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Selective Endothelial Cell Adhesion Via Mussel-Inspired Hybrid Microfibrous Scaffold Jianguang Zhang, Wei Chen, Leixiao Yu, Mingjun Li, Falko Neumann, Wenzhong Li, and Rainer Haag ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00017 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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ACS Applied Nano Materials

Selective Endothelial Cell Adhesion Via MusselInspired Hybrid Microfibrous Scaffold Jianguang Zhang, ‡, # Wei Chen, ‡, §, # Leixiao Yu, ‡ Mingjun Li, ‡ Falko Neumann, ‡ Wenzhong Li, ‡ and Rainer Haag *‡ ‡

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany.

§

Department of Pharmaceutical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 21009, People’s Republic of China.

ABSTRACT: Endothelialization of polymer substrate is limited by unspecific cell adhesion. Herein, a biodegradable microfibrous scaffold with a reversibly thermo-switchable property was developed to dynamic regulate endothelial progenitor cell adhesion by exposing or concealing cRGD motif to the surface with thermo-sensitive moiety (cRGD-PNIPAM) and antifouling moiety linear polyglycerol (LPG). Owing to reversible αvβ3 integrin-cRGD interaction and ligand presentation, the accelerated endothelial cell adhesion and spreading was achieved. Under the static and dynamic condition, pre-stained endothelial cells were quickly attached to the surface at 25 °C via the integrin-cRGD interaction and the cRGD was the head group of the stretched PNIPAM below the LCST of PNIPAM. With the increase of the temperature to 37 °C, a quick detachment of cells from the surface was observed due to cRGD moiety shielded by the antifouling LPG layer. As compared to current strategies for endothelialization, for example, loaded drugs or growth factors, such tunable dynamic system based on “switchable surfaces” may unlock new application in in situ targeted cell recruitment and might become useful in regenerative medicine. KEYWORDS: thermosensitive, endothelial cells, in situ recruitment, mussel adhesives, cell adhesion, αvβ3 Integrin. INTRODUCTION Biodegradable scaffolds that mimic the native extracellular matrix (ECM) in vivo provide favourable mechanical support and the biomicroenvironment for cell anchorage, proliferation, and activity, which is a promising strategy for tissue engineering applications.1-3 Among

the

various

fabrication

techniques

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3D

scaffolds,

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electrospinning has been considered as a simple and efficient method for producing 3D fibrous scaffolds with larger surface area to enhance the topographical interaction with cells.4,5 Furthermore, the diameter and the pore size of electrospun microfiber networks can be further adapted to mimic the structural property of native ECM and control the cell behavior.6,7 Accordingly, cells can change their adhesion and migration behavior on the fiber in order to adapt to the different microenvironments.8 Cell adhesion onto scaffolds is a preliminary and crucial step for cell–material communication, which determines whether the scaffolds will achieve a successful longterm tissue repair and regeneration.9-12 The oligopeptide arginine-glycine-aspartic acid (RGD) exists in most ECM proteins and can bind to the integrin cell adhesion receptor to initiate cell adhesion in many surface-modified substrates.13 Recently, it was reported that the cell adhesion behavior could be regulated by switchable functional surfaces based on the RGD motif, which is a new promising strategy for biointerface engineering. Such as supramolecular system,14 photo-sensitive system,15-17 enzymatical system,18 magnetical system,19 and epitope-imprinted system.20 Although the dynamic interaction could be achieved in these studies, they were limited by UV irradiation and using 2D surfaces, particularly difficult for implantable vessel regeneration. Hence, more advanced and sophisticated biointerfaces are required for mimicking the dynamic presence of adhesive factors in the ECMs with guiding cellular function.

