Promoted Cytocompatibility of Silk Fibroin Fiber Vascular Graft through Chemical Grafting with Bioactive Molecules

GUAN Guo-ping(關國平)，ELAHI Md Fazley，WANG Lu(王 璐)* ，SHEN Gao-tian(沈高天)，CHEN Xiao-hui(陳肖會)，ZHOU Hao(周 浩)，XU Rui(徐 睿)

1 Key Laboratory of Textile Science and Technology，Ministry of Education，Donghua University，Shanghai 201620，China

2 College of Textiles，Donghua University，Shanghai 201620，China

Introduction

Natural silk from Bombyx mori consists of two kinds of proteins，core fibrils protein and coating glue protein，namely fibroin and sericin.Both of them comprise 18 natural amino acids，such as serine， tyrosine， and threonine［1］.After removing the coating glue protein (sericin)，fibroin fiber could be obtained from natural silk.Because of its robust mechanical properties and satisfactory biocompatibility，it has been used as medical sutures for several decades［2］.Moreover，regenerated silk fibroin (RSF)could be produced by dissolving the fibroin fiber into a tertiary aqueous solution and dialysis，and then used to prepare a number of RSF based biomaterials for clinical bone and soft tissue repair，such as 3D scaffolds，films，nonwoven mat，and hydrogel［3］.Therefore， silk fibroin has been increasingly studied in a broader field of biomaterials in the recent years［4-5］.

However， with limitation of textile science and engineering，silk fibroin fiber based biomedical prostheses have been little studied，especially for preparing devices in a smaller dimension.Therefore， with the development of textile equipment and technologies，a few fibroin fiber based implants have been tried to explore and fabricate，such as vascular graft［6-8］.

An ideal medical device is required to have satisfying mechanical properties，but also favorable biocompatibility.But it has been reported that the biocompatibility of the fibroin fiber based implants has not been satisfactory as anticipated.Therefore，in order to improve the biocompatibility，including antithrombotic properties as well as cytocompatibility of the vascular graft，surface modification may be required.Even though studies on surface modification of biomaterials derived from RSF have been carried out extensively，little research on surface modification on silk fibroin fiber and its products has been found.Therefore，the goal of the present study is to modify the surface of a silk fibroin fiber vascular graft (SF)with bioactive molecules， in order to improve the cytocompatibility of the graft.

1 Materials and Methods

1.1 Preparation of SF

Six“A”grade natural silk from Bombyx mori was selected to weave silk grafts，which were followed by a degumming process with 0.5% (by weight)Na2CO3aqueous solution to obtain the SF［6］.

1.2 Surface amination of the SF

It has been reported that the outer layer of silk fibroin fiber is mainly composed of serine and tyrosine，therefore chemical groups presented on the surface of fibroin fiber include —OH，—COOH，and —NH2［9］.So it provides possibility to use these chemical groups as active sites for functional surface modification of the fibroin fiber.3-aminopropyl-triethoxysilane(APTES)is a kind of coupling agent with amino group(—NH2)，and usually used for surface modification of biomaterials.Furthermore，its amino group (—NH2)could be introduced into substrate surface through chemical reaction with—OH of substrate surface［10-11］.So the surface amination of the SF would be achieved by the following hypothesis(Fig.1).

Fig.1 Schematic of surface amination of the SF

The graft of 1 g was soaked in the 250 mL APTES solution with concentration of 0.12 g/mL，at the temperature of 25 ℃，for 28 h with shaking of 100 r/min.Furthermore，the graft was then rinsed for 3 times，each for 5 min，with distilled water under ultrasonic condition，at 37 ℃.And then it was dried for use.

