Regulating Coupling Efficiency of REDV by Controlling Silk Fibroin

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Regulating coupling efficiency of REDV by controlling silk fibroin structure for vascularization Danyu Yao, Ge Peng, Zhiyong Qian, Yimeng Niu, Haifeng Liu, and Yubo Fan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00553 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017

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ACS Biomaterials Science & Engineering

Regulating coupling efficiency of REDV by controlling silk fibroin structure for vascularization Danyu Yao,1 Ge Peng,1 Zhiyong Qian,1 Yimeng Niu,1 Haifeng Liu,*,1,2 Yubo Fan*,1,2,3 1

Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education,

School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, People’s Republic of China 2

Beijing Advanced Innovation Centre for Biomedical Engineering, Beihang University,

Beijing 102402, People’s Republic of China 3

National Research Center for Rehabilitation Technical Aids, Beijing 100176,

People’s Republic of China

*Corresponding author: Haifeng Liu Tel.: 86-10-82338456; Fax: 86-10-82338456 Email address: [email protected] Address: School of Biological Science and Medical Engineering, Beihang University, Xue Yuan Road No. 37, Haidian District, Beijing 100191, People’s Republic of China.

*Corresponding author: Yubo Fan Tel.: 86-10-82339428; Fax: 86-10-82339428 Email address: [email protected] Address: School of Biological Science and Medical Engineering, Beihang University, Xue Yuan Road No. 37, Haidian District, Beijing 100191, People’s Republic of China.

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Abstract: Controlled and rapid vascularization of engineered tissues remains one of the main challenges for tissue engineering. The immobilization of peptides and other bioactive molecules on the scaffolds has been demonstrated to be able to improve vascularization. However, the density of peptides modified on the scaffold surface is an important factor influencing vascularization. Thus, regulating the coupling efficiency of peptides may be an effective way to adjust vascularization. In this study, two-dimensional (2D) silk fibroin (SF) films and three-dimensional (3D) porous SF scaffolds with different secondary structure were prepared and coupled with REDV peptide. Compared with the high crystalline scaffolds, more peptides were bounded on the scaffolds with low crystalline both in 2D and 3D forms, with the result that more endothelial cells adhered on the low crystalline SF scaffolds. In addition, the in vivo angiogenic assays demonstrated that the low crystalline scaffolds showed higher blood vessel density after 28 days of implantation, which was 1.4 times as much as that of the high crystalline group. The results indicated that the peptide density could be controlled by SF structure, and the low crystalline SF scaffolds modified with REDV peptide could be a potential candidate for inducing angiogenesis in tissue engineered applications.

Keywords: Silk fibroin structure, Coupling efficiency, REDV, Vascularization

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1. Introduction Silk fibroin (SF) has been widely used as surgical suture material, cell culture matrix and tissue engineering scaffold due to its excellent biocompatibility and desirable mechanical properties.1 Nevertheless, the surface of SF scaffolds needs to be improved for better performance in these applications, especially in tissue engineering. This engineering demands rapid vascularization, which is much influenced by the cell-matrix interactions, for the regeneration of tissues within implants.2 Creating desired interfaces between implanted scaffolds and surrounding tissues has thus become increasingly important in tissue regeneration.

One of the approaches to improve interactions between the implants and organism is to make the scaffolds more “attractive” for target cells. Thus, strategies to functionalize the surface of scaffolds have been developed to improve cell adhesion and enhance tissue neogenesis within these scaffolds.3 Blood vessels are established by two processes, angiogenesis and vasculogenesis. Both processes require endothelial cells (ECs) to form the capillary lumen or vessel primordial.4 Thus, the ECs-matrix interactions are crucial in the vascularization of implanted scaffolds. REDV (Arg-Glu-Asp-Val) tetrapeptide has drawn much attention in the area of surface modification of materials.5 REDV can specifically bind to the α4β1 integrin, which is limited to a small number of cell types. The peptide shows specific affinity with ECs, which can be used to enhance vascularization.6