Poly(N-isopropylacrylamide)

(PNIPAM)

is

a

well-established

thermo-

responsive material and the reversibly switchable ability using PNIPAM based materials could be easily controlled by the temperature to realize the hydrophilic and hydrophobic transitions around their lower critical solution temperature (LCST).21, 22 PNIPAM based copolymers also have been widely used to generate new cell-sheet engineering surface and switchable cell culture substrates.23-27 Integrin αvβ3, as a transmembrane receptor for arginine-glycine-aspartic (RGD) tripeptide sequence, plays an essential role for angiogenesis and endothelialization.28 It is expressed at very low levels on epithelial cells, but it is overexpressed on the activated and circulating endothelial cells.29 Therefore, targeted manipulation of integrin αvβ3-RGD interactions on the polymer substrate may contribute to the improvement of endothelialization. Song Li et al. have reported some studies for endothelialization and vascularization based on electrospun fibrous scaffolds.30-33 ACS Paragon Plus Environment

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Herein, we designed a novel biodegradable 3D electrospun microfibrous scaffold, whose surface was engineered with antifouling linear polyglycerol (LPG) and thermo-responsive PNIPAM via catechol chemistry for reversibly control cell adhesion and regulate a triggered presentation of cell-adhesive ligands dynamically controlled cell adhesion (Scheme 1). In addition, the cyclic (RGDfK) was selected as cell-adhesive ligand in this study, which exhibited a highly specific affinity for αvβ3 in the nanomolar range (IC50 = 2.6 nM) but was inactive for αvβ5 and cyclic (RGDfK) peptide represents a feasible way to restrict its conformational space and increase its bioactivity and receptor selectivity.34 Our previous studies have demonstrated that LPG surface modifications exhibited efficient antifouling properties towards proteins and cells.35-37 The surface modified with combinations of LPG and cyclic (RGDfK)-functionalized PNIPAM (cRGD-PNIPAM) was able to confer the dynamic presence of RGD ligand on the surface for controlled cell adhesion. The process of cell adhesion and release behaviors on the surface could be reversibly performed from 25 °C to 37 °C. Moreover, endothelial progenitor cells, which can express αvβ3 integrin to form high affinity cRGD-integrin binding, have played a major role in endothelial cell homing and adhesion.38,39 This multifunctional scaffold can provide an endothelial cell selective adhesion to achieve in situ targeted cell recruitment, which might be applied for accelerated endothelialization. RESULTS AND DISSCUSSION It has been reported that polydopamine (PDA), which is formed by oxidative polymerization of dopamine monomers can be coated on various substrates to fabricate multifunctional coatings with high stability and be extensively used for further surface modification via Michael-type addition or Schiff-base reaction based on catechol group with a thiol/amino-group.40-45 In this study, the microfibrous PCL meshes were prepared by electrospinning with an average thickness of 3 µm and a fiber diameter of ca. 1.3 µm, which was observed by SEM (Figure 1A). The FPCL were facially decorated with PDA coating by immersing fibrous meshes into the dopamine MOPS solution overnight. The PDA coating was highly homogenous on the fiber surface, and the morphology of the fibers did not altered after coating (Figure 1B). Before PDA modification, the water contact angle (WCA) of the PCL fibrous surface was over 120°. However, the PDAcoated PCL (FPCL) fibrous surface became highly hydrophilic and the water droplet rapidly absorbed by the surface during the contact angle measurement. The diameter of ACS Paragon Plus Environment


microfiber increased from 1.3 ± 0.2 µm to 2.8 ± 0.5 µm after PDA coating (Figure 1C). In addition, SEM images might not provide enough information on how PDA coating affects pore size distribution, which was a very important parameter for cell adhesion efficiency.46-50 The porosity of microfiber increased from 81.5 ± 0.8 % to 88.2 ± 0.6 % after PDA coating as shown in Figure 1D. These results indicated that the scaffolds can provide enough surface area for cell attachment after PDA coating. Thiolated cRGD-PNIPAM was synthesized by RAFT polymerization using 2(dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester as a chain transfer agent, which was followed by cRGD coupling via amide chemistry and ammonolysis of the thiocarbonyl moiety (Scheme S1). To reduce the weight loss, we predissolved the 10 mg cRGD into 1 mL of Milli-Q water. The products of thiolated cRGDPNIPAM was final purified by freeze-drying, yield 50%. We used 200 µg mL-1 and 100 µg mL-1 for NMR and UV-Vis spectrum, respectively. The rest products were all used for further coating study. According to the 1H-NMR (Figure S1) and UV-Vis spectra (Figure S2), thiolated cRGD-PNIPAM was successfully synthesized with Mw of 7,500 g·mol-1 (PDI = 1.2, GPC). As determined by UV transmittance, the LCST of cRGD-PNIPAM (200 µg mL-1) was evaluated at around 29 °C (Figure S3). Thiolated cRGD-PNIPAM and aminocapped LPG were used for further modification to the FPCL. The modification procedure was carried out by immersing the fiber meshes into the mixed polymer solution overnight. The fiber meshes were modified with different concentration ratios of LPG and PNIPAM on the fiber surface: only LPG (FPCL-LPG), 5/5 of LPG/PNIPAM (FPCL-LPG-PNIPAM), 5/1 of LPG/cRGD-PNIPAM (FPCL-LPG-PNIPAMcRGD1) and 5/2 of LPG/cRGD-PNIPAM (FPCL-LPG-PNIPAMcRGD2) mixtures (Table

