Programmed Release of Multimodal, Cross-Linked Vascular

Subscriber access provided by Nottingham Trent University

Surfaces, Interfaces, and Applications

Programmed Release of Multi-modal, Crosslinked Vascular Endothelial Growth Factor and Heparin Layers on Electrospun Polycaprolactone Vascular Grafts Dongfang Wang, Xiaofeng Wang, Zhi Zhang, Lixia Wang, Xiaomeng Li, Yiyang Xu, Cuihong Ren, Qian Li, and Lih-Sheng Turng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10621 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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 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 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.

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 33 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

1 2

Programmed Release of Multi-modal, Crosslinked Vascular Endothelial Growth Factor

3

and Heparin Layers on Electrospun Polycaprolactone Vascular Grafts

4 5

Dongfang Wang a,b,c,d,1, Xiaofeng Wang a,b,1, Zhi Zhang c,d, Lixia Wang a,b, Xiaomeng Li a,b,

6

Yiyang Xu b,c,d, Cuihong Ren a,b, Qian Li b**, and Lih-sheng Turng c,d*

7 8

a

9

China

School of Mechanics and Engineering Science, Zhengzhou University, Zhengzhou 450001, P. R.

10

b

11

University, Zhengzhou 450001, P. R. China

12

c

Department of Mechanical Engineering, University of Wisconsin, Madison, WI, USA

13

d

Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, USA

14

1

These authors contributed equally to this paper.

National Center for International Research of Micro-Nano Molding Technology, Zhengzhou

15 16

Keywords: Programmed and sustained drug release; Endothelialization; Antithrombogenicity;

17

Surface functionalization; Angiogenesis.

18

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

1

Abstract

2

Viable tissue-engineering small-diameter vascular grafts should support rapid growth of an

3

endothelial cell layer and exhibit long-term antithrombogenic property. In this study, multi-

4

layers of various bioactive molecules, such as VEGF and heparin, on an electrospun

5

polycaprolactone (PCL) scaffold have been developed through repeated electrostatic adsorption

6

self-assembly (up to 20 layers), followed by genipin crosslinking. Programmed and sustained

7

release of biomolecules embedded within the multi-layered structure can be triggered by matrix

8

metallopeptidase 2 (MMP-2) enzyme in vitro. The result is an early and full release of VEGF to

9

promote rapid endothelialization on the intended vascular grafts, followed by a gradual but

10

sustained release of heparin, for long-term anticoagulation and antithrombogenicity. This method

11

of forming biologically responsive, multi-modal delivery of VEGF and heparin is highly suitable

12

for all hydrophobic surfaces and provides a promising way to meet the critical requirements of

13

engineered small-diameter vascular grafts.

14 15 16

2


Page 2 of 33

Page 3 of 33 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

1


1. Introduction

2

Currently, artificial blood vessels with diameters smaller than 6 millimeters—the kind

3

needed for bypass surgeries—are not commercially available1. However, such a product could

4

transform treatment for cardiovascular diseases, which are the leading cause of death globally2.

5

The major reason for their lack of availability is the high risk of luminal thrombosis, which is

6

caused by the lack of an endothelial layer, and anastomotic intimal hyperplasia3. Vascular tissue

7

engineering has become an important topic in biomedical engineering, and various biodegradable

8

materials have been employed to fabricate artificial blood vessels. However, these materials tend

9

to suffer from a slow endothelialization rate and a high risk of thrombosis4. Therefore, how to

10

effectively promote endothelialization and inhibit intimal hyperplasia for antithrombogenicity

11

have been the subject of study for many years5–7.

12

There have been many effective ways to regulate endothelial cells by surface modification.

13

For example, polypeptides—including RGD, VEGF, REDV, and YIGSR—have been used to

14

target and promote rapid endothelialization8,9. In particular, VEGF can be combined with a

15

specific receptor (VEGFR) on the cytomembrane to promote endothelial cell adhesion,

16

proliferation, differentiation, migration, and vascularization by the VEGF/VEGFR-2 signal

17

pathway10. Also, some researchers have tried to promote antithrombogenicity by grafting

18

anticoagulant factors, including nitric oxide (NO) donor and heparin11,12. Although rare side

19

effects of heparin therapy, such as thrombocytopenia, bleeding events, and osteopenia13, do exist,

20

it had an incontrovertible effect on inhibiting thrombus formation by catalytically increasing the

21

affinity of antithrombin III (AT III) to thrombin in the process of tissue regeneration14, while

22

impairing angiogenesis through the inhibition of microRNA-10b15. It should be noted that

23

endothelialization and antithrombogenicity are not two unrelated and isolated processes, and that 3



1

the traditional single-factor delivery has not proven to be clinically efficacious in pathological

2

states16. During the re-endothelialization process, the risk of thrombosis is still present. This is

3

because the endothelial cells can often fall off the endothelium of the regenerated tissue scaffold

4

under the combined actions of mechanical force, shearing from blood flow, and hyperplasia of

5

smooth muscle cells, which will eventually lead to in-graft restenosis 4,17. Thus, it is essential to

6

introduce rapid endothelialization and long-term antithrombogenicity into artificial blood vessels

7

to fabricate viable and functional grafts18. Nevertheless, there are many technical challenges that

8

hamper the growth of a complete endothelial layer and the ability to maintain long-term

9

anticoagulant capacity. Strategies that respect the complex nature of diseases will have a better

10

chance at being successful. It will be of interest to study the delivery of opposing factors (i.e.,

11

VEGF and heparin) to modulate several stages of the pathology over time19, and to see if they

12

will bring more benefits than possible deficiencies compared to single heparin therapy.

13

To address the challenges mentioned above, one possible strategy is to construct a

14

biologically responsive, multi-modal delivery system that utilizes opposing factors to create a

15

stable activation gradient that can release VEGF (an angiogenic factor) and heparin (an

16

anticoagulant and antiangiogenic factor) in a programmed and sustained fashion at the tissue

17

regeneration site to achieve multiple therapeutics in a temporal manner16,19. To this end, we have

18

designed and fabricated a new multi, crosslinked layer consisting of VEGF and heparin on an

19

electrospun polycaprolactone (PCL) matrix to circumvent the problems of poor protein activity

20

and burst release, while achieving rapid endothelialization and long-term antithrombogenicity.

21

The fabrication process of the multi-layered structure on a PCL matrix includes the

22

following: (a) electrospinning of PCL to obtain a biomimetic substrate, (b) hydrophilic treatment

23

of the PCL scaffold surface by plasma, followed by the introduction of amino groups through 4


Page 4 of 33

Page 5 of 33 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


1

polyethyleneimine (PEI) grafting, (c) self-assembly and deposit of layers of VEGF and heparin

2

alternatively through electrostatic adsorption, (d) genipin crosslinking to obtain a stable multi-

3

layered structure, and (e) programmed release of VEGF and heparin in vitro triggered by

4

metallopeptidase 2 (MMP-2). As will be shown below, this programmed and sustained release

5

behavior of the multi, crosslinked layer on the PCL scaffolds promotes rapid endothelialization

6

and exhibits antithrombogenicity performance.

