Bladder Acellular Matrix Graft Reinforced Silk Fibroin Composite

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Bladder acellular matrix graft reinforced silk fibroin composite scaffolds loaded VEGF with aligned electrospun fibers in multiple layers Zhaobo Li, Qiangqiang Liu, Hongsheng Wang, Lujie Song, Huili Shao, Minkai Xie, Yuemin Xu, and Yaopeng Zhang ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 10 Mar 2015 Downloaded from http://pubs.acs.org on March 17, 2015

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ACS Biomaterials Science & Engineering

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Bladder acellular matrix graft reinforced silk fibroin composite scaffolds loaded VEGF with aligned electrospun fibers in multiple layers Zhaobo Li1, Qiangqiang Liu1, Hongsheng Wang2, Lujie Song3, Huili Shao1, Minkai Xie3, Yuemin Xu3,*, Yaopeng Zhang1,* 1

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

College of Materials Science and Engineering, Donghua University, Shanghai 201620, P. R. China 2

College of Chemistry, Chemical Engineering and Biotechnology, Donghua

University, Shanghai 201620, P.R. China 3

Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s

Hospital, Shanghai 200233, P. R. China [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; Corresponding Author *Yaopeng Zhang Phone: +86-21-67792954. Fax: +86-21-67792855 E-mail: [email protected] State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, 2999 North Ren-min Road, Songjiang District, Shanghai 201620, P.R. China *Yuemin Xu Phone: +86-21-64369181-58698 E-mail: [email protected] Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, P. R. China Multiplelayered composite scaffolds of silk fibroin/BAMG

ACS Paragon Plus Environment


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ABSTRACT

2

Bombyx mori silk is of great interest to people for its outstanding mechanical and biological

3

properties. However, the traditional electrospun regenerated silk fibroin (RSF) scaffolds from

4

aqueous solution were weak and had limited applications. This study was to fabricate reinforced

5

scaffolds with well-aligned RSF fibers electrospun on a layer of native extracellular matrix,

6

bladder acellular matrix graft (BAMG). The silk fibroin fibers were well-aligned as a grill in

7

multiple layers. Both the BAMG and the grill structure significantly improved the tensile

8

properties and suture retention of the composite scaffolds, which can be sutured well with tissue

9

during implantation. In vitro assay indicates that the scaffolds had a good biocompatibility.

10

Porcine iliac endothelial cells (PIECs) attached and proliferated well on the vascular endothelial

11

growth factor (VEGF) loaded scaffolds compared with those without VEGF. Moreover, the

12

grill-like structure guides PIECs well along the aligned fiber.

13

KEYWORDS: electrospinning; well-aligned RSF fibers; BAMG; VEGF; PIECs

14 15

1. INTRODUCTION

16

In recent years, electrospinning has attracted much attention as a simple and effective

17

technique to fabricate a porous structure of fibers with diameters ranging from tens of

18

nanometers up to micrometers, which has a consequent large surface-to-volume ratio to the

19

natural extracellular matrix (ECM) 1-4. Silkworm silk has been used as suture for decades due to

20

its good biomaterial properties 5. As a natural protein, silk fibroin is one of the most widely

21

studied biomaterials because of its biocompatibility, slow biodegradability and good mechanical

22

properties 6-10. Regenerated silk fibroin (RSF) was electrospun as tissue engineering scaffolds to

23

mimic ECM, which can be used for cell proliferation and drug delivery and release 11-13. Multiplelayered composite scaffolds of silk fibroin/BAMG


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Conventionally electrospun RSF scaffolds are usually randomly collected in a form of

2

nonwoven mats with low orientation and poor mechanical properties, which limits their

3

applications 14. In order to improve the poor mechanical properties of RSF scaffolds electrospun

4

from aqueous solutions, people have adopted many methods, such as water vapor annealing 15, 16,

5

immersion in organic solvents

6

However, the scaffolds still need reinforcement both in dry and wet states. This is very important

7

especially in some occasions that the scaffolds would be sutured, such as in urethra or bladder 6.

8

In addition, the safety of the agents added into the scaffolds needs further investigation. Aligned

9

fibers with improved mechanical properties were electrospun and collected using kinds of special

10

equipment with many weaknesses, such as poor adhesive force between the fiber layers, only

11

parallel to one direction, limited length or existing in the form of yarn. Recently, Jiang reported a

12

simple and effective method to fabricate multilayered electrospun RSF scaffolds with

13

well-aligned fibers overlapping in different angles

14

reinforce the scaffolds compared with the random fibers

15

desired anisotropy 23, 24.