1). It has been reported that the

hydrophilic/hydrophobic properties of the fiber surface could be thermo-switchable by utilizing lower critical solution temperature (LCST) of thermo-responsive PNIPAM.51,52 As show in Figure 2, FPCL with only LPG modification on the fiber surface displayed similar wettability as the PDA-coated fiber surface (Figure 1B), in which the water droplets were rapidly absorbed, regardless whether the temperature was kept at 25 °C or 37 °C. In contrast, the wettability of fiber surfaces with combined modifications of cRGD-PNIPAM exhibited evident changes. The water droplets were absorbed by the surface at 25 °C, but suspended at 37 °C with WCAs of 14.5° ± 3.5°, 20.3° ± 5.2°, 40.5° ± 4.8° for FPCL-LPG-PNIPAMcRGD1, FPCL-LPGPNIPAMcRGD2, and FPCL-LPG-PNIPAM, respectively. The morphology of the fibers did not ACS Paragon Plus Environment

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change after coating with different ratios of linkers. The results confirmed the thermoresponsiveness of the multi-functionalized fiber surface can reversibly switch by static water contact angle measurements between 25 °C and 37 °C (Table 1). Also, a higher PNIPAM decoration increased the surface hydrophobicity at 37 °C. FT-IR spectra of different scaffolds were shown in Figure S4. The spectrum of all the scaffold exhibited main peaks at 2949 cm-1 and 1727 cm-1 corresponding to the PCL peaks for asymmetric CH2 stretching and carbonyl stretching, respectively.53 The C=C stretches and N-H bending vibration of PDA were evident around 1600 cm-1.54 The absorbance at 3400 cm-1 was attributed to the hydroxyl of LPG.55 The main peaks of PNIPAM were 3292 cm-1 for secondary amide N-H stretching and 1540 cm-1 for secondary amide C=O stretching, respectively.56 The results indicated the successful formation of LPG and PNIPAM on the FPCL surface. Mechanical strength test revealed that there was no statistically significant changes of tensile strength and elongation at break after modification, suggesting the flexibility of PCL microfibers was still retained (Figure S5). The availability of surface grafted cRGD peptide on microfiber surfaces was characterized by immunofluorescent staining. Significant fluorescent signal could be observed on both of the FPCL-LPG-PNIPAMcRGD1 and FPCL-LPG-PNIPAMcRGD2 surfaces after incubation with their corresponding Alexa Fluor-488conjugated with a commercial human integrin αvβ3 as shown in Figure S6. It was found that the amounts of bound peptide on the FPCL-LPG-PNIPAMcRGD surfaces did not show impressive reduction. Specifically, we hypothesized that the αvβ3 positive cells would quickly attach on the thermo-responsive fiber surface with cRGD moieties at 25 °C, because the cRGD moieties becomes available on the surface to integrin binding at the headgroup of the stretched PNIPAM below the LCST. Above the LCST, the PNIPAM shrinks, leaving only the antifouling LPG exposed on the surface, which would result in a fast release of the cells. Furthermore, this process could be reversed by thermo-control (Scheme 1B). To test the specificity of the microfibrous scaffolds, we set up a parallel cell adhesion by comparing the FPCL-LPG-PNIPAMcRGD2, FPCL-LPG-PNIPAMcRGD1 and FPCL-LPGPNIPAM at 25 °C and 37 °C, respectively. Bare FPCL and FPCL modified with LPG were used as positive and negative controls. We used αvβ3 integrin positive human endothelial progenitor cell line (green fluorescently pre-stained HUVECs) and one αvβ3 integrin negative epithelial cell of human lung carcinoma cell line (blue fluorescently pre-stained A549) (Figure 3A). The average attached cell number of HUVECs (targeted cells) was 4ACS Paragon Plus Environment