7 8

2. Materials and Methods

9

2.1. Materials

10

PCL, N,N-dimethylformamide (DMF), chloroform (CF), polyethyleneimine (PEI), and

11

gelatin were all purchased from Aladdin (Shanghai, China). VEGF, MMP-2, and heparin were

12

purchased from Sigma–Aldrich (Wisconsin, WI, USA).

13

2.2. Electrospinning of PCL Fibers

14

The PCL fiber scaffold was produced by electrospinning as described in our previous study,

15

and the diameter of the grafts was adjusted to 3 mm20. The PCL was dissolved in the solution of

16

CF and DMF (v/v = 3:7, 15% (W/V)). The voltage, temperature, and needle-to-collector distance

17

were 18 kV, 25°C, and 18 cm, respectively. A roller collector was used for this experiment.

18

2.3. Surface Functionalization

19

Figure 1 shows the structure of a multi, crosslinked, layered structure on the electrospun

20

PCL surface and the programmed and sustained release profile of VEGF and heparin, which

21

form the biologically responsive extracellular matrix (ECM). The surface functionalization 5



1

process was as follows. The electrospun PCL scaffold was first soaked in alcohol and sonicated

2

for 30 minutes, vacuum dried, and then treated with oxygen plasma for 10 minutes to enhance its

3

surface hydrophilicity via a plasma etcher (PlasmaEtch PE-200) (RF power 100 W, oxygen flow

4

rate 20 cm3/min1). Then, to introduce enough amino groups (NH3+), the scaffold was

5

immediately immersed in a solution of PEI (5 mg/ml), shaken in the enclosed chamber for 16

6

hours, and washed three times with pure water. Next, the PCL scaffold was alternately soaked in

7

a polycationic gelatin solution (5 mg/ml, pH 4.5) and a polyanionic heparin solution (5 mg/ml,

8

pH 7.0) for 30 minutes each, leading to the self-assembled electrostatic adsorption of gelatin and

9

heparin molecules on the PCL scaffold surface. The soaking process was repeated 14 times to

10

obtain seven “gelatin-heparin” bi-layered structures, and the samples were named PGH (for

11

PCL-gelatin-heparin). Then, extra steps of soaking the treated sample with the VEGF solution

12

(10 ng/ml) in PBS were added to cap the PGH scaffold with two “gelatin-heparin-VGEF” tri-

13

layered structures. The VEGF-coated PGH samples were named PGHV (for PCL-gelatin-

14

heparin-VEGF). Finally, the PGH and PGHV scaffolds were crosslinked for 48 hours using a

15

genipin solution (2 mg/ml) to obtain a stable multi, crosslinked, layered structure; they were

16

named cPGH and cPGHV, respectively.

6


Page 6 of 33

Page 7 of 33 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


1 2

Fig. 1 Schematic illustration of the multi, crosslinked, layered structure and the programmed and

3

sustained release profile of VEGF and heparin triggered by the MMP-2 enzyme.

4 5

2.4. Characterization Methods

6

2.4.1. Scanning Electron Microscopy (SEM)

7

The morphologies of the treated scaffold surfaces were analyzed by a scanning electron

8

microscope (LEO GEMINI 1530, Zeiss, Germany) with an accelerating voltage of 10 kV. Prior

9

to SEM, the scaffolds were coated with gold for 40 s.

10

2.4.2. Atomic Force Microscopy (AFM)

11

The surface topographies of the treated surfaces were characterized in tapping mode at room

12

temperature by an atomic force microscope (AFM) (Agilent 7500, Agilent Instruments, USA). 7



1

PPP-CONTR (PointProbe® Plus, NANOSENSORS™) AFM probes, with a nominal force

2

constant value of 0.2 N/m, were used. Image analysis was done using the CSPM Imager software.

3

2.4.3. Fourier Transform Infrared Spectroscopy (FTIR)

4

To characterize the FTIR spectra of the modified samples, the recorded in transmittance

5

(ATR) mode was used in the range of 4000–400 cm-1 with a resolution of 4 cm-1 (Tensor 27,

6

Bruker, Germany).

7

2.4.4. X-ray Photoelectron Spectrometer (XPS)

8

To perform the surface elemental analysis of different samples, an X-ray photoelectron

9

spectrometer with a focused, monochromatic K-alpha X-ray source and a monoatomic/cluster

10

ion gun (Thermo Scientific) was used. The AVANTAGE software was used for data acquisition

11

and analysis.

12

2.4.5. Water Contact Angle (WCA) Tests

13

To analyze the wettability, the treated samples were measured by a contact angle instrument

14

(Dataphysics, OCA 15) using 2 μL of DI water droplets at room temperature. Four parallel

15

samples were tested for each group, and the average value was reported with standard deviation

16

(±SD).

17

2.5. In Vitro VEGF Release Study

18

The in vitro VEGF release study was analyzed as described in a previous study21. MMP-2

19

was used to cleave the crosslinked sites of the multi-layer biomolecular structure22. The amount

20

of VEGF released from the treated samples was quantified using a VEGF Quantitative Factor

21

ELISA kit (Thermo, WI, US). Briefly, after washing with phosphate buffer saline (PBS), cPGHV 8


Page 8 of 33

Page 9 of 33 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


1

samples were incubated in 2 ml of medium (supplemented with MMP-2, 50 ng/ml) at 37°C and 5%

2

CO2. The medium was completely replaced at each time point (i.e., 1, 2, 3, 5, 7, 9, 11, 13, and 15

3

days). The released medium was collected and analyzed by a microplate reader, and the release

4

curve was obtained according to the standard curve.

5

2.6. In Vitro Heparin Release Study

6

The sulfonic groups of heparin on the surface of treated scaffold samples form complexes

7

with toluidine blue O (TBO) dye23. The method described by Smith was selected to determine

8

the surface density of the immobilized heparin24. Briefly, the cPGHV samples were incubated in

9

1.5 ml of PBS solution (50 ng/ml MMP-2, pH 7.4) at 37°C and shaken (60 rpm) for 1, 3, 5, 7, 10,

10

14, 21, 28, 35, 42, 49, and 56 days. The released medium was replaced with fresh buffer at fixed

11

time intervals. The slow-release solution was placed in a 2 ml centrifuge tube, and 1 ml of

12

toluidine blue staining solution (0.2 wt% NaCl, 0.01M HCl, 0.04 wt% toluidine blue) was added.

13

The mixture was then shaken at room temperature for 4 hours. Next, the tube was centrifuged at

14

3500 rpm for 10 minutes. The supernatant was removed and the pellet was washed with DI water.

15

Subsequently, 1 ml of an ethanol/0.1 M NaOH mixture (v/v = 4/1) was added to the centrifuge

16

tube to dissolve the dye–heparin precipitate. A quantity of 150 μl of solution was placed into a

17

96-well plate, and the absorbance of the solution at 530 nm was measured using an ultraviolet

18

spectrophotometer. The heparin release amount was calculated using a standard curve.