17, 18

, extension

6, 19, 20

or adding some reinforcing agents

12, 20, 21

.

14

. Moreover, aligned fibers can not only 22

, but also guide the cell growth with

16

Most of the researchers have focused on adding reinforced agents or improving the orientation

17

degree to improve the mechanical properties of the scaffolds, but ignored combining the

18

scaffolds with another strong biomaterial. Bladder acellular matrix graft (BAMG) is a kind of

19

biomaterials

20

physicochemical properties and great mechanical reliability

21

BAMG and Jiang’s scaffolds with well-aligned RSF fibers to fabricate tough composite

22

scaffolds.

used

in

urethral

reconstruction

due

to its excellent

Multiplelayered composite scaffolds of silk fibroin/BAMG


biocompatibility,

25-27

. This study combined the


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For tissue engineering scaffolds, it is important to promote vasculogenesis and angiogenesis 28, 29

2

during tissue regenerating

. Vascular endothelial growth factor (VEGF), a kind of protein,

3

induces proliferation, sprouting and tube formation of endothelial cells, which plays an essential

4

role in the formation of new blood vessels. Due to its short half-life time and rapid clearance,

5

VEGF should be specially delivered and released in tissue engineering scaffolds for applications

6

30, 31

.

7

In this paper, we fabricated RSF/BAMG composite scaffolds with well-aligned RSF fibers

8

loaded with VEGF by electrospinning. Both the BAMG and the grill structure of the RSF mats

9

are expected to reinforce the scaffolds. We adopted water vapor annealing to transform the water

10

soluble scaffolds to water-insoluble scaffolds. The morphology, pore size distribution,

11

microstructure, and mechanical properties of the scaffolds were investigated. The

12

biocompatibility of the scaffolds and the effect of released VEGF with porcine iliac endothelial

13

cells (PIECs) seeded were also demonstrated.

14 15

2. MATERIALS AND METHODS

16

2.1. Materials

17

Bombyx mori cocoons were produced in Tongxiang, China. A molecular weight cutoff 14,000

18

± 2000 D cellulose semipermeable membranes were purchased from Yuanju Co., Ltd (Shanghai,

19

China). BAMG was prepared from sacrificed pigs as previously reported 26. VEGF, trypsin and

20

Dulbecco Modified Eagle's Medium (DMEM) were purchased from Gibco Life Technologies

21

Co., USA. BSA and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)

22

were purchased from Sigma-Aldrich, USA. PIECs were purchased from institute of biochemistry



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and cell biology (Chinese Academy of Sciences, China). Other analytical grade chemicals were

2

purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

3

2.2. Preparation of RSF/VEGF Electrospinning Dopes

4

B. mori cocoons were boiled in 0.5 wt% Na2CO3 solution for 30 min twice to remove sericin.

5

The dried degummed silk was then dissolved in 9.0 M LiBr aqueous solution at 40 °C for 2 h,

6

dialyzed against deionized water for 3 days and concentrated. VEGF was dissolved in BSA

7

aqueous solution with a concentration of 20 µg/mL and stored at -20 °C, since BSA binds the

8

VEGF and make it stable in aqueous solution. One hundred µL VEGF solution was added into

9

16 mL RSF aqueous solution (20 wt%) to get a mixed solution, which was then concentrated to

10

33 wt% in forced airflow at 5 °C for further electrospinning.

11

12 13

Figure 1. Schematic drawing of (a) the blend electrospinning and (b~e) the preparation process

14

of multilayered RSF/BAMG composite scaffolds with aligned RSF fibers Multiplelayered composite scaffolds of silk fibroin/BAMG



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2.3. Fabrication of Composite Scaffolds and Post-treatment

2

Fig.1 is the schematic drawing illustration of the electrospinning process (Fig.1a). Firstly, the

3

dried BAMG was wetted by saline to improve its conductivity and stretched smoothly on the

4

aluminum foil, which was adhered on the cylinder. Due to the asymmetric surfaces of BAMG,

5

the serosal surface with dense structure contacted the aluminum foil, while the muscular surface

6

with nanofibers faced air to collect RSF fiber (Fig.1b). VEGF-RSF aqueous solution was spun

7

using a 0.6 mm diameter needle at a flow rate of 1.2 mL/h. The experiments were operated at a

8

voltage of 20 kV, a distance from spinneret to collector of 10 cm and the rotating speed of the

9

cylinder was 2000 RPM. During the electrospinning process, we collected RSF layer for one

10

hour, then rotated the aluminum foil with an angle of 90º on the cylinder to get another RSF