fold higher than that of A549 (non-targeted cells) on FPCL-LPG-PNIPAMcRGD2 and FPCLLPG-PNIPAMcRGD1 at 25 °C. The average cell attachment efficiencies for HUVECs were 64%, 52% and 20%, 14% for FPCL-LPG-PNIPAMcRGD2, FPCL-LPG-PNIPAMcRGD1 at 25 °C and 37 °C, respectively. There was no specific difference between the HUVECs and A549 on FPCL and FPCL-LPG. FPCL-LPG and FPCL exhibited cell-repellent and cell adhesion, respectively, and the different temperatures did not significantly affect the cell attachment. Moreover, based on thermo-responsive hydrophilic and hydrophobic transition of PNIPAM, the cell-attachment number of HUVECs and A549 on FPCL-LPG-PNIPAM at 37 °C were 2-fold higher than at 25 °C, respectively. There was also no specific difference between the HUVECs and A549 on FPCL-LPG-PNIPAM. The average cell attachment efficiency for HUVECs was 42% at 37 °C, which was lower than FPCL-LPGPNIPAMcRGD2 (64%) and FPCL-LPG -PNIPAMcRGD1 (52%) at 25 °C (Figure 3B-C). These results indicated that cRGD was the head group of the stretched PNIPAM at 25 °C (below the LCST of PNIPAM) demonstrated enhancement in cell adhesion and the cell-repellent was observed at 37 °C (above the LCST of PNIPAM) due to cRGD moiety shielded by the antifouling LPG layer. If there was no RGD modification, the surface of FPCL-LPGPNIPAM will become hydrophobic at 37 °C (above the LCST) that can induce cell attachment and become hydrophilic at 25 °C (below the LCST) that can detach the cells. Moreover, the cRGD modified microfibrous scaffolds were more specifically and efficiently to recognize endothelial cells in cell mixtures at 25 °C, when the cRGD moieties becomes available on the surface, compared with pure hydrophobic interaction using thermo-trigged PNIPAM alone at 37 °C. To evaluate the ability of in situ reversible endothelial cell attachment and release based on the thermo-responsive fiber surface, the dynamic cell adhesion/release tests were performed in a microfluidic-based chip, which can better mimic the natural vascular environment. In addition, serum contains proteins that could be absorbed on the surface and induce nonspecific cell adhesion in the chips, thus serum-free condition was used for the following experiments, which was conducive to confirm the specific interactions between cRGD moieties and αvβ3 positive cells. Briefly, the sterilized samples were attached to the bottom of the device channel, and the temperature of the channel was controlled by a thermo-control platform (Scheme S2, Movie S1). Depicted in Figure 4, FPCL-LPG exhibited cell-repellent properties at both temperatures of 25 °C and 37 °C, as ACS Paragon Plus Environment