19

2.7. Platelet Adhesion

20

To investigate the antithrombogenicity of the treated PCL scaffolds, platelet-rich plasma

21

(PRP) extracted from fresh pig blood was used to perform the platelet adhesion tests 25. Briefly,

22

the blood was first centrifuged at 1500 rpm for 15 minutes to obtain PRP, then 500 μL of PRP 9



1

were added to the samples, followed by incubation at 37°C for two hours. Next, the samples

2

were rinsed with PBS and fixed with PBS containing 2.5 wt% glutaraldehyde for 48 hours.

3

Thereafter, the samples were dehydrated using a series of ethanol washes (50%, 70%, 80%, 90%,

4

and 100%). Prior to SEM observation, the samples were dried in a desiccator overnight for 48

5

hours.

6

2.8. Biocompatibility Evaluations

7

2.8.1. Human Umbilical Vein Endothelial Cell (HUVEC) Culture

8

HUVECs were cultured on untreated and treated scaffolds at 37°C in a 5% CO2 and 95% air

9

environment26. To study the angiogenic capability of surface-functionalized grafts and the

10

individual effect of each factor employed, RPMI1640 containing 10% fetal bovine serum media

11

was used instead of the EGM-2 BulletKit, as the latter contains multiple factors. Additionally,

12

culture media will be consumed during cell culture, which means that, after transplantation, those

13

factors wouldn’t exist to have a long-term effect. Immobilization of targeted factors on the graft

14

surface is conducive to long-term patency and function. The cells grew and proliferated on the

15

fibers in 24-well plates for 7 days, and the medium was replaced every day. Prior to cell seeding,

16

the samples were immersed in 75% alcohol overnight, and then washed with PBS three times.

17

Afterwards, the films were sterilized by ultraviolet light for 8 hours on each side with PBS, and

18

then soaked in the complete medium for 2 h to aid protein adhesion and cell attachment. The

19

density was 2×104 cells per well. All tests were done with at least three replicates.

20

2.8.2 Cell Viability (Live/Dead Assay)

21

Cell viability was characterized using a Live/Dead Viability/Cytotoxicity Kit (KeyGEN

22

BioTECH) after culturing for 7 days. Green fluorescent calcein-AM and red fluorescent 10


Page 10 of 33

Page 11 of 33 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


1

propidium was used to image the living and dead cells, respectively. Briefly, the medium was

2

first removed from the scaffolds, followed by washing the scaffolds and cells with PBS. Next,

3

the staining solution was added directly to the scaffold and incubated for 30 min. After washing

4

with PBS, the samples were observed using a fluorescence inverted microscope (DMI3000,

5

Leica).

6

2.8.3 Cell Viability (CCK-8)

7

Cell proliferation was assessed at days 1, 3, and 5 using a CCK-8 assay (Dojindo, Japanese).

8

A quantity of 200 μL of medium containing 20 μL of CCK-8 was added directly onto the

9

scaffolds, followed by incubating for 2 h. Next, 100 μL of the medium was transferred into the

10

96-well plate, and the absorbance at 450 nm was measured using UV spectrophotometer to

11

detect the number of cells.

12

2.8.4 Cell Morphology (Cytoskeleton Assay)

13

The shape and cytoskeleton of the ECs on day 3 were characterized by CF™ 568 phalloidin

14

staining. Briefly, the samples were first fixed by incubating the scaffolds on ice with a 3.75%

15

formaldehyde solution in PBS for 15 min. Next, bovine serum albumin (BSA) was used to block

16

the samples for 30 min at room temperature, followed by incubation in 200 μL of PBS

17

containing 5 μL fluorescent phalloidin for 30 min. Then the samples were washed again and

18

incubated in 200 μL of 4’,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature,

19

followed by imaging with a Zeiss LSM880 confocal microscope system.

20

2.8.5 Cell–Scaffold Interaction

21

The interaction between the cells and the substrate on day 7 was observed using SEM.

22

Briefly, the samples stained with phalloidin/DAPI were washed with PBS and dehydrated 11



1

through a series of ethanol washes (50%, 70%, 80%, 90%, and 100%), followed by sufficient

2

drying in a vacuum oven overnight. They were then imaged using SEM.

3

2.9. Endothelial Cells (EC) Angiogenesis

4

To study the effect of the VEGF and heparin released from the cPGHV scaffolds on EC

5

angiogenesis, 1.5 x 106 endothelial cells/ml were encapsulated in 5 mg/ml of matrigel. A

6

quantity of 10 μl of matrigel was spotted in the middle of the 24-well plate and incubated for 5

7

min at 37°C for solidification. Endothelial cell media released from the PCL scaffolds was then

8

applied. Four days later, immunostaining was performed by CD-31PE, and the structures were

9

photographed using a Nikon confocal microscope27. cPGH and pure PCL scaffolds without

10

modification of VEGF and heparin were used as control samples.

11

2.10. Statistical Analysis

12 13

The mean ± standard deviation (SD) was used to characterize the data, and a single-factor analysis of variance was used to express the statistical analyses.

14 15

3. Results and Discussion

16

3.1. Surface Morphology

17

The SEM and AFM results on the surface morphology of untreated and treated PCL

18

scaffolds are shown in Figures 2 and 3, respectively. It can be seen that the untreated PCL

19

scaffold had a relatively smooth morphology (Figures 2A and 3A). After self-assembled

20

electrostatic adsorption and multilayer deposition, the surface roughness of the PGH and PGHV

21

samples increased (Figures 2B–C), and there were nanoscale features on the surface of the fibers 12


Page 12 of 33

Page 13 of 33 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


1

(cf. inset of Figures 2B–2C). Meanwhile, the surface topography showed a layered, stacked

2

structure (Figures 3B–3C), indicating the formation of the layer-by-layer, self-assembled

3

structure on the fiber surface. This was because the PCL surface treated with oxygen plasma and

4

the subsequent PEI grafting resulted in a large number of amino groups. Then, the two

5

oppositely charged materials, i.e., polycationic gelatin (positive) and polyanionic heparin

6

(negative), underwent a physical adsorption process via repeated electrostatic immersions. That

7

is, the two biomolecules were deposited layer-by-layer through self-assembled electrostatic

8

interactions between the +NH3 groups of gelatin and the −COOH groups of heparin, which

9

resulted in the layered surface topography28,29. In the first 14 layers, the bi-layered structure was

10

formed by alternating polycationic gelatin and polyanionic heparin layer adsorption. Then one

11

extra layer of VEGF was added on top of the gelatin and heparin layers and the procedure was

12

repeated once more to create two “gelatin-heparin-VGEF” tri-layered structures, utilizing the –

13

OH group of heparin to attract VEGF molecules30. As a result, the thickness and number of self-

14

assembled layers of PGHV increased and the multi layers formed. After the layer-by-layer

15

assembly, the remaining amino groups from the gelatin segment provided a binding site for the

16

final genipin crosslinking to endow the coating structure with stability.