11

layer (Fig.1c). After 6 h, the composite scaffolds (Fig.1e) were fabricated with BAMG and

12

aligned electrospun RSF fibers in six layers (Fig.1d). The thickness of BAMG and each RSF

13

layer were about 30 and 20 µm, respectively. Because the water volatilizes slowly compared

14

with organic solvents, the adhesive force between adjacent RSF layers and BAMG was enhanced

15

by the effect of water. Finally, the composite scaffolds were peeled off from the collector after

16

15 days drying in air. For comparison, bare RSF scaffolds with aligned fibers in multiple layers

17

were fabricated on aluminum foil directly at the same conditions as above. We also prepared the

18

composite RSF/BAMG scaffolds with randomly collected fibers, which have the same total

19

thickness of 150 ± 30 µm as the scaffolds shown in Fig.1e. All spinning parameters including

20

spinning time were the same as above.

21 22

The as-spun scaffolds were post-treated by water vapor annealing at 37 °C and 90 % relative humidity for 36 h to induce the conformation transition of RSF



32, 33

. The method is

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environmentally friendly and effective to keep VEGF active. Then the samples were dried in air

2

and stored at -20 °C for further study.

3

2.4. Morphology Observation

4

The morphology of the composite scaffolds and BAMG were observed by SEM (JEOL

5

JSM-5600LV, Japan) at a voltage of 10 kV. The fibers diameter distribution was analyzed using

6

Image Tool software and more than 100 counts were randomly selected for each sample.

7

The bare RSF scaffolds with aligned fibers in multiple layers were cut to 3 cm× 3 cm squares

8

for pore size and pore size distribution measurements, during which each sample was wetted by

9

Calwick with a surface tension of 21 dyn/cm (PMI Porous Materials Int.) and measured using a

10

CFP-1100-AI capillary flow porometer (Porous Materials Int. USA) 34.

11

2.5. Mechanical Test

12

Tensile and suture retention tests of the composite scaffolds (35 mm × 5 mm) were operated

13

using an Instron 5969 material testing machine at 25 ± 5 °C and 50 ± 5 % of relative humidity in

14

dry and wet states. To measure the samples in wet states, all the samples were soaked beforehand

15

in simulated body fluid (SBF) for 30 min to be fully saturated and achieve equilibrium 35. Tensile

16

test was performed at a gauge length of 20 mm with an extension rate of 3 mm/min. The

17

thickness of each sample was measured 10 times using a CH-1-S thickness gauge (Shanghai

18

Liuling Instruments Co., Shanghai, China).

19

Fig. 2 shows the process of suture retention test of the composite scaffolds. Suture retention

20

strength was measured as reported 36, the upper end of the sample was threaded with a suture (5–

21

0 Polyglactin, Ethicon, USA) and connected to the upward clamp, while the lower end was fixed

22

by the downward clamp of the Instron 5969 material testing machine. The distance was 2 mm




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from the hole threaded with the suture to the upper edge of the sample. Other experiment

2

conditions were the same as tensile test.

3 4

Figure 2. Schematic for the suture retention test of the composite scaffolds

5

2.5. Fourier Transform Infrared Spectroscopy (FTIR)

6

RSF scaffolds were characterized through Fourier Transform Infrared Spectroscopy using a

7

spectrophotometer (Nicolet Nexus 670) with ATR apparatus, operating in the 4000−400 cm−1

8

range with a resolution of 4 cm−1 and 37 acquisitions per minute. The quantitative secondary

9

structure analysis was carried out by performing deconvolution of the spectra using the PeakFit

10

software (Version 4.12, SeaSolve Software Inc., San Jose, CA) 37.

11

2.6. Wide Angle X-ray Diffraction

12

Wide angle X-ray diffraction (WAXD) curves were obtained at the BL15U1 beamline in

13

Shanghai Synchrotron Radiation Facility (SSRF) with an energy ring of 3.5 GeV and a beam

14

current of 220 mA. The wavelength of the X-rays and the sample spot size were 0.7746 Å and 10

15

× 10 µm2, respectively. The data was analyzed using FIT2D software (version 12.077, Andy

16

Hammersley/ESRF, Grenoble, France). The crystallinity of the sample was estimated by

17

separating the Bragg reflections from the broad short range order background.