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Recently, numerous studies have shown that the immobilization of certain peptides on the scaffold surface enhances osteoblasts7 and ECs8 adhesion. Moreover, Chollet et al. found that the density of peptides immobilized on the scaffolds surface was a crucial parameter influencing ECs adhesion and focal contact formation.9 The number of focal contacts was improved with increased peptide densities grafted on the scaffolds surface. Thus, improving the peptide density on the scaffolds surface can be an available approach to catch more ECs and promote scaffold vascularization. In addition, according to Chollet’s work, the peptide density on PET could be adjusted by controlling COOH anchor. The number of carboxyl and hydroxyl chain-ends displayed on the material surface decided the amount of coupling peptide. Different materials were modified on the PET surface, thus providing different binding site density for peptide covalent attachment. The material surface properties played an important role in material modification.

The presence of serine, aspartic, tyrosine and other reactive amino acids in SF gives a chance for surface modification to tailor the SF scaffold for a desired application.10 The serine and tyrosine residues in degummed silk can be sulfated by chlorosulfonic acid in pyridine, resulting in hydrolysis of the SF backbone.11 Aspartic, glutamic and lysine residues can participate in carbodiimide coupling reaction, a standard method for amino acid modification.12 These amino acids are widely distributed in the crystalline and non-crystalline regions, as well as the terminal of molecular chains.13 SF scaffolds mainly consist of the β-sheet secondary structure (silk II), metastable structure (silk I) and the random coil structure.14 The silk I structure is unstable and 4


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can be converted to silk II structure upon exposure to heat or stretching. The silk II structure is thermodynamically stable and difficult to change. The crystalline β-sheet domains prevent the penetration of water and resulting in stable properties.10 The properties of SF scaffolds, such as mechanical properties, degradation properties and thermal properties, vary according to the content of β-sheet crystals.15 Several methods have been developed to induce β-sheet structure in SF scaffolds, resulting in SF scaffolds with different crystalline contents.16 Through these processes, SF scaffolds may reach a crystalline content of ~55% (methanol treated) or a content of ~30% (temperature controlled freeze).

Considering the influences of the secondary structure of SF scaffolds to its properties, we hypothesize that the crystalline structure may play a role in the efficiency of SF scaffold modification. The aim of this study is to probe the influence of SF structure to the coupling efficiency of REDV on SF scaffolds. We modified SF scaffolds with different secondary structure by grafting REDV peptides onto the scaffold surfaces, improving their EC capture abilities and promoting vascularization in vivo. We prepared SF scaffolds with different crystalline structure in two-dimensional (2D) and three-dimensional (3D) forms. The secondary structure and peptides coupling efficiency were detected, as well as the ECs capture ability of scaffolds. Finally, we evaluated the vascularization abilities of 3D porous SF scaffolds in vivo.

2. Materials and methods 5



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2.1. Preparation of SF solutions. Bombyx mori fibroin solution was prepared according to a previously published procedure.15b Bombyx mori silk fibers were supplied from Rugao Chunqiu Textile Co. Ltd (Jiangsu, China). Natural silk was boiled for 30 min in the aqueous solution of 0.02 M Na2CO3 (Sinopharm Chemical Reagent Co., Ltd., China) and rinsed with deionized water to remove the sericin proteins. The rinse process was repeated 5 times. The extracted SF was dried in a 60oC oven (Shanghai fullmark Experimental Equipment Co. Ltd ， China) and dissolved in 9.3 M LiBr (Shanghai Aladdin biochemical Polytron Technologies Inc, China) solution at 60oC for 4 h, yielding a 20% (w/v) solution. The SF solution was dialyzed against distilled water using Slide-a-Lyzer dialysis cassettes (Pierce, molecular weight cutoff 3500) for 3 days to remove salt. After dialyzing, the SF solution was centrifuged at 9000 rpm for 20 min at 4oC to remove aggregates formed during the process. Centrifuged SF solution was sterilized by the low temperature intermittent sterilization. The final concentration of aqueous SF solution was ~ 6 wt%, determined by weighing the remaining solid after drying.