17

After genipin crosslinking, the surface roughness of the cPGH and cPGHV samples further

18

increased (Figures 2D–3E), and the layered structure on the scaffolds became more rugged with

19

profound sub-micron-scale features (Figures 3D–3E). Furthermore, from the SEM images of

20

treated fibers, it can be seen that the multi, crosslinked layers coated the fiber surface. Also, for

21

all of the treated samples, the overlapping joint of the fibers was encompassed by the multi,

22

crosslinked layers, which was beneficial for the improvement of the mechanical properties

23

(Figures 2B–2E)31. This was because the layered structure was strengthened by the genipin 13



1

crosslinking reaction of the amino segments between the different gelatin layers32,33. Compared

2

to the surface of cPGH, the dimensions of the rugged topography of cPGHV were larger. This

3

was because the VEGF molecules were encapsulated by the layered structure. The two adjacent

4

gelatin layers were crosslinked by genipin, and heparin underwent an H-bonding interaction with

5

the −OH of the VEGF macromolecules, which formed a stronger interaction between the heparin

6

and gelatin phases30. Furthermore, the crosslinking process made these surface structures more

7

cohesive, which further increased the stiffness and surface roughness22. It is also believed that

8

the heterogeneous crosslinked structure formed by the self-assembled, multi, crosslinked layers

9

promoted cell adhesion while efficiently encapsulating growth factors for a responsive, multi-

10

modal release behavior34.

11 12

Fig. 2 SEM images of (A) untreated PCL and the treated scaffolds (B) PGH, (C) PGHV, (D)

13

cPGH, and (E) cPGHV.

14


Page 14 of 33

Page 15 of 33 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


1 2

Fig. 3 AFM images of (A) untreated PCL and the treated scaffolds (B) PGH, (C) PGHV, (D)

3

cPGH, and (E) cPGHV.

4

3.2. Surface Chemistry

5

Figure 4A shows the FTIR spectra of the heparin, gelatin, untreated PCL, and treated PCL

6

scaffold samples. Compared with heparin, gelatin, and pure PCL nanofibers, the treated PGH,

7

PGHV, and cPGHV samples all showed absorption peaks at 2860 cm-1 and 2940 cm-1, which

8

represented –CH2– stretching. All treated samples showed a peak at 1720 cm-1, corresponding to

9

the absorbance of the carbonyl group. The three treated groups presented a band at 3400 cm-1

10

and a new peak at 1660 cm-1, corresponding to O–H, N–H stretching, and an amide C=O,

11

respectively, which came from the gelatin and heparin. Furthermore, the samples showed the

12

sulfate group at 1000 cm-1, which originated from the antisymmetric vibrations of the C−O−S

13

groups of heparin, indicating that the heparin was successfully encapsulated in the multi,

14

crosslinked layers35,36. 15



1

XPS was used to further characterize the surface elements of the modified samples (cf.

2

Figure 4B). From the survey scans and atom percentage statistical data (inset in Figure 4B), it

3

can be seen that only C and O were detected on PCL, accounting for 85.23% and 14.77%,

4

respectively. After the self-assembled multi layers of gelatin and heparin, N, S, and Na were

5

detected on the treated samples, which confirmed the grafting of heparin and gelatin. S was

6

detected on the surface of PGH, PGHV, cPGH, and cPGHV at a rate of 3.6%, 3.56%, 4.98%, and

7

3.36%, respectively, suggesting the successful introduction of bioactive components37,38.

8

Therefore, the results of the FTIR spectroscopy and XPS demonstrated the successful fabrication

9

of multi, crosslinked layers and the effective loading of growth factors on the surface of the

10

electrospun PCL scaffolds.

11 12

Fig. 4 (A) The FTIR spectra of PCL, PGH, PGHV, cPGH, and cPGHV samples, and (B) XPS

13

analysis of the different scaffolds.

14 15

3.3. Release Profile

16


Page 16 of 33

Page 17 of 33 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


1

Figure 5 shows the multi-modal, programmed and sustained release profile of VEGF and

2

heparin in vitro triggered by MMP-239. The VEGF release amounts of the cPGHV on days 1, 3, 5,

3

and 7 were 17%, 32%, 51%, and 70%, respectively. The release time lasted for 12 days. On the

4

other hand, the heparin release amounts at each recorded time node remained stable, and the total

5

release time was around 50 days. Neither had burst release phenomena due to the multi,

6

crosslinked layer structure. The multi-modal, programmed and sustained drug release profiles

7

could be divided into two stages: the rapid endothelialization stage and the long-term

8

anticoagulation stage. The first stage presents a dual function, including the initial rapid release

9

of a certain amount of VEGF, which promoted endothelial cell growth, proliferation, and

10

angiogenesis. Meanwhile, the sustained release of a small amount of heparin inhibited the initial

11

coagulation caused by the inflammatory response. In the second stage, the long-term release of

12

heparin was beneficial for promoting anticoagulant and antithrombotic effects, and regulating

13

angiogenesis, thereby contributing to long-term patency and vascular tissue regeneration40.

14 15

Fig. 5 The programmed and sustained release profiles of VEGF and heparin triggered by MMP-2.

16

17



1

The polycation gelatin and polyanion heparin biomacromolecules were encapsulated into

2

self-assembled layers by electrostatic adsorption, forming an alternating, two-phase, layered

3

structure that relied on the bonding of the –COOH of the gelatin and the NH3+ of the heparin.

4

During the last steps of the self-assembly process, VEGF biomolecules were attracted and

5

encapsulated into layers, thanks to the –OH of the heparin, thus forming a multi-layered

6

structure30. Furthermore, the remaining amino group of the gelatin segment provided the binding

7

site for genipin crosslinking. The electrostatic interactions, covalent bonding, and hydrogen

8

bonding resulted in a multi-layer, crosslinked, intermolecular network with a protruding micro-

9

nano interface and a controlled release of VEGF and heparin41. The release of VEGF and heparin

10

at the crosslinked sites were sequentially triggered from the outer layer to the inner layer, under

11

the action of the MMP-2 enzyme, which acted as an on–off switch. By controlling the process

12

parameters, namely, the adsorption time, concentration, number of layers, and crosslinking

13

degree of the self-assembled layer, multi-modal, programmed and sustained release profiles were

14

achieved. The multi-modal drug delivery system, including the rapid initial release of VEGF and

15

long-term release of heparin effectively promoted endothelial cell affinity, regulates

16

angiogenesis, and enhances antithrombogenicity of the treated PCL scaffolds.