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very few HUVECs attached to the surface during the thermo-cycles. As a control, the results of the cell-adhesion ability on FPCL without further modification indicated that cells could successfully attach to the fiber surface. Furthermore, the different temperatures did not significantly affect the cell attachment on FPCL. To the contrary, the cells were effectively attached on the fiber surface when the flow was applied on the FPCL-LPGPNIPAMcRGD1 and FPCL-LPG-PNIPAMcRGD2 at 25 °C, and the attached cells were quickly released from the surface as the temperature increased to 37 °C. Furthermore, the cell attachment capability decreased to the low level as well as FPCL-LPG at 37 °C. On the other hand, FPCL-LPG-PNIPAM also exhibited cells attach and release property, but opposite tendency from 25 °C to 37 °C compared with cRGD-mediated fiber surface. The results confirmed that the cell attachment and release were thermo-reversible on the fiber surface because of the thermo-switchable property of PNIPAM incorporated with the antifouling property of LPG and cRGD triggered cell adhesion. The quantification tests revealed that the number of cells attached by the cRGD-modified surface at 25 °C was 2.0-2.5-fold higher than the FPCL-LPG-PNIPAM surface at 37 °C. The attached cells on the surface of FPCL-LPG-PNIPAM were merely held by weak hydrophobic interactions with the cells. Also, the surface with a higher rate of cRGD-functionalization induced a stronger cell attachment by cRGD-mediated attachment. However, when the temperature increased to 37 °C, the cell number on FPCL-LPG-PNIPAMcRGD1 and FPCL-LPG-PNIPAMcRGD2 were quite similar to the low density, which meant the cell attachment behavior was predominantly affected by the antifouling LPG. Once again, the cell number on FPCL-LPG-PNIPAMcRGD1 and FPCL-LPGPNIPAMcRGD2 returned to a similar level compared with the initially attached cell number at 25 °C and released again by rising the temperature to 37 °C, respectively (Figure 5). The cells attachment and release efficiency on FPCL-LPG-PNIPAMcRGD2, FPCL-LPG-PNIPAMcRGD1 and FPCL-LPG-PNIPAM exhibited stable tendencies during the 2 cycles. On the first cycle, the average cell-attachment efficiency of FPCL-LPG-PNIPAMcRGD2 and FPCL-LPG-PNIPAMcRGD1 was 65% and 56%, which exhibited the same level as in the static test. In comparison with FPCL-LPGPNIPAM showed a lower attachment efficiency, suggesting that at both cRGD-mediated scaffolds showed a more effective attachment, especially under dynamic conditions. The average cell-release efficiency was 87% and 85% for FPCL-LPG-PNIPAMcRGD2 and FPCL-LPGPNIPAMcRGD1, respectively, when changing the temperature from 25 °C to 37 °C. The average cell-release efficiency of FPCL-LPG-PNIPAM was 68%, when changing the ACS Paragon Plus Environment


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temperature from 37 °C to 25 °C. These results suggested that cRGD-mediated surface can efficiently and reversibly induce the cell-attachment and cell-release by switching the temperature from 25 °C to 37 °C (Figure S7). In order to study whether cells can grow on our engineered 3D fibrous scaffolds after thermo-induced cRGD-mediated attachment to achieve in situ recruitment, the cells that were attached on FPCL-LPG-PNIPAMcRGD1 and FPCL-LPG-PNIPAMcRGD2 were preincubated for 1 h at 25 °C and then incubated at 37 °C for another 1 h. The fluorescence images revealed that the morphology of most cells started to change and distributed them evenly and spread among the microfibrous matrix (Figure 6A-C). In addition, we evaluated viability of attached HUVECs on different fibrous scaffolds utilizing CCK-8 assay on 24 h and 3 days. It was found that the metabolic activity of HUVECs on both scaffolds increased with time (Figure 6D). These findings indicated that our designed scaffold enhance selective cell adhesion at room temperature by cRGD-mediated attachment for the initial stages of cell recognition and minimize the non-specific absorption to the scaffolds based on antifouling property of LPG. Moreover, the 3D fibrous

scaffold

can

provide

good

microenvironment

for

further

increasing

endothelialisation, as well as widening its application in the vascular regenerative medicine.

CONCLUSION In summary, we have successfully developed a biodegradable, electrospun PCL scaffold using the combined functions of thermo-switchable cRGD-PNIPAM and antifouling LPG to reversibly control the adhesion and release of endothelial cells on the microfiber substrate. We demonstrated that the functionalized scaffolds can control the selective endothelial cell adhesion in a dynamic manner and the ratio of the adhesive factor on the surface is related to the attachment efficiency onto the scaffolds. Moreover, the cRGDmediated cell attachment can accelerate targeted endothelial cells spread and growth and formation of monolayer compared with pure hydrophobic and hydrophilic interaction using thermo-trigged PNIPAM alone, especially under dynamic conditions. Therefore, this 3D microfibrous scaffold with its advanced multifunctional surface would provide a promising platform for in situ targeted endothelial cell recruitment and endothelialization


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of polymer substrate. We propose that this multifunctional scaffold is a particularly promising candidate for formation of vascular in the future.