1.3 Chemical grafting with bioactive molecules

Heparin and RSF have been extensively used in biomaterials for many years for improved antithrombotic properties and cell adhesion，respectively［12-13］.Therefore，in the present study，both of them were introduced onto the aminated surface of the SF respectively.As to heparin，not only the antithrombotic property of the graft should be improved，but the cytocompatibility of the graft should not be tampered.However，both molecules could hardly crosslink with the aminated surface directly，so activation of both with 1-ethyl-3-(3-dimethylaminopropyl)carbodiie hydrochlide (EDC·HCl)/N-hydroxysuccinimide (NHS)was conducted firstly (Figs.2 and 3).The activated heparin solution was obtained as follows:(1)prepare the reactive solution according to the weight ratio:sodium heparin∶EDC·HCl∶NHS =1∶1∶2;(2)react for 15 min at 25 ℃，then obtain the activated heparin solution.The activated RSF solution was obtained as follows:(1)prepare the reactive solution according to the weight ratio: RSF∶EDC·HCl∶NHS =1∶1∶2;(2)react for 15 min at 25℃，then obtain the activated RSF solution.

Fig.2 Activation of heparin

Fig.3 Activation of RSF

Secondly， both activated bioactive molecules were respectively crosslinked to the aminated surface of the SF(Figs.4 and 5).The temperature，duration，and concentration of heparin solution were 25 ℃，1 h，and 2.5 g/L，respectively.Moreover，the temperature，duration，and concentration of RSF solution were 37 ℃，4 h，and 6% by weight，respectively.

1.4 MTT assay for cell viability on the modified graft

Porcine iliac endothelial cells (PIECs)(No.GN 015，Cell Bank of the Chinese Academy of Sciences，Shanghai)were seeded on one piece of graft disc with 14 mm in diameter in a well of 24-well tissue culture plates (TCPs).The concentration of the seeded cells was 1.4 ×104cells per well.The time points were chosen at 5 d.The aminated graft and blank wells were used as controls.Every sample was cultured in triplicate (n=3).Cell viability was checked using MTT assay，in which the graft and cells were incubated with 5 g/L MTT and 100 μL medium for 4 h.The medium was then extracted and 100 μL dimethyl sulfoxide (DMSO)was added for dissolving the crystals，at 37 ℃for 15 min with shaking.Then 100 μL of the solution were pipetted into a well of 96-well TCP and the absorbance was measured at 492 nm using a spectrophotometer(MK3，Thermo，USA).

1.5 Statistical analysis

Data were expressed as mean ± standard deviation (SD).Statistical differences were determined by one-way analysis of variance (ANOVA) and the difference was considered significant when p was less than 0.05 (p ＜0.05).

2 Results and Discussion

2.1 Gross examination of the SF

The SF we obtained is ivory，soft，and flexible.A dyeing procedure described previously［14］and microscopy were used to check the degumming results (Figs.6 and 7)，respectively.The results showed that the graft became light yellow from ivory after dyeing，indicating that the removal of sericin was complete(Fig.6).Moreover，the microphotographs showed the yarns were changed from bundles of fibers to separated ones because of the removal of sericin (Fig.7).

Fig.6 Images of (a)the degummed graft and (b)the dyeing result (scale bar=100 μm)

Fig.7 Microphotographs of the yarns (a)bundles of fibers and (b)separated fibers

2.2 Surface amination of the SF

The results of ATR-FTIR showed that the graft was successfully aminated by amination reaction with APTES (Fig.8).In Fig.8，the —O—H stretching vibration peak at 3 282 cm-1of the hydroxyl group (—OH)on the silk fibroin fiber surface disappeared after treatment，and the —N—H stretching vibration peak of the amino group (—NH2)and the —C—H absorption peak in the —Si(OCH2CH3)3of APTES appeared at 3 346 cm-1and 2 930 cm-1，respectively.Furthermore，two —Si—O— stretching vibration peaks appeared at 1 139 cm-1and 1 036 cm-1，respectively.One was due to the—Si—O— of APTES，the other may be caused by the reaction between —OH on the fiber surface and —Si(OCH2CH3)3of APTES.These findings demonstrated that amino groups(—NH2)in APTES were successfully introduced onto the surface of the silk fibroin fiber after amination reaction with APTES.

