Preparation and characterization of biomimetic mesoporous bioactive

glass-silk fibroin composite scaffold for bone tissue engineering

Caihong Lei1,2, Xinxing Feng3, Yayang Xu1,2, Yuerong Li1,2, Hailin Zhu1,2, Jianyong Chen1,2*

1The Key Laboratory of Advanced Textile Materials and Manufacturing Technology, Xiasha Higher

Education Zone, Zhejiang Sci-Tech University, Hangzhou, 310018, China

2Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education，

Zhejiang Sci-Tech University，Hangzhou, 310018, China

3The quartermaster research institute of the general logistics department of the PLA, Beijing, China

*To whom correspondence should be addressed.

E-mail: cjy@zstu.edu.cn (Jianyong Chen)

Keywords: three-dimensional scaffolds; mesoporous bioactive glass; silk fibroin; structure; bioactivity

Abstract. Three-dimensional (3D) mesoporous bioactive glass (MBG) scaffolds were obtained by

using the demineralized bone matrix (DBM) and P123 as co-templates through a dip-coating method

followed by evaporation induced self-assembly (EISA) process. 3D mesoporous bioactive glass-silk

fibroin (MBG/SF) composite scaffolds were prepared by immersing MBG scaffolds into SF solutions

with different concentration. Transmission electron microscopy (TEM), field mission scanning

electron microscope (FESEM), fourier transform infrared spectroscopy (FT-IR) and wide angle X-ray

diffraction (WA-XRD) were used to analyze the inner pore structures, pore sizes, morphologies and

composition of the scaffolds. The in vitro bioactivity of the scaffolds was evaluated by soaking in

simulated body fluid (SBF). The results showed that the MBG and MBG/SF composite scaffolds with

the interconnected macroporous network and mesoporous walls could be obtained by this method. In

addition, both the MBG scaffolds and the MBG/SF composite scaffolds have excellent

apatite-forming bioactivity. Therefore, this method provides a simple way to prepare scaffolds for

bone tissue engineering.

Introduction

It is well known that bone is a composite material composed of inorganic component such as

nanocrystalline hydroxyapatite and organic phase such as collagen[1]

. Therefore, the development of

polymer-ceramic hybrids has been proposed to prepare biomimetic bone materials and minimize the

above drawbacks [2]

. Recently, a new generation of bioactive glass referring to as mesoporous

bioactive glass (MBG) has been developed, which has highly ordered mesopore channel structure

with a pore size ranging from 2 to 50nm and high specific surface area [3]

. Because of these

outstanding properties, this type of material possesses excellent in vitro and vivo biocompatibility and

apatite mineralization. Organic polymer silk fibroin (SF) derived from silkworm Bombyx mori has

been widely used in biomedical aspect thanks to its outstanding properties such as biocompatibility,

controllable biodegradability, minimal inflammatory reactions[4]

. However, pure SF scaffolds also

lack osteoconductivity. Therefore, combining MBG with SF can be an effective way to prepare

composite scaffolds for bone regeneration.

Demineralized bone matrix (DBM) is one of the materials which has been studied extensively in

bone tissue regeneration and it shows to be of porous structures[5]

. To the best of our knowledge, there

is rarely about using DBM as macroporous template to obtain 3D MBG scaffolds. Therefore, in this

work, a new procedure is described to obtain 3D MBG scaffolds with 58S37C5P (S, C, and P

represents SiO2, CaO and P2O5; 58, 37, and 5 is the molar ratio of SiO2, CaO and P2O5 in percentage,

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respectively) by using DBM and P123 as co-templates though a dip-coating method followed by an

evaporation induced self-assembly (EISA) process. After that, MBG/SF composite scaffolds could be

obtained by immersing MBG scaffolds into SF solutions with different concentration. The structures,

morphologies and in vitro bioactivity of the composite scaffolds were investigated.