ASSOCIATED CONTENT

Supporting Information The supporting information is available free of charge on the ACS Publication website at DOI: 10.1021/acsami.6bxxxxx. Experimental

section;

Additional

figures

cited

in

this

article,

synthesis

and

characterization of compounds; used characterization methods; Figure S1-S7; Scheme S1S2 Movie S1. Dynamic cell adhesion test. The cell flow through the chip at 25 °C and fluorescence excited at 488 nm.

AUTHOR INFORMATION Corresponding Author *

E-Mail: [email protected]

Author Contributions #

J. Zhang and W. Chen contributed equally to this work and share the co-first authorship.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was funded by SFB 1112 and the Focus Area Nanoscale of Freie Universität Berlin. J.Z., L.Y., and M.L. also thank the China Scholarship Council (CSC) from P. R. China. We would like to acknowledge the assistance of the Core Facility BioSupraMol ACS Paragon Plus Environment


supported by the DFG. We would like to thank Dr. Pamela Winchester for language polishing this manuscript.

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Campo, A.; Garcia, A. J., Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials. Nat. Mater. 2015, 14 (3), 352-360. (11) Park, J.; Kim, D. H.; Kim, H. N.; Wang, C. J.; Kwak, M. K.; Hur, E.; Suh, K. Y.; An, S. S.; Levchenko, A., Directed migration of cancer cells guided by the graded texture of the underlying matrix. Nat. Mater. 2016, 15 (7), 792-801. (12) Jeon, H.; Koo, S.; Reese, W. M.; Loskill, P.; Grigoropoulos, C. P.; Healy, K. E., Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces. Nat. Mater. 2015, 14 (9), 918-923. (13) Ye, K.; Wang, X.; Cao, L.; Li, S.; Li, Z.; Yu, L.; Ding, J., Matrix Stiffness and Nanoscale Spatial Organization of Cell-Adhesive Ligands Direct Stem Cell Fate. Nano Lett. 2015, 15 (7), 4720-4729. (14) An, Q.; Brinkmann, J.; Huskens, J.; Krabbenborg, S.; de Boer, J.; Jonkheijm, P., A supramolecular system for the electrochemically controlled release of cells. Angew. Chem., Int. Ed. 2012, 51 (49), 12233-12237. (15) Kadem, L. F.; Holz, M.; Suana, K. G.; Li, Q.; Lamprecht, C.; Herges, R.; Selhuber-Unkel, C., Rapid Reversible Photoswitching of Integrin-Mediated Adhesion at the Single-Cell Level. Adv. Mater. 2016, 28 (9), 1799-1802. (16) Salierno, M. J.; Garcia-Fernandez, L.; Carabelos, N.; Kiefer, K.; Garcia, A. J.; del Campo, A., Phototriggered fibril-like environments arbitrate cell escapes and migration from endothelial monolayers. Biomaterials 2016, 82, 113-123. (17) Weis, S.; Lee, T. T.; del Campo, A.; Garcia, A. J., Dynamic cell-adhesive microenvironments and their effect on myogenic differentiation. Acta Biomaterialia 2013, 9 (9), 8059-8066. (18) Roberts, J. N.; Sahoo, J. K.; McNamara, L. E.; Burgess, K. V.; Yang, J.; Alakpa, E. V.; Anderson, H. J.; Hay, J.; Turner, L. A.; Yarwood, S. J.; Zelzer, M.; Oreffo, R. O.; Ulijn, R. V.; Dalby, M. J., Dynamic Surfaces for the Study of Mesenchymal Stem Cell Growth through Adhesion Regulation. Acs Nano 2016, 10 (7), 6667-6679. (19) Wong, D. S.; Li, J.; Yan, X.; Wang, B.; Li, R.; Zhang, L.; Bian, L., Magnetically Tuning Tether Mobility of Integrin Ligand Regulates Adhesion, Spreading, and Differentiation of Stem Cells. Nano Lett. 2017, 17 (3), 1685-1695.