17

3.4. Platelet Adhesion

18

Platelet adhesion was investigated to characterize the effect of surface treatment on the anti-

19

coagulation of untreated and treated PCL samples (cf. Figures 6A–E). The number of platelets

20

adhered on the surface of differently treated samples and the water contact angle are shown in

21

Figure 6F and 6G, respectively. For the untreated PCL sample (Figure 6A), the platelet showed a

22

dendritic morphology with extruding pseudopodia. After heparin was introduced into the multi-

23

layers, the platelets of PGH and PGHV showed a round shape, reflecting the less activated state 18


Page 18 of 33

Page 19 of 33 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


1

of the adhered platelets. When the multi-layers were crosslinked by genipin, the adhered platelets

2

of cPGH and cPGHV had the same morphology. From the statistical results, it can be seen that

3

the highest number of adhered platelets was on the untreated PCL (Figure 6F), while the treated

4

samples showed many fewer adhered platelets with no significant differences.

5 6

Fig. 6 Platelet adhesion on (A) untreated PCL and the treated scaffolds (B) PGH, (C) PGHV, (D)

7

cPGH, and (E) cPGHV. (F) The number of platelets adhered on the surface of differently treated

8

samples. (G) The water contact angles of differently treated samples.

9 19



1

The platelets were more likely to adhere on the hydrophobic surface of electrospun PCL

2

scaffolds14. For the PGH and PGHV groups, a better anticoagulant effect was attributed to

3

heparin encapsulated in the multi-layers. However, conceivably, as time goes on, the

4

anticoagulant effect will gradually decrease due to the breakdown of the loosely self-assembled

5

coating. However, for the crosslinked cPGH and cPGHV samples, this low adhesion

6

phenomenon will last much longer due to the adsorption of heparin, which is an effective

7

anticoagulant factor due to its high negative charge density25,42. Thus, with the controlled and

8

sustained release profiles, the heparin release triggered by MMP-2 could last for 50 days, thereby

9

effectively promoting the long-term antithrombogenicity of the treated and crosslinked PCL

10

scaffolds.

11

3.5. HUVEC Viability and Proliferation

12

The viability of HUVECs on different PCL fibers was investigated using a live/dead assay.

13

As shown in Figure 7A, HUVECs were able to grow on all samples after seven days of cell

14

culture. From the statistical results (Figure 7B) of cell viability, it can be seen that the cell

15

viability on an untreated PCL scaffold was about 80%. After gelatin, heparin, and VEGF were

16

self-assembled to form the multi-layers on the PCL scaffolds, it was about 94% and 96% for

17

PGH and PGHV, respectively. Furthermore, when the multi-layers were crosslinked by genipin,

18

the cell viability of cPGH and cPGHV was around 93% and 98%, respectively. The difference

19

between the genipin crosslinked groups and the non-crosslinked groups was small, which

20

indicates that there was no inhibition of cell growth behavior by crosslinking with genipin43. The

21

quantitative cell proliferation results at days 1, 3, and 5 are shown in Figure 7C. The results of

22

the CCK-8 assay indicated that HUVECs on untreated PCL scaffolds proliferated relatively

23

slowly. After gelatin and heparin biomacromolecules were self-assembled into the multi, 20


Page 20 of 33

Page 21 of 33 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


1

crosslinked layers, PGH showed an improved cell proliferation profile compared to untreated

2

PCL, with a cell population 2.2 times that of untreated PCL at day 5. A significant improvement

3

was achieved when VEGF was encapsulated. PGHV showed a cell population 2.8 times that of

4

untreated PCL at day 5, suggesting that the VEGF further promoted HUVEC proliferation, as

5

previously reported9.

6

Compared to the non-crosslinked groups, the cell proliferation profiles of cPGH and

7

cPGHV groups had similar trends and showed further slight improvement. The adhesion and

8

proliferation of HUVECs on the multi, crosslinked layers were cellular-activity mediated by the

9

signals and specific proteins of gelatin and heparin44. The interfaces provided a microstructure

10

and signaling biomacromolecules similar to the in vivo environment for the cells44,45.

11

Furthermore, a programmed and sustained release of VEGF—based on the multi, crosslinked

12

layers—combined with VEGFR on the surface of the ECs, promoted cell adhesion and

13

proliferation. Thus, up to 14 days into the release cycle, the early and strong release of VEGF

14

was able to promote the rapid endothelialization of the PCL scaffold10.

21



1 2

Fig. 7 (A) Fluorescence images of HUVECs cultured on untreated and treated PCL samples for 7

3

days. Live cells were stained with green and dead cells were stained with red. (B) Statistical

4

results of cell viability from the live/dead assay at day 7. (C) Statistical results of cell

5

proliferation from the CCK-8 assay at days 1, 3, and 5.

6 7

3.6. Cytoskeleton and Morphology of HUVECs

8

The cytoskeleton/nuclei morphology of HUVECs is shown in Figure 8, and the SEM images

9

of HUVECs cultured on different PCL samples for 7 days, showing the interaction between the

10

cells and the scaffolds, are shown in Figure 9. The cytoskeletons of the cells were stained red

11

with phalloidin, while cell nuclei were stained blue with DAPI. As can be seen, compared to the 22


Page 22 of 33

Page 23 of 33 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


1

TCP samples that showed typical endothelial cell morphology, the morphology on the untreated

2

PCL samples and treated PCL samples were different. The cell sizes on the untreated samples

3

were smaller than cells on the treated samples. After the gelatin and heparin were self-assembled

4

into the multi, crosslinked layers, the PGH and cPGH groups showed significantly larger cell

5

sizes than the cells on PCL. The cells were also more stretched and spread out on the scaffolds.

6

The cells on the PGHV and cPGHV groups showed extremely prosperous growing states, with a

7

typical spindle-like cell morphology. The average aspect ratio of the cells on the multi,

8

crosslinked layers was largest, and it was significantly larger than those grown on TCP. All of

9

these results strongly suggest that the programmed and sustained release mechanism based on

10

multi, crosslinked layers greatly improved the biocompatibility of the PCL scaffold.

11

23



1

Fig. 8 Fluorescence images showing the cytoskeleton of HUVECs cultured on untreated and

2

treated PCL samples for 3 days: (A) PCL, (B) PGH, (C) PGHV, (D) cPGH, (E) cPGHV, and (F)

3

TCP.

4 5

The cellular–substrate interactions at day 7 were imaged using SEM. As shown in Figure 9,

6

the endothelial cells covered the untreated PCL surface, but the endothelialization area was less.

7

For the PGH and cPGH groups, cells were flattened and the endothelialization area increased.

8

Remarkably, when VEGF was encapsulated into the interfaces, the endothelialization area

9

increased significantly, and many cells were connected to each other, thus forming a larger area.

10

Especially for the cPGHV groups, the lamellipodia and filopodia were tightly attached to the

11

scaffolds, thus suggesting strong interactions between cells and scaffold. Furthermore, an

12

endothelial cell monolayer formed preliminarily, indicating that the programmed and sustained

13

VEGF release successfully promoted endothelialization4,46.