18

2.7. Proliferation of PIECs on RSF Scaffolds Multiplelayered composite scaffolds of silk fibroin/BAMG


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Circular scaffolds samples (15 mm in diameter) with or without VEGF were cut and put in

2

24-well plates, then sterilized in 75 vol.% ethanol vapor for 5 h. PIECs were seeded with a

3

density of 5 × 103 cells/well and incubated at 37 °C/ 5 % CO2. Negative control groups are

4

coverslips and tissue culture plates (TCPs). Cell proliferation was determined by MTT method.

5

The details of the MTT assay process can be found in the supporting information. The

6

post-treated electrospun RSF scaffolds without or with VEGF were designated as RSF-WGF and

7

RSF-GF, respectively.

8

PIECs were seeded at a density of 8 × 103 cells/well and cultured for 3 days for spreading and

9

cytoskeletal structure observation using SEM and a SP-5 II (LEIKA Co., Germany) laser

10

scanning confocal microscope (LSCM). PIECs were fixed with 2.5 % glutaraldehyde and 4 %

11

paraformaldehyde for 2 h at 4 °C for SEM and LSCM, respectively. For SEM observation, the

12

fixed samples were rinsed twice with phosphate buffer solution (PBS) and dehydrated in a

13

graded series of ethanol 38. Then the dry samples were sputtered with platinum and observed at a

14

voltage of 10 kV. For LSCM observation of cell cytoskeletal structure, the fixed samples were

15

rinsed by PBS, respectively incubated in 0.1 % (v/v) Triton-X 100 and 1 % (v/v) BSA for 5 and

16

30 min, respectively. Then the samples were stained by Rhodamine labeled phalloidin and

17

observed at a wavelength of 561 nm. 3D cytoskeletal morphology images of PIECs were

18

acquired from a stack of successive slices in the z dimension and analyzed with Imaris 6.2

19

software (Bitplane Co., Switzerland).

20

2.9. Statistics and Data Analysis

21

Statistical analysis was performed in triplicate and presented as mean ± standard deviation. In

22

experimental evaluations, statistically significant differences between groups depended on a

23

p-value < 0.05, which was performed by one-way ANOVA tests 39. Multiplelayered composite scaffolds of silk fibroin/BAMG



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3. RESULTS AND DISCUSSION 3.1. Morphology of Composite Scaffolds

4 5

Figure 3. SEM images of the muscular (a) and serosal (a") surfaces of BAMG, RSF surface of (b)

6

composite scaffolds, and corresponding diameter distributions of the RSF fibers (a' and b'), and

7

(b") the pore size distribution of the post-treated RSF scaffolds.

8 9

Fig. 3 shows the SEM images of the muscular (a) and serosal (a") surfaces of BAMG and RSF

10

layer (b) of the composite scaffolds. As shown in Fig. 3 (a) and (a"), we can find that the serosal

11

surface of BAMG was much smoother than the muscular surface, which made us choose the

12

muscular surface with nanofibers structure to collect the RSF fibers during the electrospinning

13

process. Fig. 3b shows that the electrospun fibers had a good orientation with a grill-like

14

structure. The fibers diameter distribution of the muscular surface (Fig. 3a') of BAMG and the Multiplelayered composite scaffolds of silk fibroin/BAMG


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post-treated composite scaffolds (Fig. 3b') were 193 ± 77 nm and 1.9 ± 0.6 µm, respectively.

2

Fig. 3b" and Table SI 1 show the pore size distribution and pore size of the post-treated bare RSF

3

scaffolds with aligned RSF fibers, which were conducted by automated capillary flow porometer

4

system software. The thickness, average, largest and smallest pore size of the RSF scaffolds were

5

about 87.5 µm, 4.4 µm, 14.1 µm and 5.5 µm, respectively. It is crucial for tissue engineered

6

scaffolds to have highly porous network with interconnected pores, because the microscale and

7

nanoscale porous structure are most favorable to facilitate the passage and exchange of nutrients

8

and gases, which are important for cellular growth and tissue regeneration 40. Further, the mean

9

pore size of the RSF scaffolds was more than 20 times bigger than other electrospun scaffolds 34,41

10

(pore size of 0.2µm)

11

because our RSF fiber has larger diameter (1.9 µm) than the electrospun fiber in literature (391

12

nm) 41.