2.2. Preparation of SF films and SF porous scaffolds. SF aqueous solution was cast round slides to generate SF films with random coil structure (SF-C). SF films were treated with gradient methanol (Beijing Chemical Plant, China) with a concentration of 50%, 65%, 80% and termed MA50, MA65, MA80, respectively. Porous SF scaffolds with different secondary structure were prepared according our previous work.15a 3D porous SF scaffolds were fabricated by lyophilization (L-SF) and temperature controlled freeze process. The SF solution was diluted to 3.5 wt% 6


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and poured into molds and frozen at -70oC for 2h. A part of frozen SF samples were moved into a -4oC refrigerator (Beijing Fu Yi Electrical Appliance Co., Ltd., China) for 48 h before taken out without any further treatment (TC-SF). And the others were freeze-dried for 48h before treated with methanol (MA-SF). SF scaffolds treated with 50% and 65% methanol could not keep their porous structure. Thus, only scaffolds treated with 80% methanol were used in the subsequent study.

2.3. Peptide modification of SF scaffolds. The REDV peptides were synthesized by GL Biochemistry (>99% purity, Shanghai). The peptide was dissolved at 1 mM/L in ultrapure water, and stored at -80°C. REDV peptide modified SF scaffolds were prepared through carbodiimide coupling reaction. Briefly, the SF scaffolds were immersed in EDC-NHS (Tokyo Chemical Industry, Japan) solution for activation. Then, the activated scaffolds were immersed in REDV peptide solution and incubated for 2 h. The unbounded peptides were removed thoroughly by Milli-Q water. After REDV peptide modification, the MA50, MA65 and MA80 were termed MAR50, MAR65 and MAR80. MA-SF scaffold and TC-SF scaffolds were termed MAR-SF, TCR-SF scaffolds, respectively.

The coupling efficiency of REDV peptides was assayed using a microplate reader (Thermo Scientific, USA). Rhodamine-labeled REDV peptides (GL Biochemistry, China, excitation wavelength 560 nm, emission wavelength 584 nm) were grafted on SF scaffolds in the same way as described above. REDV peptides solution was diluted to 0.25µM/ml with water. For 3D porous scaffolds, every 200µl SF solution was

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added into a mold to fabricated porous scaffold. 3D scaffold was immersed into 200µl REDV peptides solution for REDV peptides modification. For each 2D SF film, 50µl SF solution was cast on a slide with a diameter of 8 mm. SF film was immersed into 50µl REDV peptides solution for REDV peptides modification. The water used to remove unbounded peptides was collected with a centrifuge tube (V1). Standard curve of the concentration of rhodamine-labeled REDV peptides was acquired through gradient dilution of standard solution. The concentration of the collected water (unbounded peptides) was determined according to the standard curve (C1). The original amount of REDV peptide was W0, thus, the efficiency of coupling peptides (E) was monitored by the follow formula:

E=1-C1╳V1/W0

(1)

2.4. Characterization of SF scaffolds. ATR-FTIR: Samples were cut into slices. The structure analysis of scaffolds was detected with a FTIR 7600 spectrophotometer (lambda scientific, Australia). For each measurement, 32 scans were recorded with a resolution of 4 cm−1 with the wavenumber ranging from 400 to 4000 cm−1.

XRD: The crystal structures of SF scaffolds were obtained by X-ray diffraction (XRD, D8 Advance, Bruker, Germany) with Cu-Kα radiation in the range of 10–40o at 6o/min.

Morphology: The SF scaffold topography was observed by scanning electron microscopy (SEM, FEI QUANTA FEG250, USA). The SF samples were pasted on the specimen stage with a conducting resin and sputtered with gold for 90 s. 8


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Water contact angle measurement: Static water surface contact angles of SF films were measured using a contact angle analyzer (Shanghai powereach digital technology equipment Co. Ltd., China). The droplet of 1 µl ultrapure water was deposited on the surface of SF films.17 Each sample was repeated 5 times.