Materials and methods

Preparation and characterization of MBG scaffolds. Demineralized bone matrix (DBM) was

prepared according to the method adapted from Chen et al[5]

. Porous mesoporous bioactive glass

(MBG) scaffolds were prepared by using DBM and P123 as co-templates. Firstly, the

MBG-58S37C5P precursor solution was prepared following the method previously reported[6]

. DBM

samples were completely immersed into the MBG-58S37C5P precursor solution for 30min. Then

they were transferred into a Petri dish and the excess solution was squeezed out. After evaporating the

solvent at room temperature for 12h, the same procedure was repeated for several times. Then the

samples were dried for several days in air at room temperature followed by vacuum drying at 40℃ for

48h. When completely dried, they were calcined at 700℃ (ramp of 1℃/min) for 5h to obtain the final

MBG scaffolds. The mesoporous-channel structure was characterized by Transmission Electron

Microscopy (TEM, JEM2010, JEOL, Japan).The porous morphology was observed by Field

Emission Scanning Electron Microscope (FE-SEM, Hitachi S-4800, Japan).

Preparation and characterization of MBG/SF composite scaffolds. The composite scaffolds were

fabricated by immersing the prepared MBG scaffolds into aqueous SF solutions. In this study, SF

solutions were prepared from cocoons of the silkworm B. mori with concentration of 3%, 6% and 8%

(w/v), respectively, in which the prepared MBG scaffolds were immersed for 15min. The prepared

composite scaffolds will be referred to as MBG/3SF, MBG/6SF and MBG/8SF, respectively. The

composite scaffolds were cross-linked in ethanol for 10min followed by vacuum drying at 40℃ for 1

day to remove any residual ethanol. The pore structures and surface morphologies of pore wall of the

composite scaffolds were characterized by FE-SEM. The chemical composition was characterized by

Fourier Transform Infrared Spectra (FT-IR, Nicolet 5700, Thermo Electron, America).

Scaffolds soaking in simulated body fluid. The assessment of the in vitro biomineralization of the

scaffolds was carried out in the simulated body fluid (SBF) at 37℃ in static condition for different

time with refreshing SBF solution each day. SBF was prepared according to the procedures described

by Kokubo[7]

and the scaffolds were held in sealed plastic flasks in order to minimize the change in

pH and contamination. After soaking, the scaffolds were removed from SBF, carefully rinsed with

ethanol for several times and dried in air at room temperature for further characterization. The

formation of hydroxyapatite on the surface of the scaffolds was studied as a function of time. The

surface morphology and phase composition of the scaffolds after soaking in SBF solution were

characterized FE-SEM, FT-IR and WA-XRD.

Results and discussion

Morphologies and microstructures of MBG scaffolds and MBG/SF composite scaffolds. Fig.1

showed the typical TEM image of MBG scaffolds. Highly ordered hexagonal mesoporous channels

observed in TEM indicated that inorganic scaffolds with ordered pore arrangements could also be

obtained through this method. Fig.2 showed the representative FE-SEM images of MBG and

MBG/SF composite scaffolds. It could be seen that MBG scaffolds exhibited a high porous structure

with pore sizes ranging from 200 to 500 μm (Fig.2 A1). The higher magnification FE-SEM images

showed that the surface of MBG scaffolds was smooth and had numerous collapsed cracks due to the

inherent brittle nature of this kind of material (Fig.2 A3). And after SF solutions were coated, the

macroporous structure was still maintained for all MBG/SF composite scaffolds and the pore surface

was also smooth (Fig.2 B3, C3 and D3). However, collapsed cracks reduced strikingly due to the effect

of SF solutions and there was no obvious difference in pore sizes.

10 Silk, Protective Clothing and Eco-Textiles

Fig. 1 TEM image of the prepared MBG scaffolds with highly ordered mesopore-channel structure

It is believed that the macropore diameter and the macropore interconnections of three-dimensional

scaffolds should be larger than 50-100 µm[8]

. These interconnected macrospores are beneficial to

tissue ingrowth, vascularization in vivo and the damaged tissues can be effectively regenerated.