13

, which was favorable for cell attachment and proliferation. This is

3.2. Mechanical Properties of the Composite Scaffolds

14

15 16

Figure 4. Stress–strain curves of the post-treated composite scaffolds in dry (a) and wet (b)

17

states, respectively. Multiplelayered composite scaffolds of silk fibroin/BAMG



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The total thickness of the composite scaffolds was 120 ± 20 µm. Fig. 4 shows the stress-strain

3

curves of the post-treated RSF/BAMG composite scaffolds in (a) dry and (b) wet states. From

4

Fig. 4a and Table SI 2, we can see the breaking strength and the elongation at break of the

5

post-treated composite scaffolds with aligned fibers were 22.7 MPa and 18.8 %, which were

6

much higher than those (14.4 MPa and 12.0 %) of the RSF/BAMG composite scaffolds with

7

random fibers prepared at the same conditions in our previous study. The difference of the

8

breaking strength between the composite scaffolds with aligned and random fibers was

9

significant with a p-value < 0.01 (Fig. SI 1). This indicates that the composite scaffolds were

10

evidently reinforced by the aligned fibers. Furthermore, the breaking strength of the composite

11

scaffolds with aligned-fibers was about 3 times as high as that (7.6 MPa) of the bare RSF

12

scaffolds with aligned fiber in multiple layers prepared in the same way14. This proves that the

13

composite scaffolds were significantly reinforced by BAMG. The initial modulus of the

14

composite scaffolds was 997 MPa, which was higher than the nanofibers reinforced by single

15

wall carbon nanotubes42 (705 MPa). All that mentioned proves that both the BAMG and

16

well-aligned fibers had a positive contribution to improve the mechanical properties of the

17

composite scaffolds. In wet state, the breaking strength of the composite scaffolds was 5.9 MPa

18

(Fig. 4b and Table SI 2), which was evidently better than bare well-aligned RSF scaffolds (1.1

19

MPa)14 and the RSF/BAMG composite scaffolds with random fibers (3.9 MPa) in our previous

20

study. The p-value was smaller than 0.01, which meant that there was a significant difference

21

among the samples tested in wet state (Fig. SI 1). The elongation at break of the composite

22

scaffolds in wet state was larger than that in dry state, which will facilitate the suturing of the

23

composite scaffolds with the tissue in clinical applications. In addition, Yang et al43 used silk Multiplelayered composite scaffolds of silk fibroin/BAMG


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fibroin/poly(l-lactide-co-caprolactone) blend dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol

2

(HFIP) to electrospin nanofibrous scaffolds for tendon tissue engineering. In Yang’s scaffolds,

3

the fibers were only aligned along one direction. The breaking strength of the scaffolds along the

4

fiber alignment direction (39.1 MPa) was significantly different from the breaking strength of the

5

scaffolds perpendicular to the fiber alignment direction (3.4 MPa) in dry state. However, the

6

elongation at break in those two directions was 58.8 % and 212.1%, respectively. The application

7

of the scaffolds was limited due to the significant differences of the mechanical properties in the

8

parallel and perpendicular directions. The breaking strength was higher than the RSF/BAMG

9

composite

scaffolds

due

to

its

great

thickness

of

600

µm and

the

blend

of

10

poly(l-lactide-co-caprolactone). McClure et al 22 fabricated polycaprolactone (PCL)/silk fibroin

11

and polydioxanone (PDO)/silk fibroin scaffolds with aligned fibers by electrospinning and tested

12

in wet state. After immersion in methanol, the breaking strength and elongation at break of the

13

PCL/SF and PDO/SF were 4.8 and 7.6 MPa, 0.5 % and 0.6 % in parallel to the mandrel rotating

14

direction. While in the perpendicular to the mandrel rotating direction, they were 0.7 and 0.7

15

MPa, 2.7 % and 2.7 %, respectively. The breaking strength was high while their elongation at

16

break was poor in parallel to the mandrel rotating direction. Jose et al

17

electrospinning apparatus to generate aligned scaffolds with silk fibroin/PEO blend by oscillating

18

the deposition signal (ODS) of multiple collectors. The axially breaking strength and elongation

19

at break of the meshes produced from the SF/PEO blend were 17.6 MPa and 4.0 % in dry state,

20

while they were 16.5 MPa and 143 % in wet state due to the combination of PEO. For some

21

random fiber scaffolds, Liu et al

22

randomly collected nanofibers by electrospinning. After ethanol treatment, the breaking stress

23

and strain of the SF nanofibers mats were 11.15 MPa and 7.66 % in dry state, and 3.32 MPa and

45

44

adopted a new

prepared SF/CaCl2/Formic acid solutions to fabricate




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174.0 % in wet state, respectively. Gandhi et al 20 used silk fibroin/formic acid/single walled

2

carbon nanotubes (CNT) solution to electrospin scaffolds with random and aligned fibers. The

3

results showed that breaking strength and elongation at break of aligned scaffolds after

4

immersion in methanol and stretching were 58.0 MPa and 0.1 %. Though the breaking strength

5

was high, the elongation at break was too small to use in clinical applications. Moreover, the

6

fibers were only aligned along one direction in the scaffolds reported in the literatures20, 22, 43, 44.