2.5. In vitro evaluation of scaffolds. Cell culture: HUVECs were used to evaluate the in vitro biocompatibility of these scaffolds. HUVECs were isolate from human umbilical cord veins collected from Haidian Maternal & Child Health Hospital (Beijing, China). Human umbilical cords collection and use were approved by the Ethics Committee of Haidian Maternal & Child Health Hospital. Informed consent was provided according to the Declaration of Helsinki. HUVECs were obtained from human umbilical vein under aseptic conditions. The HUVECs were detached from the umbilical vein lumen by short trypsinization with 1 mg/ml of collagenase II (Sigma, USA) at 37°C for 15 min. The detached cells were resuspended and seeded on culture dishes. The cells were cultured in endothelial cell medium (ECM, Invitrogen, USA). When primary ECs became 70-80% confluent, they were detached from the dishes with 0.25% trypsin (Invitrogen, USA) and expanded at a splitting rate of 1:4. The second passage cells were then used for further study. All cells were maintained at 37oC in an incubator with 5% humidified CO2. The cultures were replenished with flash medium at 37oC every 3 days. HUVECs detached from the dishes were re-suspended in ECM after being centrifuged. The cells were identified by their cobblestone morphology and confirmed by staining with a specific antibody to CD31

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as described previously.18 Cells were detached from the dishes and seeded into the scaffolds at a density of 5×104 cells per sample.

Cell attachment: Cell attachment on the SF scaffolds was measured by confocal microscopy. After culturing for 24 h, the cell-seeded scaffolds were washed 3 times with sterile PBS to remove the unattached cells and steeped in 4% paraformaldehyde (Sigma-Aldrich, USA) for 30 min and washed with PBS. The rewashed samples were permeabilized with 0.1% Triton X-100 (Biotopped, China) for 15 min, and stained with fluorochrome (F-actin filaments (1:100 dilution, Sigma, USA), vinculin (1:100 dilution, Abcam, UK) and DNA (1:1000 dilution, Sigma, USA)) for fluorescent microscopy. For quantification of attached cells, we counted the number of stained cell nucleus of six random view fields (under 10× magnification) of each sample. The density of attached cells was reported as the average number of each view field and expressed as mean values ± the standard deviation.

Cell viability: CCK-8 assay was applied to detect cell viability. SF scaffolds with seeded cells were transferred into a new 24-well plate. The cells were treated with 10% CCK-8 (Beyotime, China) in DMEM media for 2 hours at 37oC. The absorbance at 450 nm was measured using a microplate reader. Cell number was correlated to OD. All samples were run in triplicate.

2.6. In vivo evaluation of scaffolds. Subcutaneous implantation: All procedures were carried out in strict accordance with guidelines for the Care and Use of Laboratory Animals of Beijing Municipal Science & Technology Commission. The 10


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protocol was approved by the Ethics Review Committee for Animal Experimentation of the Peking University (SYXK (Beijing) 2006-0025). Surgeries were performed under phenobarbital anesthesia, and all efforts were made to minimize suffering.

The in vivo vascularization analysis was performed using SD male rats (200-250g). Each SD rat was subcutaneously implanted with irradiation sterilized scaffolds (8mm in diameter, 2mm in height). Scaffolds were sterilized and immersed in PBS 2 hours before implantation and then implanted into the subcutaneous space on the dorsal region. The subcutaneous implantation was under general anesthesia of 1% Pentobarbital Sodium.19 On 7, 14 and 28 days post-implantation, animals were euthanized and the specimens (n = 4 from each time point) along with the adjacent tissues were collected and fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for further examinations.20

Hematoxylin and eosin staining: The harvest specimens were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for 48h at room temperature and embedded in paraffin blocks after a series of xylene (Sinopharm Chemical Reagent Co., Ltd.) and graded ethanols (Sinopharm Chemical Reagent Co., Ltd.) dehydrate. Paraffin sections were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, USA) to assess cell infiltration and granulation tissue formation area. The tissue slices were observed by Inverted microscope and photographed.

