557.

(12)

Wade, R. J.; Bassin, E. J.; Gramlich, W. M.; Burdick, J. A. Nanofibrous Hydrogels with Spatially Patterned Biochemical Signals to Control Cell Behavior. Adv. Mater. 2015, 27, 1356-1362.

(13)

Kraehenbuehl, T. P.; Langer, R.; Ferreira, L. S. Three-Dimensional Biomaterials for the Study of Human Pluripotent Stem Cells. Nat. Methods 2011, 8, 731-736.

(14)

Xie, J.; Macewan, Ќ. M. R.; Ray, Ќ. W. Z.; Liu, W.; Siewe, D. Y.; Xia, Y. Radially

Aligned

Electrospun

Nanofibers

and

Tissue

Regeneration

Applications. ACS Nano 2010, 4, 5027-5036. (15)

Xie, J.; Wang, C. H. Electrospun Micro- and Nanofibers for Sustained Delivery of Paclitaxel to Treat C6 Glioma in Vitro. Pharm. Res. 2006, 23, 1817-1826.

(16)

Demirci, S.; Celebioglu, A.; Aytac, Z.; Uyar, T. pH-Responsive Nanofibers with Controlled Drug Release Properties. Polym. Chem. 2014, 5, 2050-2056.

(17)

Chen, M.; Li, Y. F.; Besenbacher, F. Electrospun Nanofibers-Mediated


Page 13 of 24

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


On-Demand Drug Release. Adv. Healthc. Mater. 2014, 3, 1721-1732. (18)

Han, D.; Steckl, A. J. Triaxial Electrospun Nanofiber Membranes for Controlled Dual Release of Functional Molecules. ACS Appl. Mater. Interfaces 2013, 5, 8241-8245.

(19)

Niu, C.; Meng, J.; Wang, X.; Han, C.; Yan, M.; Zhao, K.; Xu, X.; Ren, W.; Zhao, Y.; Xu, L.; Zhang, Q.; Zhao, D.; Mai, L. General Synthesis of Complex Nanotubes by Gradient Electrospinning and Controlled Pyrolysis. Nat. Commun. 2015, 6, 7402-7402.

(20)

Chen, L. F.; Huang, Z. H.; Liang, H. W.; Guan, Q. F.; Yu, S. H. Bacterial-Cellulose-Derived Carbon nanofiber@MnO2 and Nitrogen-Doped Carbon Nanofiber Electrode Materials: An Asymmetric Supercapacitor with High Energy and Power Density. Adv. Mater. 2013, 25, 4746-4752.

(21)

Cui, C.; Qian, W.; Yu, Y.; Kong, C.; Yu, B.; Xiang, L.; Wei, F. Highly Electroconductive Mesoporous Graphene Nanofibers and Their Capacitance Performance at 4 V. J. Am. Chem. Soc. 2014, 136, 2256-2259.

(22) Liu, H.; Cao, C. Y.; Wei, F. F.; Huang, P. P.; Sun, Y. B.; Jiang, L.; Song, W. G. Flexible Macroporous Carbon Nanofiber Film with High Oil Adsorption Capacity. J. Mater. Chem. A 2014, 2, 3557. (23)

Guler, M. O.; Stupp, S. I. A Self-Assembled Nanofiber Catalyst for Ester Hydrolysis. J. Am. Chem. Soc. 2007, 129, 12082-12083.

(24)

Blakeney, B. A.; Tambralli, A.; Anderson, J. M.; Andukuri, A.; Lim, D. J.; Dean, D. R.; Jun, H. W. Cell Infiltration and Growth in a Low Density, Uncompressed

Three-Dimensional

Electrospun

Nanofibrous

Scaffold.

Biomaterials 2011, 32, 1583-1590. (25)

Jin, L.; Wang, T.; Feng, Z. Q.; Zhu, M.; Leach, M. K.; Naim, Y. I.; Jiang, Q. Fabrication and Characterization of A Novel Fluffy Polypyrrole Fibrous Scaffold Designed for 3D Cell Culture. J. Mater. Chem. 2012, 22, 18321.

(26)

Si, Y.; Yu, J.; Tang, X.; Ge, J.; Ding, B. Ultralight Nanofibre-Assembled Cellular Aerogels with Superelasticity and Multifunctionality. Nat Commun 2014, 5, 5802-5802.



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 14 of 24

(27) Bi, H.; Yin, Z.; Cao, X.; Xie, X.; Tan, C.; Huang, X.; Chen, B.; Chen, F.; Yang, Q.; Bu, X.; Lu, X.; Sun, L.; Zhang, H. Carbon Fiber Aerogel Made from Raw Cotton: A Novel, Efficient and Recyclable Sorbent for Oils and Organic Solvents. Adv. Mater. 2013, 25, 5916-5921. (28)

Assfour, B.; Leoni, S.; Seifert, G.; Baburin, I. A. Packings of Carbon Nanotubes New Materials for Hydrogen Storage. Adv. Mater. 2011, 23, 1237-1241.

(29)

Bäckdahl, H.; Helenius, G.; Bodin, A.; Nannmark, U.; Johansson, B. R.; Risberg, B.; Gatenholm, P. Mechanical Properties of Bacterial Cellulose and Interactions with Smooth Muscle Cells. Biomaterials 2006, 27, 2141-2149.

(30)

Zhou, K.; Thouas, G. A.; Bernard, C. C.; Nisbet, D. R.; Finkelstein, D. I.; Li, D.; Forsythe, J. S. Method to Impart Electro- and Biofunctionality to Neural Scaffolds Using Graphene-Polyelectrolyte Multilayers. ACS Appl. Mater. Interfaces 2012, 4, 4524-4531.