7

In short, the RSF/BAMG composite scaffolds possessed isotropous mechanical properties in

8

both parallel and perpendicular directions. There were no organic solvent or other agents left in

9

the scaffolds with reinforced mechanical properties, which may not be friendly to the cells in

10

human body.

11

Table 1. Suture retention of RSF/BAMG composite scaffolds and bare RSF scaffolds with

12

aligned fibers in multiple layers in dry and wet states Sample Composite scaffolds Bare RSF scaffolds

Suture retention (N) Dry state Wet state 4.3 ± 0.7 1.9 ± 0.3 1.1 ± 0.3 0.4 ± 0.1

13 14

Table 1 shows that the suture retentions of the post-treated composite and bare RSF scaffolds

15

were 4.3 and 1.1 N in dry state, and 1.9 and 0.4 N in wet state, respectively. It proves that the

16

composite scaffolds were evidently reinforced by the BAMG compared with the bare RSF

17

scaffolds, which was coincident with the result of tensile test. The suture retention strength of the

18

composite scaffolds was more than or closely to 2.0 N in dry and wet states, which was generally

19

accepted for the requirement of suturing with tissue in implantation 36.



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In a word, the grill-like composite scaffolds of RSF and BAMG had significantly great

2

mechanical properties due to the combination of BAMG and well-aligned fibers. The reinforced

3

composite scaffolds may satisfy the needs for tissue engineering applications.

4

3.3. Secondary Structure and Crystallinity Analysis

5 6

Figure 5. Determination of silk secondary structure and crystallinity. (a) FTIR spectra curves

7

and corresponding deconvoluted FTIR-ATR profiles of amide I of (b) as-spun and (c)

8

post-treated scaffolds; (d) WAXD diffractograms and corresponding deconvoluted crystal

9

structures of (e) as-spun and (f) post-treated scaffolds, respectively.

10 11

Table 2. Quantitative analysis of the secondary structure and crystallinity of the RSF scaffolds β-sheet

α-helix/random coil

β-turn

content/%

content/%

content/%

As-spun

21.1

64.1

14.8

38.2

Post-treated

40.5

48.4

11.1

53.8

Sample

12 Multiplelayered composite scaffolds of silk fibroin/BAMG


Crystallinity/%


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Page 16 of 30

Li Page 16 1

FTIR is universally applied to investigate the secondary structure of silk fibroin

46, 47

. FTIR

2

spectra of as-spun and post-treated RSF scaffolds were shown in Fig.5. The secondary structure

3

information can be obtained by analyzing the shape and position of different bands in Amide I

4

region of the spectrum. For Amide I 48, 49, the random coil/α-helical conformations give bands in

5

the range of 1640–1660 cm−1, the β-sheet and β-turn conformations occurs in the range of 1618–

6

1640 cm−1 and 1660-1700 cm−1, respectively. Fig. 5a shows that at 1627 and 1645 cm−1 there

7

were obvious peaks occurred in the post-treated scaffolds which presented the β-sheet and

8

random coil/α-helical conformations compared with the as-spun scaffolds. For Amide III, 1233

9

and 1266 cm−1 are assigned as the characteristic peaks of the random coil/α-helical and β-sheet 37

10

conformations

. Fig. 5 (b and c) and Table 2 show the deconvoluted FTIR-ATR profiles of

11

Amide I and quantitative analysis of the secondary structure. It indicates that the content of

12

β-sheet conformation increased from 21.1 to 40.5 % by water vapor annealing, while the content

13

of random coil/α-helix was decreased from 64.1 to 48.4 %, which meant water vapor annealing

14

promoted random coil/α-helix transforming into β-sheet. The change of secondary structure

15

observed here might relate to the mechanical properties of the RSF scaffolds. The large breaking

16

strength could be attributed to the high β-sheet content, which related to the crystallization of

17

RSF 15,32.