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Table 1. Compositions of PNIPAM- and LPG-modified PCL microfibers and static water contact angles measured at 25 °C and 37 °C. Samples FPCL FPCL-LPG FPCL-LPG-PNIPAM b FPCL-LPG-PNIPAMcRGD1 FPCL-LPG-PNIPAMcRGD2

LPG (w/v %) 0 0.5 0.5 0.5 0.5

cRGD-PNIPAM (w/v %) 0 0 0 0.1 0.2

Contact angle (°) a 25 °C 37 °C 129.2 ± 7.2 128.6 ± 4.5 0c 0c c 0 40.5 ± 4.8 0c 14.5 ± 3.5 0c 20.3 ± 5.2

a

The results were presented as an average value of five measurements at different areas of each sample. b The weight ratio of PNIPAM/LPG was set as 1/1 in the modification process. c The water droplets were absorbed by the surface and the details are shown in Figure 2.



Scheme 1. (A) Illustration of PCL microfibers modified with cRGD-functionalized PNIPAM (cRGD-PNIPAM) and linear polyglycerol (LPG) through the universal polydopamine (PDOPA) coating on the fiber surface by Michael-type addition and Schiff-base reaction, respectively. The two linkers can be covalently immobilized on to the fiber surfaces. (B) Thermo-switchable cell attachment and release behavior on the advanced functional fiber surface: thermo-switchable cRGD-PNIPAM for dynamic cell attachment via cRGD-mediated interaction at 25 °C (below the LCST); and LPG for antifouling purpose at 37 °C (above the LCST).


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Figure 1. SEM image of (A) PCL microfibers, and (B) PDA-coated PCL microfibers with their corresponding images of water contact provided as inlets in each image. The black arrows represent the PDA homogeneously coated on the surface of microfibers. Scale bar: 5 µm. (C), (D) The diameter and porosity of microfiber before and after PDA coating. All data were expressed as the means ± standard deviations (s.d) with independent experiments (n = 100). The statistical analysis was performed using one-way analysis of variance (ANOVA) with the Tukey significant difference post hoc test using Origin 9.0 software. The data is indicated with (*) for p < 0.05.



Figure 2. SEM images and water contact angle images on the fiber surface modified with LPG, cRGD-PNIPAM, and PNIPAM at 25 °C and 37 °C. Scale bar: 5 µm.


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Figure 3. (A) Specific identification of the designed scaffolds at 25 °C and 37 °C based on prestained with cell traceTM CFSE and Violet, respectively. Two different cell lines, HUVECs (green) were used as a positive control and A549 (blue) was chosen as a negative control. Scale bar = 100 µm. (B) Quantification analysis of HUVECs and A549 attachment at 25 °C and 37 °C. (C) Comparison of attach efficiency between HUVECs and A549 at 25 °C and 37 °C. Bare PCL fibers and PCL fibers modified with LPG were used as positive and negative controls, respectively. (Ten images with cells counted for each condition. Data plotted as mean ± SD)



Figure 4. Fluorescence images of HUVECs attached on the different fiber surfaces at 25 °C and 37 °C during two cycles. A) FPCL-LPG, B) FPCL-LPG-PNIPAMcRGD1, C) FPCL-LPGPNIPAMcRGD2, D) FPCL-LPG-PNIPAM, E) FPCL. Bare PCL fibers and PCL fibers modified with LPG were used as positive and negative controls, respectively. Scale bar: 5 µm.


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Figure 5. Quantification analysis of HUVECs attachment on the different fiber surfaces performed with two cycles at 25 °C and 37 °C.



Figure 6. Fluorescence images of viable HUVECs on the fibers after two steps of incubation: 1 h incubation at 25 °C for cRGD-mediated cell attachment on the surface, and another 1 h incubation at 37 °C for further cell growth labelled with cell traceTM CFSE. (A) FPCL-LPGPNIPAMcRGD1, (B) FPCL-LPG-PNIPAMcRGD2, and (C) FPCL. Scale bar: 20 µm. (D) CCK-8 of HUVECs viability after 24 h and 3 d culture on the FPCL, FPCL-LPG-PNIPAMcRGD1 and FPCLLPG-PNIPAMcRGD2, respectively, after dynamically controlled cell adhesion.