(31)

Moutos, F. T.; Freed, L. E.; Guilak, F. A Biomimetic Three-Dimensional Woven Composite Scaffold for Functional Tissue Engineering of Cartilage. Nat. Mater. 2007, 6, 162-167.

(32)

Thorvaldsson,

A.; Stenhamre,

H.;

Gatenholm,

P.; Walkenstrom,

P.

Electrospinning of Highly Porous Scaffolds for Cartilage Regeneration. Biomacromolecules 2008, 9, 1044-1049. (33)

Sun, B.; Long, Y. Z.; Yu, F.; Li, M.-M.; Zhang, H. D.; Li, W. J.; Xu, T. X. Self-Assembly of a Three-Dimensional Fibrous Polymer Sponge by Electrospinning. Nanoscale 2012, 4, 2134.

(34)

Baker, B. M.; Shah, R. P.; Huang, A. H.; Mauck, R. L. Dynamic Tensile Loading Improves the Functional Properties of Mesenchymal Stem Cell-Laden Nanofiber-Based Fibrocartilage. Tissue Eng. Part A 2011, 17, 1445-1455.

(35)

Huang, A. H.; Farrell, M. J.; Kim, M.; Mauck, R. L. Long-Term Dynamic Loading Improves the Mechanical Properties of Chondrogenic Mesenchymal Stem Cell-Laden Hydrogels. Eur. Cells Mater. 2010, 19, 72-85.

(36)

Dzau, V. J.; Gnecchi, M.; Pachori, A. S. Enhancing Stem Cell Therapy through


Page 15 of 24

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


Genetic Modification. J. Am. Coll. Cardiol. 2005, 46, 1351-1353. (37)

Baker, B. M.; Mauck, R. L. The Effect of Nanofiber Alignment on the Maturation of Engineered Meniscus Constructs. Biomaterials 2007, 28, 1967-1977.

(38)

Peltz, C. D.; Perry, S. M.; Getz, C. L.; Soslowsky, L. J. Mechanical Properties of the Long-Head of the Biceps Tendon Are Altered in the Presence of Rotator Cuff Tears in a Rat Model. J. Orthop. Res. 2009, 27, 416-420.

(39)

Neuman, R. E.; Logan, M. A. The Determination of Hydroxyproline. J. Biol. Chem. 1950, 184, 299-306.

(40)

Feng, Z. Q.; Wang, T.; Zhao B.; Li, J. C.; Jin, L. Soft Graphene Nanofibers Designed for the Acceleration of Nerve Growth and Development. Adv. Mater. 2015, 27, 6462-6468.



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. Design, processing and the architecture of the 2D-NFs (a) and 3D NFs (b). (b) Schematic diagram of the fabrication steps for 3D NFs. (1) Electrospinning nanofibers into ethanol solution. (2) The obtained mixed solution was replaced with DI water by adding DI water 3-5 times with rigorous shaking. (3) 3D NFs prepared by freeze drying. (inset in b) Optical photograph of freeze dried 3D NFs in diverse shapes.


Page 16 of 24

Page 17 of 24

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 2. Optical photographs, SEM images and pore size distributions of NFMs (a-c), and 3D nanofibers (d-f).



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 3. (a-c) Fluorescence images of hMSCs cultured for 5 days on TCPs, NFMs and 3D NFs-S3. (d-f) SEM images of hMSCs cultured for 5 days on TCPs, NFMs and 3D NFs-S3. (g) The attachment and (h) proliferation of hMSCs cultured on specialized day on the TCPs, NFMs and 3D NFs-S3. Proliferation values were calculated by the number of cells after one day culture. Data are expressed as mean ±SD, n = 3, these results indicate that cells could infiltrate into the scaffold and freely achieve growth and proliferation in the 3D ECM-like microenvironment, and obtain a real 3D culture in vitro.


Page 18 of 24

Page 19 of 24

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 4. (a) SEM image of 3D eNFs (the inset is the optical photograph of a 3D eNF block).

(b) TEM image of single core-shell PPy nanofiber. (c and d) Compressive

strain-stress curve and optical picture of 3D eNFs before and after squeeze in air and DI water, respectively.



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 5. The compressive strain-stress curves of 3D eNF during ten cycles in the air. The axis range of compressive strain in the figure is from 0-40%, and the axis range of compressive stress in the figure is from 0-5 KPa.


Page 20 of 24

Page 21 of 24

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 6. Schematic of a bioreactor for cell culture under dynamic loading condition.



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 7. (a1, a2, b1, b2 and c1, c2) Fluorescence images of hMSCs cultured for 5 days onto/into 2D PPy-NFs and the 3D eNF scaffolds in static and dynamical states, the bars are 50µm. (a3, b3, c3) SEM image of hMSCs cultured for 5 days onto/into the 2D PPy-NFs and

3D eNFs in static and dynamical states. ) (d) Proliferation of hMSCs

cultured for 14 days onto/into 2D PPy-NFs and the e-NFs in static and dynamic states. (e) Collagen expression levels of hMSCs cultured 14 days onto/into the 2D PPy-NFs, 3D e-NFs with static and dynamic state.


Page 22 of 24

Page 23 of 24

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 8. RUNX2 expression of MSCs after 14 days culture in both dynamic and static condition, as well as positive controls marked with “+”. Scale bar: 50 µm.



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

Graphic for manuscript


Page 24 of 24

Fabrication and Characterization of Three Dimensional Core-Shell

Recommend Documents