Fig.8 ATR-FTIR of the graft aminated by APTES

Moreover，SEM was carried out to observe the surface changes of the grafts before and after amination (Fig.9).A glue-like coating could be found on the surface in Fig.9(b)，and the surface seems a bit smoother than that before amination.

Fig.9 SEM microphotographs of the fibroin fibers of the grafts (a)before and (b)after amination

Comparison of mechanical properties of the grafts before and after amination has been carried out by testing probe bursting strength，so as to determine the influence of the amination on the mechanical properties of the grafts.The results showed that no significant loss of mechanical properties could be found after amination (n≥3，p ＞0.05)(Fig.10).

Fig.10 Comparison of the probe bursting strengths of SF and SF-APTES

2.3 Chemical grafting with bioactive molecules

Both bioactive molecules including heparin and RSF have been linked to the surfaces of the aminated grafts，respectively(Fig.11).The results of ATR-FTIR showed that the —O—H stretching vibration peak at 3 281 cm-1of the hydroxyl group(—OH) on the silk fibroin fiber surface appeared after treatment with activated heparin，and became wider than before.In addition，—S—O stretching vibration peak at 1 225 cm-1appeared.This suggested that heparin molecules had already been linked onto the surface of the aminated graft.As to the graft modified with activated RSF，ATR-FTIR spectrum showed that the—O—H stretching vibration peak at 3 277 cm-1of the hydroxyl group (—OH)appeared as well with a slight shift，suggesting that RSF had been successfully linked onto the surface of the aminated graft.

Fig.11 ATR-FTIR of the graft crosslinked with heparin and RSF

Figure 12 showed SEM microphotographs of the grafts treated with heparin and RSF，respectively.There were no obvious changes on the surface of the graft treated with heparin.However，granules could be found on the surface of the graft treated with RSF.The granules might be formed by assembling the polypeptides of RSF.

Fig.12 SEM microphotographs of the grafts treated with(a)heparin and (b)RSF，respectively

Likewise，probe bursting strength has been employed to evaluate the mechanical properties of the graft after treatment with bioactive molecules.The results showed that there were no significant differences between the grafts before and after treatment (n=3，p ＞0.05)with heparin and RSF (Fig.13)，indicating that the treatment did not cause any significant loss of its mechanical properties.

Fig.13 Comparison of probe bursting strengths of SFAPTES，SF-HEP，and SF-RSF

2.4 MTT assay

The results of MTT assay were shown in Fig.14.The viability of cells on both grafts treated with heparin and RSF had been higher than those of cells on the aminated ones (p ＜0.05).However，there was no significant difference of viability between the grafts treated with heparin and RSF (p ＞0.05).This suggested that surface modification of silk fibroin fiber graft with either heparin or RSF could improve the cytocompatibility.More importantly，the heparin plays a role in improving not only the cytocompatibility， but also antithrombotic performance［12］.Therefore， it provides a possibility to develop a vascular graft with favorable properties regarding both cyto-and hemo-compatibility.

Fig.14 MTT assay (TCP，n=3，p ＜0.05)

3 Conclusions

In the present work，SF was woven in plain construction，degummed with hot 0.5% (by weight)Na2CO3aqueous solution，aminated with APTES and chemically grafted with bioactive molecules such as heparin and RSF.Furthermore，the grafts were characterized by ATR-FTIR，microscopy，cell culture，and MTT assay.Moreover，probe bursting strength was utilized to evaluate the mechanical properties of the graft before and after treatment.The results revealed that amino groups could be successfully introduced onto the surface of the SF through amination with APTES.The viability of cells on both grafts linked with heparin and RSF have been higher than those of the aminated ones (p ＜0.05).In addition，no significant loss of mechanical properties was found after amination and treatment with bioactive molecules.This suggested that it was feasible to develop a fibroin fiber vascular graft with favorable cyto- and hemo-compatibility through chemical grafting.The present study might provide insights into development of small diameter vascular graft with antithrombotic and endothelialization-promoting properties.

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