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For TOC Only



Scheme 1. (A) Illustration of PCL microfibers modified with cRGD-functionalized PNIPAM (cRGD-PNIPAM) and linear polyglycerol (LPG) through the universal polydopamine (PDOPA) coating on the fiber surface by Michael-type addition and Schiff-base reaction, respectively. The two linkers can be covalently immobilized on to the fiber surfaces. (B) Thermo-switchable cell attachment and release behavior on the advanced functional fiber surface: thermo-switchable cRGD-PNIPAM for dynamic cell attachment via cRGD-mediated interaction at 25 °C (below the LCST); and LPG for antifouling purpose at 37 °C (above the LCST). 898x544mm (96 x 96 DPI)


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Figure 1. SEM image of PCL microfibers (A), and PDA-coated PCL microfibers (B) with their corresponding images of water contact provided as inlets in each image. The black arrows represent the PDA homogeneously coated on the surface of microfibers. Scale bar: 5 µm. (C), (D) The diameter and porosity of microfiber before and after PDA coating. All data were expressed as the means ± standard deviations (s.d) with independent experiments (n = 100). The statistical analysis was performed using one-way analysis of variance (ANOVA) with the Tukey significant difference post hoc test using Origin 9.0 software. The date is indicated with (*) for p < 0.05. 186x149mm (150 x 150 DPI)



Figure 2. SEM images and water contact images on the fiber surface modified with LPG, cRGD-PNIPAM, or PNIPAM at 25 °C and 37 °C. Scale bar: 5 µm. 225x123mm (150 x 150 DPI)


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Figure 3. A) Specific identification of the designed scaffolds at 25 °C and 37 °C based on pre-stained with cell traceTM CFSE and Violet, respectively. Two different cell lines, HUVECs (green) were used as a positive control and A549 (blue) was chosen as a negative control. Scale bar = 100 µm. B) Quantification analysis of HUVECs and A549 attachment at 25 °C and 37 °C. C) Comparison of attach efficiency between HUVECs and A549 at 25 °C and 37 °C. Bare PCL fibers and PCL fiber modified with LPG were used as positive and negative controls, respectively. (Ten images with cells counted for each condition. Date plotted as mean ± SD) 494x559mm (150 x 150 DPI)



Figure 4. Fluorescence images of HUVECs attached on the different fiber surfaces at 25 °C and 37 °C during two cycles. A) FPCL-LPG, B) FPCL-LPG-PNIPAMcRGD1, C) FPCL-LPG-PNIPAMcRGD2, D) FPCL-LPG-PNIPAM, E) FPCL. Bare PCL fibers and PCL fiber modified with LPG were used as positive and negative controls, respectively. Scale bar: 5 µm. 117x132mm (220 x 220 DPI)


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Figure 5. Quantification analysis of HUVECs attachment on the different fiber surfaces performed with two cycles at 25 °C and 37 °C. 584x304mm (300 x 300 DPI)



Figure 6. Fluorescence images of viable HUVECs on the fibers after two steps of incubation: 1 h incubation at 25 °C for cRGD-mediated cell attachment on the surface, and another 1 h incubation at 37 °C for further cell growth labelled with cell traceTM CFSE. (A) FPCL-LPG-PNIPAMcRGD1, (B) FPCL-LPG-PNIPAMcRGD2, and (C) FPCL. Scale bar: 20 µm. (D) CCK-8 of HUVECs viability after 24 h and 3 d culture on the FPCL, FPCL-LPGPNIPAMcRGD1 and FPCL-LPG-PNIPAMcRGD2, respectively, after dynamically controlled cell adhesion. 186x120mm (300 x 300 DPI)


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TOC 371x193mm (96 x 96 DPI)


Selective Endothelial Cell Adhesion Via Mussel ... - ACS Publications

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