Immunofluorescence staining: In order to characterize the blood vessel formation, sections were stained for CD34. Endogenous peroxidase activity was blocked by 11



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incubating sections in 3% H2O2 (Dongfang Chemical Factory, China) for 15 min. Antigen retrieval was performed using citrate buffer pH 6 (Google Bio, China) in a microwave oven at 98oC for 15 min. Slides were rinsed and treated with PBS containing 10% goat serum for 20 min to reduce nonspecific background staining. Then primary antibody against the endothelial cell marker, CD34 (1:100 dilution, Abcam, UK) was added and incubated overnight at 4oC, followed by secondary antibody labeled with Fluorescein isothiocyanate (Beijing Emarbio Science & Technology Co., LTD) at 37oC for 20min. Vessels were counted under an inverted microscope (AxioVert A1, Carl Zeiss, Germany).21 The vessels formed within implants were evaluated by quantification of five random view fields (under 20× magnification) of the stained sections. The density of vessel was reported as the average number of vessels and expressed as mean values ± the standard deviation.22

2.7. Statistical Analysis. Data were presented as means ± standard deviation. Statistical analysis was conducted using one-way ANOVA followed by a Student-Newman-Keuls test. Results were considered to be statistically significant at *p ≤ 0.05.

3. Results and discussion 3.1．．Characterization of 2D SF films Difference in the structure of SF films prepared from the various treatments was determined by FTIR-ATR and XRD assays (Figure 1E, F). According to the XRD curves, the crystallinity of SF films increased after methanol treatment (Figure 1F). The absorption of amide I at 1610-1630 cm-1

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and absorption of amide II at 1510-1520 cm-1 are characteristic of silk II structure FTIR spectrum.23 The SF film without methanol treatment was used as control (SF-C) and exhibited random coil structure. All curves of the methanol treated films appeared peaks at 1620 cm-1 and 1515 cm-1, indicating the β-sheet structure. And the peaks became increasing sharper from MA50 to MA80, indicating the increase of crystalline structure. Deconvolution of the infrared spectral region in the amide I (1595–1705 cm-1) region was performed by Peakfit software to assess the contents of different secondary structures. The β-sheet content of SF-C was 33.11±0.47 percent. And the content increased to 47.98±1.01, 51.16±1.16 and 54.91±1.38 percent for MA50, MA65 and MA80, respectively. It was consistent with previous studies, which showed that while the methanol concentration increased from 50% to 80%, the contents of silk II structure that mainly constituted of β-sheet structure, increased with the methanol concentrations.24

Morphologies of SF films were observed by SEM (Figure 1A-D). Before treated with methanol, SF films exhibited a smooth surface. All methanol treated SF films showed rougher surface features compared with the untreated films, which might be caused by the change of SF protein conformation from random coil to the β-sheet crystalline structure during the methanol treatment process.25 Interestingly, the film morphology seemed smoother with increasing methanol content, which might be caused by the increasing orderly structure. It has been demonstrated that the rough surface of these films was conducive for cell adhesion.26

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Peptide density was determined by grafting rhodamine labelled REDV peptides and measuring the OD values of the waste solution of as-treated scaffolds by a micro-plate reader (Figure 1G). The coupling efficiency of MA50, MA65 and MA80 was 62.94± 7.96, 54.13 ± 5.43 and 42.07 ± 8.25%, respectively. The peptide density was calculated and the peptide density of MAR50, MAR65 and MAR80 was 156.52± 19.79, 134.61±13.5 and 104.62±20.52 pmol/mm2. The difference of coupling efficiency might be owned to the different contents of crystalline structure. Compared with silk I and random coil structure, the silk II structure was more stable.27 The methyl groups and hydrogen groups of opposing sheets in β-sheets interacted with each other and formed the inter-sheet stacking in the crystals. The strong hydrogen bonds and van der Waals forces led to a stable structure.14 The energy barrier of silk I structure was much lower than that for silk II.28 Silk II was mainly composed of β-sheet structure which was a more stable structure than β-turn structure, the main structure of silk I.23 The less stable structure might offer more coupling sites or lower energy barrier,28 which led to higher modification efficiency.