14

24


Page 24 of 33

Page 25 of 33 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


1

Fig. 9 SEM images of HUVECs cultured on untreated PCL and treated PCL samples for 7 days:

2

(A) PCL, (B) PGH, (C) PGHV, (D) cPGH, and (E) cPGHV.

3

3.7. EC Angiogenesis

4

To investigate the effect of heparin and VEGF on the HUVEC vascular network formation,

5

scaffolds made of PCL, cPGH, and cPGHV and prepared for EC angiogenesis after days 1, 2,

6

and 4 are shown in Figure 10, along with scaffolds immunostained by CD-31PE. As can be seen

7

from the images, the HUVECs distributed in the matrigel at day 1 for all samples, and some

8

HUVECs stretched. In the cPGH and cPGHV groups, a small number of networks appeared. As

9

the culture time increased, more HUVECs stretched and a small number of networks appeared in

10

the PCL group. Meanwhile, in the cPGH group, the already existing networks started to

11

disappear, and some HUVECs stretched at the same time, but did not connect with each other.

12

This was because the release of heparin impaired angiogenesis through the inhibition of

13

microRNA-10b15. However, in the cPGHV group, the number of networks increased, and they

14

exhibited a tendency of being stretched. By day 4, several vascular networks appeared on the

15

pure PCL scaffold. But in the cPGH group, most of the network structure disappeared. There

16

were only some nodes, which might have been caused by the adverse effect of heparin47.

17

Remarkably, a fiber structure developed and the HUVECs formed patent vascular networks in

18

vitro for the cPGHV group. This was likely because of the programmed release of VEGF, which

19

was useful for the stretching of the cells in the flexible matrigel base48. The early and full release

20

of VEGF through the multiple crosslinked layers of biomolecules on the electrospun PCL

21

scaffold reinforced the angiogenesis ability of the ECs49, even though the inhibition ability of

22

heparin was active. This indicated that the VEGF played a leading role in the early

23

endothelialization stage. It is believed that the long-term release of heparin would regulate 25



Page 26 of 33

1

angiogenesis and modulate several stages of the pathology over time15. The results above

2

indicate that cPGHV could potentially serve as a tissue engineering scaffold material to help

3

replace damaged blood vessels and promote angiogenesis.

4 5

Fig. 10 The PCL, cPGH, and cPGHV scaffolds stimulated EC angiogenesis after days 1, 2, and 4.

6 7

4. Conclusions

8

In summary, multi, crosslinked layers of biomolecules on electrospun PCL scaffolds were

9

fabricated in this study for the surface functionalization of PCL toward its application as small-

10

diameter vascular grafts. This was done via the multi-modal, programmed and sustained release

11

of VEGF and heparin to achieve rapid endothelialization and long-term antithrombogenicity. The

12

biomacromolecules—including

polycationic

gelatin

and

26


polyanionic

heparin—were

Page 27 of 33 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


1

encapsulated into the multi-layers by layer-by-layer self-assembled electrostatic adsorption, and

2

VEGF was subsequently absorbed into the top layers. Finally, multi, crosslinked layers with

3

better micro-nano structure integrity were obtained by genipin crosslinking the enzyme-

4

uncoupling-protein-2 segment of gelatin. The successful modification was verified via FTIR and

5

XPS, and the SEM and AFM results indicated the successful preparation of the multi,

6

crosslinked layers. The surface roughness increased as more components were grafted onto the

7

PCL surface, and the hydrophilicity increased due to the increased number of hydrophilic groups.

8

The programmed and sustained release profiles of the resulting multi, crosslinked layers were

9

triggered at the crosslinking sites via MMP-2 in vitro. The multi-modal drug delivery system,

10

including the initial and strong release of VEGF lasted for 12 days to boost rapid

11

endothelialization, while the sustained release of heparin lasted up to 50 days to ensure long-term

12

antithrombogenesis and regulated angiogenesis. Platelet adhesion decreased sharply after the

13

encapsulation

14

antithrombogenicity due to the sustained heparin release.

of

heparin,

and

the

endothelialization

surface

maintained

long-term

15

In vitro, HUVEC cultures revealed that all of the treatments had a positive effect on the

16

biocompatibility of PCL scaffolds. The cell viability and cell proliferation improved when VEGF

17

and heparin were encapsulated. The morphology of the HUVECs cultured on the multi,

18

crosslinked layers exhibited favorable cell morphologies and strong cell–substrate interactions.

19

More importantly, the programmed and sustained release of VEGF and heparin triggered by

20

MMP-2 was effective at promoting biocompatibility, angiogenesis of endothelial cells, and

21

antithrombogenicity of small-diameter vascular grafts.

22 23

Conflicts of interest 27



1

There are no conflicts to declare.

2 3

Acknowledgments

4

The authors would like to acknowledge the support of the Wisconsin Institute for Discovery

5

(WID) at the University of Wisconsin–Madison, the National Center for International Joint

6

Research of Micro-Nano Molding Technology in China, the China Scholarship Council (CSC),

7

the financial support of the International Science & Technology Cooperation Program of China

8

(2015DFA30550), and the Key Project of Science and Technology of the Education Department

9

of Henan Province, China (19A430003). The corresponding author at the University of

10

Wisconsin–Madison would like to acknowledge the partial support by the NHLBI of the National

11

Institutes of Health (grant number U01HL134655) and the Kuo K. and Cindy F. Wang

12

Professorship.

13

28


Page 28 of 33

Page 29 of 33 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

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


References (1) (2) (3) (4) (5) (6) (7) (8) (9)

(10)

(11) (12) (13) (14)