18

WAXD is often used to investigate the crystalline structure of RSF materials. Fig. 5d shows

19

the WAXD and crystallinity analysis of the (e) as-spun and (f) post-treated RSF scaffolds. As

20

shown in Fig. 5d, the one-dimensional diffractograms of the as-spun scaffolds showed only one

21

broad peak centered around 4.07 Å corresponding to amorphous structure, while the post-treated

22

scaffolds exhibited characteristic peaks at 2.09, 2.26, 3.60 and 4.48 Å of silk I and II

23

meant that the water vapor annealing had significantly changed the conformation and Multiplelayered composite scaffolds of silk fibroin/BAMG


50, 51

. It

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Li Page 17 1

crystallinity of RSF fibers. The crystallinity of the as-spun and the post-treated RSF scaffolds

2

was analyzed in Fig. 5 (e and f), which shows that the area of amorphous region was evidently

3

decreased after water vapor annealing. Table 2 shows that crystallinities of the as-spun and

4

post-treated RSF scaffolds were 38.2 % and 53.8 %, respectively. The high crystallinity was

5

attributed to the temperature of water molecule action, which changed the conformation of RSF

6

15, 52

7

mechanical properties of RSF fibers.

8

3.5. In Vitro Cell Culture Study

9

Fig. 6 shows the cell proliferation of PIECs cultured on the post-treated scaffolds with or

10

without VEGF for 7 days, respectively. As shown in Fig.6, the OD value increased obviously

11

during the 7 days culture, which revealed that PIECs could grow well on all the groups.

12

Compared with the coverslips, the optical density (OD) values of other experimental groups were

13

high. This proves that the RSF scaffolds exhibited good cytobiocompatibility. The scaffolds

14

loaded with VEGF (RSF-GF) showed faster cell proliferation than those without VEGF

15

(RSF-WGF), which indicates that VEGF was successfully encapsulated. The loaded VEGF

16

remained its bioactivity and released VEGF could promote fast proliferation of PIECs on the

17

VEGF-loaded scaffolds1.

. Meanwhile, an increase of the crystallinity might have good effects on the stability and

18

Furthermore, the cell spreading and cytoskeletal structure were observed by SEM and LSCM

19

(2D and 3D) in Fig. 7. SEM images show that PIECs could adhere and stretch well on both the

20

RSF-GF and RSF-WGF samples. Compared with the RSF-WGF scaffolds (Fig. 7a), there are

21

obviously more cell attachment on the RSF-GF scaffolds (Fig. 7b), which is consistent with the

22

trend of MTT assay results presented in Fig. 6. As shown in the LSCM 2D images, it can be seen

23

that the PIECs cultured on the aligned fibrous scaffolds exhibited a kind of contact guidance by Multiplelayered composite scaffolds of silk fibroin/BAMG



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 18 of 30

Li Page 18 1

growing parallel to the RSF fibers, which proved that the well-aligned fibers could guide the

2

cells growth along the orientation direction

3

structured tissues, e.g. ligament, nerve or muscle.

53

. That might be useful in the engineering of

4 5

Figure 6. MTT OD value of PIECs cultured on the post-treated electrospun scaffolds for 7 days

6

(*P < 0.05).

7

The LSCM 3D images (Fig. 7a'' and b'') prove that PIECs could not only grow on the surface

8

of the scaffolds (Fig. 7a and b), but also infiltrate into the scaffolds. Because the scaffolds

9

possessed bigger pore diameter (Fig. 3b" and Table SI 1) than the nanofibrous scaffolds

10

fabricated by Zhang (average pore size of 0.3 µm)41, which offered good permeability for cells.

11

From the LSCM 2D images, we can see that the well-stretched cells were fusiform with a length

12

of about 80 µm and a width of about 8 µm. Before cells attaching and stretching on the scaffolds,

13

the cells were circular with a size smaller than 8 µm. It might be possible for PIECs to enter the

14

scaffolds through big pores. This is very important because the cells grew and distributed in the

15

scaffolds with large pores are easier to integrate with tissue once the scaffolds degrade gradually.

16

If the pore size of the scaffolds is much smaller than the cells, the cells only attach and stretch on

17

the surface of the scaffolds with dense structure 30, 34, 54. In addition, the cell adhesion and Multiplelayered composite scaffolds of silk fibroin/BAMG


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Li Page 19 1

proliferation results observed from LSCM images agreed with those of MTT assay (Fig. 6).

2

Owing to these good properties, the composite scaffolds might have great potential applications

3

for urethral, bladder, or muscle reconstruction in the future.