Mammalian cells prefer to attach on scaffold surfaces with a suitable hydrophilicity.29 Water contact angle is a macroscopic property that quantifies the hydrophilicity of the scaffolds surface. The contact angles of the methanol treated SF films were presented in Figure 2. The water contact angles of MA50, MA65 and MA80 was 50.78±2.48, 57.85±0.60 and 60.8±1.06, respectively. It seemed that the water contact angles of the three SF films increased with the β-sheet contents. A possible reason might be that the crystalline β-sheet structure hindered the permeating 14


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of water.10 When the silk II structure formed, most of the hydrophilic SF-water interactions were destroyed,28 leading to a hydrophobic surface of high crystalline SF scaffolds. Another factor that affected water contact angles was the surface morphologies of SF films. According to SEM observations (Figure 1), the film morphology became smoother with the increasing methanol contents. Although previous studies showed that the contact angles tended to increase with scaffold surface roughness,30 SF films in the present study exhibited an adverse tendency. The contact angles increased when the surface of films became smoother from MA50, MA65 to MA80. The result might be owned to the influence of the film structure, and the hydrophobic effects of high crystalline structure played the major role.

DNA staining revealed that REDV peptides constituted a good ligand for improving the cell attachment at short time intervals (24 h) (Figure 3). The cells attached on each sample increased from 282.96±68.04, 388.15±87.25 to 565.00±98.25 per square millimeter for MAR80, MAR65 and MAR50, respectively. The cell number attached on MAR50 was about 2 times as much as that of the MAR80 group, which showed that the REDV peptide immobilization resulted in an improvement of HUVEC attachment. The focal adhesion of cells on SF films with various REDV densities was showed by immunostaining of vinculin (Figure 4). The results showed that vinculin was observed in all the three films, whatever the REDV peptide density. Moreover, the cells on MAR50 group exhibited a higher number of focal contacts compare with the other groups. Thus, for the SF films with different REDV peptides densities, the

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number of attached cells and focal contacts increased with their REDV peptide densities.

3.2. Characterization of 3D SF scaffolds

The objective of tissue engineering is to develop viable 3D succedaneums of the damaged organs or tissues.31 Scaffolds used in tissue engineering should establish the architecture and the physical boundaries of the engineered tissues.31a For tissue-engineering scaffolds, highly porous structure with interconnected pore network is required for cell growth and the transport of nutrients and metabolic waste.32 Thus, 3D porous SF scaffolds were prepared in this study.

The SF scaffolds were fabricated by lyophilization and temperature controlled freeze process respectively. The L-SF was treated with methanol to acquire the water stable structure. Morphologies of both porous SF scaffolds were exhibited through SEM observation (Figure 5A, B). Both TC-SF and MA-SF scaffolds showed interconnected porous structure, which was benefit for cell attachment and cell migration. The interconnected porous structure of the SF scaffolds allowed cell migration and nutrient diffusion, providing a good foundation for vascularization.

ATR-FTIR was used for assessment of SF scaffolds structure. For the curve of MA-SF, a peak appeared at 1624 cm-1, indicative of the silk II structure (Figure 5C).23 The TC-SF showed peaks at 1651 and 1643 cm-1, indicative of silk I and random coil structure, respectively. The β-sheet contents of TC-SF and MA-SF were 31.31±0.17 and 51.30±0.96 percent, respectively.15a XRD test also indicated that the crystalline 16


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structure content of MA-SF was higher than that of the TC-SF (Figure 5D). The coupling efficiencies of REDV peptides on MAR-SF and TCR-SF scaffolds were 66.79±0.83 and 81.92±1.38 percent, respectively (Figure5 E), which showed similar performance to the 2D films. The peptide density was calculated and the peptide density of MAR-SF and TCR-SF was 33.40±0.42 and 40.96±0.69 nmol/mm3.

Concerning the biological evaluation, the main objective of this research was to study the influence of SF scaffolds with different structure grafted with different REDV densities on the behavior of HUVECs in vitro and in vivo. The cell behavior was similar on 3D porous scaffolds to 2D films. More cells were detected on the TCR-SF than that of the MAR-SF 24 h after cell seeding (Figure 6). Considering the wide distribution of cells among 3D porous scaffolds, the viability of total cells was also quantified through CCK-8 assay. According to the CCK-8 assessment, cell viability of the TCR-SF scaffolds was 1.19±0.09 times as much as that of the MAR-SF scaffolds. REDV peptide promoted the attachment of ECs,3 and the REDV densities played a role in the different performance of the two scaffolds.