Townsend, N.; Wilson, L.; Bhatnagar, P.; Wickramasinghe, K.; Rayner, M.; Nichols, M. Cardiovascular Disease in Europe: Epidemiological Update 2016. Eur. Heart J. 2016, 37, 3232–3245. Das, A. M.; Eggermont, A. M. M.; ten Hagen, T. L. M. A Ring Barrier–Based Migration Assay to Assess Cell Migration in Vitro. Nat. Protoc. 2015, 10, 904–915. Kuang, H.; Wang, Y.; Hu, J.; Wang, C.; Lu, S.; Mo, X. A Method for Preparation of an Internal Layer of Artificial Vascular Graft Co-Modified with Salvianolic Acid B and Heparin. ACS Appl. Mater. Interfaces 2018, 10, 19365–19372. Mi, H.-Y.; Jing, X.; Thomsom, J. A.; Turng, L.-S. Promoting Endothelial Cell Affinity and Antithrombogenicity of Polytetrafluoroethylene (PTFE) by Mussel-Inspired Modification and RGD/Heparin Grafting. J. Mater. Chem. B 2018, 6, 3475–3485. Liang, C.; Hu, Y.; Wang, H.; Xia, D.; Li, Q.; Zhang, J.; Yang, J.; Li, B.; Li, H.; Han, D.; Dong, M. Biomimetic Cardiovascular Stents for in Vivo Re-Endothelialization. Biomaterials 2016, 103, 170–182. Nichol, J. W.; Koshy, S. T.; Bae, H.; Hwang, C. M.; Yamanlar, S.; Khademhosseini, A. Cell-Laden Microengineered Gelatin Methacrylate Hydrogels. Biomaterials 2010, 31, 5536–5544. Ledo, A. M.; Sasso, M. S.; Bronte, V.; Marigo, I.; Boyd, B. J.; Garcia-Fuentes, M.; Alonso, M. J. Co-Delivery of RNAi and Chemokine by Polyarginine Nanocapsules Enables the Modulation of Myeloid-Derived Suppressor Cells. J. Controlled Release 2019, 295, 60–73. Zheng, W.; Wang, Z.; Song, L.; Zhao, Q.; Zhang, J.; Li, D.; Wang, S.; Han, J.; Zheng, X.L.; Yang, Z.; Kong, D. Endothelialization and Patency of RGD-Functionalized Vascular Grafts in a Rabbit Carotid Artery Model. Biomaterials 2012, 33, 2880–2891. Bigalke, C.; Luderer, F.; Wulf, K.; Storm, T.; Löbler, M.; Arbeiter, D.; Rau, B. M.; Nizze, H.; Vollmar, B.; Schmitz, K.-P.; Klar, E.; Sternberg, K. VEGF-Releasing Suture Material for Enhancement of Vascularization: Development, in Vitro and in Vivo Study. Acta Biomater. 2014, 10, 5081–5089. Zhou, J.; Ding, J.; Zhu, Z.; Xu, J.; Yi, Y.; Li, Y.; Fan, H.; Bai, S.; Yang, J.; Tang, Y.; Dong, X.; Dong, N. Surface Biofunctionalization of the Decellularized Porcine Aortic Valve with VEGF-Loaded Nanoparticles for Accelerating Endothelialization. Mater. Sci. Eng. C 2019, 97, 632–643. Yang, Z.; Yang, Y.; Zhang, L.; Xiong, K.; Li, X.; Zhang, F.; Wang, J.; Zhao, X.; Huang, N. Mussel-Inspired Catalytic Selenocystamine-Dopamine Coatings for Long-Term Generation of Therapeutic Gas on Cardiovascular Stents. Biomaterials 2018, 178, 1–10. Kim, I.; Lee, S. S.; Bae, S.; Lee, H.; Hwang, N. S. Heparin Functionalized Injectable Cryogel with Rapid Shape-Recovery Property for Neovascularization. Biomacromolecules 2018, 19, 2257–2269. Levine, M. N.; Raskob, G.; Beyth, R. J.; Kearon, C.; Schulman, S. Hemorrhagic Complications of Anticoagulant Treatment: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004, 126, 287S-310S. Bae, S.; DiBalsi, M. J.; Meilinger, N.; Zhang, C.; Beal, E.; Korneva, G.; Brown, R. O.; Kornev, K. G.; Lee, J. S. Heparin-Eluting Electrospun Nanofiber Yarns for Antithrombotic Vascular Sutures. ACS Appl. Mater. Interfaces 2018, 10, 8426–8435.

29



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

(15) Shen, X.; Fang, J.; Lv, X.; Pei, Z.; Wang, Y.; Jiang, S.; Ding, K. Heparin Impairs Angiogenesis through Inhibition of MicroRNA-10b. J. Biol. Chem. 2011, 286, 26616– 26627. (16) Browne, S.; Pandit, A. Multi-Modal Delivery of Therapeutics Using Biomaterial Scaffolds. J. Mater. Chem. B 2014, 2, 6692–6707. (17) Ditadi, A.; Sturgeon, C. M.; Tober, J.; Awong, G.; Kennedy, M.; Yzaguirre, A. D.; Azzola, L.; Ng, E. S.; Stanley, E. G.; French, D. L.; Cheng, X.; Gadue, P.; Speck, N. A.; Elefanty, A. G.; Keller, G. Human Definitive Haemogenic Endothelium and Arterial Vascular Endothelium Represent Distinct Lineages. Nat. Cell Biol. 2015, 17, 580–591. (18) Lin, Q.; Yan, J.; Qiu, F.; Song, X.; Fu, G.; Ji, J. Heparin/Collagen Multilayer as a Thromboresistant and Endothelial Favorable Coating for Intravascular Stent. J. Biomed. Mater. Res. A 2011, 96A, 132–141. (19) Yuen, W. W.; Du, N. R.; Chan, C. H.; Silva, E. A.; Mooney, D. J. Mimicking Nature by Codelivery of Stimulant and Inhibitor to Create Temporally Stable and Spatially Restricted Angiogenic Zones. Proc. Natl. Acad. Sci. 2010, 107, 17933–17938. (20) Jiang, Y.; Jiang, L.; Huang, A.; Wang, X.; Li, Q.; Turng, L. Electrospun Polycaprolactone/Gelatin Composites with Enhanced Cell–Matrix Interactions as Blood Vessel Endothelial Layer Scaffolds. Mater. Sci. Eng. C 2017, 71, 901–908. (21) Formiga, F. R.; Pelacho, B.; Garbayo, E.; Abizanda, G.; Gavira, J. J.; Simon-Yarza, T.; Mazo, M.; Tamayo, E.; Jauquicoa, C.; Ortiz-de-Solorzano, C.; Prósper, F.; Blanco-Prieto, M. J. Sustained Release of VEGF through PLGA Microparticles Improves Vasculogenesis and Tissue Remodeling in an Acute Myocardial Ischemia–Reperfusion Model. J. Controlled Release 2010, 147, 30–37. (22) Cui, H.; Zhu, W.; Holmes, B.; Zhang, L. G. Biologically Inspired Smart Release System Based on 3D Bioprinted Perfused Scaffold for Vascularized Tissue Regeneration. Adv. Sci. 2016, 3, 1600058–1600067. (23) Liu, Y.; Zhang, J.; Wang, J.; Wang, Y.; Zeng, Z.; Liu, T.; Chen, J.; Huang, N. Tailoring of the Dopamine Coated Surface with VEGF Loaded Heparin/Poly- L -Lysine Particles for Anticoagulation and Accelerate in Situ Endothelialization: Dopamine Coated Surface with VEGF Loaded H EP /PLL. J. Biomed. Mater. Res. A 2015, 103, 2024–2034. (24) Li, G.; Zhang, F.; Liao, Y.; Yang, P.; Huang, N. Coimmobilization of Heparin/Fibronectin Mixture on Titanium Surfaces and Their Blood Compatibility. Colloids Surf. B Biointerfaces 2010, 81, 255–262. (25) Yao, Y.; Wang, J.; Cui, Y.; Xu, R.; Wang, Z.; Zhang, J.; Wang, K.; Li, Y.; Zhao, Q.; Kong, D. Effect of Sustained Heparin Release from PCL/Chitosan Hybrid Small-Diameter Vascular Grafts on Anti-Thrombogenic Property and Endothelialization. Acta Biomater. 2014, 10, 2739–2749. (26) Kuttappan, S.; Mathew, D.; Jo, J.; Tanaka, R.; Menon, D.; Ishimoto, T.; Nakano, T.; Nair, S. V.; Nair, M. B.; Tabata, Y. Dual Release of Growth Factor from Nanocomposite Fibrous Scaffold Promotes Vascularisation and Bone Regeneration in Rat Critical Sized Calvarial Defect. Acta Biomater. 2018, 78, 36–47. (27) Zhang, J.; Chu, L.-F.; Hou, Z.; Schwartz, M. P.; Hacker, T.; Vickerman, V.; Swanson, S.; Leng, N.; Nguyen, B. K.; Elwell, A.; Bolin, J.; Brown, M. E.; Stewart, R.; Burlingham, W. J.; Murphy, W. L.; Thomson, J. A. Functional Characterization of Human Pluripotent Stem Cell-Derived Arterial Endothelial Cells. Proc. Natl. Acad. Sci. 2017, 114, E6072– E6078. 30