4 5

Figure 7. The spreading (SEM) and cytoskeletal structure (LSCM 2D and 3D) of PIECs cultured

6

for 3 days on the scaffolds: (a, a', a'') RSF-WFG, (b, b', b'') RSF-GF. LSCM 2D and 3D

7

photographs of PIECs (Red) labeled by Rhodamine. Multiplelayered composite scaffolds of silk fibroin/BAMG



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Page 20 of 30

Li Page 20 1 2

4. CONCLUSION

3

This study described a simple way to fabricate VEGF-loaded RSF/BAMG composite scaffolds

4

with well-aligned fibers by electrospinning. SEM image shows that the fibers had a good

5

orientation with a grill-like structure and pore size distribution shows that the scaffolds possessed

6

big pore diameter which was favorable for cellular growth. The tensile properties of the

7

composite scaffolds were significantly reinforced by BAMG and the well-aligned fibers

8

compared with the conventional electrospun RSF scaffolds. The suture retention of the

9

composite scaffolds was much more than the bare RSF scaffolds to satisfy the need for suture in

10

clinical applications. Water vapor annealing promoted the secondary structure transition and

11

crystallization of RSF fibers, which may result in the reinforcement of the scaffolds. In vitro

12

experiments show that the prepared scaffolds had good biocompatibility for cell growth and

13

VEGF-loaded scaffolds exhibited better cell proliferation than those without it. Moreover, PIECs

14

could infiltrate into the scaffolds instead of only spreading on the surface. In summary, the

15

VEGF-loaded composite scaffolds with aligned fibers in multiple layers could be considered as

16

attractive candidates for tissue engineering applications in many fields, such as nerve, urethral,

17

bladder, ligament or muscle.

18 19

Supporting Information Available. SI.pdf: Experimental details of BAMG preparation, MTT

20

assay, detailed data of pore size and tensile properties of RSF scaffolds. This information is

21

available free of charge via the internet at http://pubs.acs.org/.

22



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Li Page 21 1

ACKNOWLEDGEMENT

2

This work is supported by the National Natural Science Foundation of China (21274018,

3

81170641), the Innovation Program of the Shanghai Municipal Education Commission

4

(12ZZ065), China Postdoctoral Science Foundation (2014M551293), DHU Distinguished Young

5

Professor Program (A201302), the Fundamental Research Funds for the Central Universities

6

(2232013A3-11) and the Programme of Introducing Talents of Discipline to Universities

7

(No.111-2-04). We also acknowledge Mrs. Mengxia Chen for her help in in vitro experiments.

8 9

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List of Figure Captions

16

Figure 1. Schematic drawing of (a) the blend electrospinning and (b~e) the preparation process

17

of multilayered RSF/BAMG composite scaffolds with aligned RSF fibers

18

Figure 2. Schematic for the suture retention test of the composite scaffolds

19

Figure 3. SEM images of the muscular (a) and serosal (a") surfaces of BAMG, RSF surface of (b)

20

composite scaffolds, and corresponding diameter distributions of the RSF fibers (a' and b'), and

21

(b") the pore size distribution of the post-treated RSF scaffolds.

22

Figure 4. Stress–strain curves of the post-treated composite scaffolds in dry (a) and wet (b)

23

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Figure 5. Determination of silk secondary structure and crystallinity. (a) FTIR spectra curves

2

and corresponding deconvoluted FTIR-ATR profiles of amide I of (b) as-spun and (c)

3

post-treated scaffolds; (d) WAXD diffractograms and corresponding deconvoluted crystal

4

structures of (e) as-spun and (f) post-treated scaffolds, respectively.

5

Figure 6. MTT OD value of PIECs cultured on the post-treated electrospun scaffolds for 7 days

6

(*P < 0.05).

7

Figure 7. The spreading (SEM) and cytoskeletal structure (LSCM 2D and 3D) of PIECs cultured

8

for 3 days on the scaffolds: (a, a', a'') RSF-WFG, (b, b', b'') RSF-GF. LSCM 2D and 3D

9

photographs of PIECs (Red) labeled by Rhodamine.

10

List of Table Captions

11

Table 1. Suture retention of RSF/BAMG composite scaffolds and bare RSF scaffolds with

12

aligned fibers in multiple layers in dry and wet states

13

Table 2. Quantitative analysis of the secondary structure and crystallinity of the RSF scaffolds




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For Table of Contents Only

2

Title:

3

Bladder acellular matrix graft reinforced silk fibroin composite scaffolds loaded VEGF with

4

aligned electrospun fibers in multiple layers

5

Author:

6

Zhaobo Li1, Qiangqiang Liu1, Hongsheng Wang2, Lujie Song3, Huili Shao1, Minkai Xie3,

7

Yuemin Xu3,*, Yaopeng Zhang1,*

8 9 10

BAMG and silk fibroin mats with grill like structure were combined to reinforce the scaffolds, which exhibited good biocompatibility.

11 12



Bladder Acellular Matrix Graft Reinforced Silk Fibroin Composite

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