3.3. Vascularization in vivo The capacity to induce cell homing, promote cell attachment and proliferation is crucial for the survival of implanted scaffolds. It is well known that cell behavior is much influenced by the surface properties of the scaffolds. To further evaluate the performance of SF scaffolds with different peptide densities on vascularization in vivo, TCR-SF and MAR-SF scaffolds were implanted

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subcutaneously in rats. We assessed the samples harvested from rats at 1, 2 and 4 weeks post-implantation.

Histological sections of the MAR-SF scaffolds implanted for 1 week showed little cell infiltration (Figure 7). The cells appeared only at the periphery of MAR-SF, with no cells reaching the middle of the scaffolds. More cells appeared within the TCR-SF at the same time point, indicating better cell infiltration of this scaffold. With increasing of implantation time, the infiltrated cells increased obviously within both kinds of scaffolds. And the TCR-SF group kept better performance all the time. The REDV peptides immobilized on SF scaffolds might provide a bioactive cue for infiltrating cells, especially the endogenous ECs, to induce vascularization within the implanted scaffolds. According to the in vitro cell attachment results, more ECs attached on the scaffolds with higher REDV peptide densities. Thus, the low crystalline scaffolds with more peptides bounded on the surface (TCR-SF) showed better cell infiltration than the high crystalline group (MAR-SF).

Capillary-like structures with clear lumens within scaffolds could be found at 2 and 4 weeks (Figure 7). The CD34+ vessels within scaffolds were labeled with FITC (Figure 8). The number of the CD34+ vessels within cambium was also quantified. About 46.01± 3.07 vessels per mm2 formed in TCR-SF scaffolds after 4 weeks, while only 32.71±1.77 newly formed vessels per mm2 formed in MAR-SF scaffolds on day 28, confirming the perfect vascularization capacity of the TCR-SF scaffolds. Quantification of such vessels indicated that the TCR-SF scaffolds possessed better

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vascularization ability, verified the superiority of high peptide density on vascularization. The increase of the number of CD34+ capillaries within the TCR-SF scaffolds might be correlated with REDV peptide-related increase in cell infiltration. The faster cell infiltration might lead to better regeneration of engineered tissue and endowed the TCR-SF scaffolds with improved vascularization than that of the MAR-SF. These results suggested that low crystalline scaffolds modified with REDV peptides could well integrate with the host cells and the surrounding environments, providing a potential candidate for tissue vascularization.

4. Conclusions In this study, REDV peptides were covalently grafted onto 2D and 3D SF scaffolds with different secondary structures and resulted in scaffolds with different peptide densities. SF scaffolds with lower crystalline structure exhibited superior peptide coupling efficiency, leading to higher peptides density on the scaffold surface compared with the high crystalline scaffolds. The REDV-modified low crystalline scaffolds showed enhanced competitive forces for EC attachment and the formation of blood vessels. Both cell attachment in vitro and engineered tissue vascularization in vivo were related to the peptide density. Thus, it was a possible way to regulate vascularization through the controlling of SF structure.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31771058, 31470938, 11421202, 61227902, and 11120101001), National Key 19



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Technology R&D Program (2014BAI11B02, 2014BAI11B03, 2016YFC1100704, 2016YFC1101101), International Joint Research Center of Aerospace Biotechnology and Medical Engineering from Ministry of Science and Technology of China, 111 Project (B13003), Research Fund for the Doctoral Program of Higher Education of China (20131102130004), The transformation project for major achievements of Central Universities in Beijing (ZDZH20141000601), and Fundamental Research Funds for the Central Universities.