Page 30 of 33

Page 31 of 33 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

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


(28) Hasuda, H.; Kwon, O. H.; Kang, I.-K.; Ito, Y. Synthesis of Photoreactive Pullulan for Surface Modification. Biomaterials 2005, 26, 2401–2406. (29) Deng, Y.; Sun, Y.; Chen, X.; Zhu, P.; Wei, S. Biomimetic Synthesis and Biocompatibility Evaluation of Carbonated Apatites Template-Mediated by Heparin. Mater. Sci. Eng. C 2013, 33, 2905–2913. (30) Nakamura, S.; Kubo, T.; Ijima, H. Heparin-Conjugated Gelatin as a Growth Factor Immobilization Scaffold. J. Biosci. Bioeng. 2013, 115, 562–567. (31) Jiang, L.; Wang, L.; Wang, N.; Gong, S.; Wang, L.; Li, Q.; Shen, C.; Turng, L.-S. Fabrication of Polycaprolactone Electrospun Fibers with Different Hierarchical Structures Mimicking Collagen Fibrils for Tissue Engineering Scaffolds. Appl. Surf. Sci. 2018, 427, 311–325. (32) Yao, C.-H.; Liu, B.-S.; Chang, C.-J.; Hsu, S.-H.; Chen, Y.-S. Preparation of Networks of Gelatin and Genipin as Degradable Biomaterials. Mater. Chem. Phys. 2004, 83, 204–208. (33) Cui, L.; Jia, J.; Guo, Y.; Liu, Y.; Zhu, P. Preparation and Characterization of IPN Hydrogels Composed of Chitosan and Gelatin Cross-Linked by Genipin. Carbohydr. Polym. 2014, 99, 31–38. (34) Kolesky, D. B.; Truby, R. L.; Gladman, A. S.; Busbee, T. A.; Homan, K. A.; Lewis, J. A. 3D Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs. Adv. Mater. 2014, 26, 3124–3130. (35) Sengupta, P.; Prasad, B. L. V. Surface Modification of Polymers for Tissue Engineering Applications: Arginine Acts as a Sticky Protein Equivalent for Viable Cell Accommodation. ACS Omega 2018, 3, 4242–4251. (36) Crouzier, T.; Picart, C. Ion Pairing and Hydration in Polyelectrolyte Multilayer Films Containing Polysaccharides. Biomacromolecules 2009, 10, 433–442. (37) Tan, H.; Wang, H.; Chai, Y.; Yu, Y.; Hong, H.; Yang, F.; Qu, X.; Liu, C. Engineering a Favourable Osteogenic Microenvironment by Heparin Mediated Hybrid Coating Assembly and RhBMP-2 Loading. RSC Adv. 2017, 7, 11439–11447. (38) Shi, C.; Yuan, W.; Khan, M.; Li, Q.; Feng, Y.; Yao, F.; Zhang, W. Hydrophilic PCU Scaffolds Prepared by Grafting PEGMA and Immobilizing Gelatin to Enhance Cell Adhesion and Proliferation. Mater. Sci. Eng. C 2015, 50, 201–209. (39) Das, A.; Monteiro, M.; Barai, A.; Kumar, S.; Sen, S. MMP Proteolytic Activity Regulates Cancer Invasiveness by Modulating Integrins. Sci. Rep. 2017, 7, 14219–14231. (40) Samal, J.; Rebelo, A. L.; Pandit, A. A Window into the Brain: Tools to Assess PreClinical Efficacy of Biomaterials-Based Therapies on Central Nervous System Disorders. Adv. Drug Deliv. Rev. 2019. (41) Liu, T.; Wang, Y.; Zhong, W.; Li, B.; Mequanint, K.; Luo, G.; Xing, M. Biomedical Applications of Layer-by-Layer Self-Assembly for Cell Encapsulation: Current Status and Future Perspectives. Adv. Healthc. Mater. 2019, 8, 1800939. (42) Limtiaco, J. F. K.; Jones, C. J.; Larive, C. K. Characterization of Heparin Impurities with HPLC-NMR Using Weak Anion Exchange Chromatography. Anal. Chem. 2009, 81, 10116–10123. (43) Khan, H.; Shukla, R. N.; Bajpai, A. K. Genipin-Modified Gelatin Nanocarriers as Swelling Controlled Drug Delivery System for in Vitro Release of Cytarabine. Mater. Sci. Eng. C 2016, 61, 457–465. (44) Watt, F. M.; Huck, W. T. S. Role of the Extracellular Matrix in Regulating Stem Cell Fate. Nat. Rev. Mol. Cell Biol. 2013, 14, 467–473. 31



1 2 3 4 5 6 7 8 9 10 11 12

(45) Rozario, T.; DeSimone, D. W. The Extracellular Matrix in Development and Morphogenesis: A Dynamic View. Dev. Biol. 2010, 341, 126–140. (46) Krawiec, J. T.; Vorp, D. A. Adult Stem Cell-Based Tissue Engineered Blood Vessels: A Review. Biomaterials 2012, 33, 3388–3400. (47) Lee, D. Y.; Kim, S. K.; Kim, Y. S.; Son, D. H.; Nam, J. H.; Kim, I. S.; Park, R. W.; Kim, S. Y.; Byun, Y. Suppression of Angiogenesis and Tumor Growth by Orally Active Deoxycholic Acid-Heparin Conjugate. J. Controlled Release 2007, 118, 310–317. (48) Bakunts, K.; Gillum, N.; Karabekian, Z.; Sarvazyan, N. Formation of Cardiac Fibers in Matrigel Matrix. BioTechniques 2008, 44, 341–348. (49) Herbert, S. P.; Stainier, D. Y. R. Molecular Control of Endothelial Cell Behaviour during Blood Vessel Morphogenesis. Nat. Rev. Mol. Cell Biol. 2011, 12, 551–564.

13

32


Page 32 of 33

Page 33 of 33 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

1


For Table of Contents Only

2

33


Programmed Release of Multimodal, Cross-Linked Vascular

Recommend Documents