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Figure captions Figure 1 Characterization of SF films. A-D: SEM images of SF films. Upper right corner were corresponding enlarged views. A: SF-C; B: MA50; C: MA65; D: MA80. E: FTIR curves of SF films. F: XRD curves of SF films. G: Peptide coupling efficiency of SF films. *Statistically significant p < 0.05. Figure 2 Contact angles of SF films treated methanol water solution with different methanol content. Figure 3 Fluorescent staining of DNA (A-C) and the statistical histogram (D) for cells 24 h after cell seeding. A: MAR50; B: MAR65; C: MAR80. *Statistically significant p < 0.05. Figure 4 Fluorescent staining images of DNA (blue), F-actin (green) and vinculin (red) for cells 24h after cell seeding. A: MAR50; B: MAR65; C: MAR80. Figure 5 Characterization of 3D porous SF scaffolds. A, B: SEM images of SF scaffolds. A: TC-SF; B: MA-SF. C: FTIR curves of SF scaffolds. D: XRD curves of SF scaffolds. E: peptide coupling efficiency of SF scaffolds. Arrows refer to small pores on the wall of holes. *Statistically significant p < 0.05. Figure 6 Confocal microscopy images (A, B) and relative OD volumes of CCK-8 assay (C) of HUVECs cultured on SF scaffolds. A: TCR-SF; B: MAR-SF. Compared with the MAR-SF group, cells cultured on -4-R-SF scaffolds showed better cell attachment and significantly higher viability after 24 h of cultivation. *Statistically significant p < 0.05. Figure 7 The histological micrographs of the scaffolds implanted after 1w (A, D), 2w (B, E) and 4w (C, F). The TCR-SF scaffolds showed better cell infiltration compared with the MAR-SF group. A-C: TCR-SF; D-F: MAR-SF. Figure 8 Immunofluorescence images of SF implants (A, B) and Vessel density (C) within the granulation tissue at 28 d after implantation. A: TCR-SF; B: MAR-SF. *Statistically significant p < 0.05.

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Figures Figure 1 Characterization of SF films. A-D: SEM images of SF films. Upper right corner were corresponding enlarged views. A: SF-C; B: MA50; C: MA65; D: MA80. E: FTIR curves of SF films. F: XRD curves of SF films. G: Peptide coupling efficiency of SF films. *Statistically significant p < 0.05.

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Figure 2 Contact angles of SF films treated methanol water solution with different methanol content.

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Figure 3 Fluorescent staining of DNA (A-C) and the statistical histogram (D) for cells 24 h after cell seeding. A: MAR50; B: MAR65; C: MAR80. *Statistically significant p < 0.05.

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Figure 4 Fluorescent staining images of DNA (blue), F-actin (green) and vinculin (red) for cells 24h after cell seeding. A: MAR50; B: MAR65; C: MAR80.

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Figure 5 Characterization of 3D porous SF scaffolds. A, B: SEM images of SF scaffolds. A: TC-SF; B: MA-SF. C: FTIR curves of SF scaffolds. D: XRD curves of SF scaffolds. E: peptide coupling efficiency of SF scaffolds. Arrows refer to small pores on the wall of holes. *Statistically significant p < 0.05.

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Figure 6 Confocal microscopy images (A, B) and relative OD volumes of CCK-8 assay (C) of HUVECs cultured on SF scaffolds. A: TCR-SF; B: MAR-SF. Compared with the MAR-SF group, cells cultured on -4-R-SF scaffolds showed better cell attachment and significantly higher viability after 24 h of cultivation. *Statistically significant p < 0.05.

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Figure 7 The histological micrographs of the scaffolds implanted after 1w (A, D), 2w (B, E) and 4w (C, F). The TCR-SF scaffolds showed the best cell infiltration. A-C: TCR-SF; D-F: MAR-SF.

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Figure 8 Immunofluorescence images of SF implants (A, B) and Vessel density (C) within the granulation tissue at 28 d after implantation. A: TCR-SF; B: MAR-SF. *Statistically significant p < 0.05.

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For Table of Contents Use Only Regulating coupling efficiency of REDV by controlling silk fibroin structure for vascularization Danyu Yao, Ge Peng, Zhiyong Qian, Yimeng Niu, Haifeng Liu, Yubo Fan In this study, silk fibroin (SF) scaffolds with different crystalline structure were prepared and coupled with REDV peptides. The peptides density could be regulated through the control of SF structure. Scaffolds with lower crystalline structure possessed higher peptides density on the scaffold surface compared with the high crystalline scaffolds. Due to the influence of REDV peptide density, the REDV-modified low crystalline scaffolds showed enhanced competitive forces for ECs attachment and the formation of blood vessels.

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Regulating Coupling Efficiency of REDV by Controlling Silk Fibroin

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