Therefore, the prepared MBG scaffolds and MBG/SF composite scaffolds have similar macroporous

structures for bone tissue engineering.

A1 A3 A2

B1

C1

B2 B3

C2 C3

Advanced Materials Research Vol. 796 11

Fig. 2 FESEM images of the pore surface morphologies and the microstructures of (A) MBG scaffold,

(B) MBG/3SF composite scaffold, (C) MBG/ 6SF composite scaffold and (D) MBG/8SF composite

scaffold. (A1, B1, C1, D150; A2, B2, C2, D2100; A3, B3, C3, D3800)

Structural analysis of MBG scaffolds and MBG/SF composite scaffolds. Structural changes in the

MBG scaffolds before and after coating silk fibroin were determined by FT-IR (Fig.3). The spectra of

MBG scaffolds showed characteristic absorption bands of Si-O stretching vibrations at 1086cm-1

,

802cm-1

and Si-O-Si bending vibrations at 467cm-1

. The pure silk fibroin had absorption bands at

1650cm-1

(AmideⅠ), 1541cm-1

(AmideⅡ) and 1235cm-1

(Amide Ⅲ). MBG/SF composite scaffolds

all exhibited obvious silk-characteristic peaks and mesoporous bioactive glass-characteristic peaks.

However, no specific bands were obtained which could be duo to no chemical reaction occurring

between MBG scaffolds and silk fibroin that is incorporated. Therefore, FT-IR further confirmed that

MBG scaffolds had been coated by silk fibroin.

3500 3000 2500 2000 1500 1000 500

SF/8MBG

SF/6MBG

Ab

sort

ance

/a.u

.

Wavenumber (cm-1)

467cm-1

1086cm-1

1650cm-1 1541cm

-1

SF

802cm-1

SF/3MBG

MBG

1235cm-1

Fig. 3 FT-IR of the SF, the MBG scaffold and the MBG/SF composite scaffolds.

In vitro bioactivity of scaffolds in SBF. A significant characteristic of the scaffold materials is their

ability to bond with living bone tissue though a HCA layer formed on their surface both in vitro and

vivo. Surface morphologies of the four types of scaffolds after soaking in SBF for 3 days were

characterized by FE-SEM as shown in Fig.4. The apatite particles with ball-like shape fully covered

the pore wall surfaces for all kinds of scaffolds after soaking in SBF for 3 days. However, the

thickness of the HCA layer and the spherical particles with needlelike crystallites decreased with the

increase of SF solutions concentration. Biomineralization process included a series of reactions

proposed by Hench and co-workers[9]

. Calcium ions were firstly released from the glass network to

form negative Si-O-, which could attract the Ca2+

ions from the SBF. Then Ca2+

ions further caught

the PO43-

from the solution to form the apatite nucleus. In our research, SF layers may affect this

process, so the quantity and sizes of spherical particles showed a few differences.

D1 D2 D3

12 Silk, Protective Clothing and Eco-Textiles

Fig. 4 FESEM images of (A) MBG scaffold, (B) MBG/3SF composite scaffold, (C) MBG/ 6SF

composite scaffold and (D) MBG/8SF composite scaffold after soaking in SBF for 3 days. (A1, B1, C1,

D1800; A2, B2, C2, D2 6000)

Besides, FT-IR spectroscopy measurements and WA-XRD patterns were also used to characterize

the formation of apatite on the surface of scaffolds. For the samples of MBG, MBG/3SF, MBG/6SF

and MBG/8SF scaffolds, after soaking in SBF for 3 days, the bands of the phosphate group at 603 and

560cm-1

can be assigned to the crystalline HCA (Fig.5). Therefore, the crystalline HCA layer can be

formed on the surface of the scaffolds with different SF concentration after soaking in SBF for 3 days.

WA-XRD patterns of the scaffolds after soaking in SBF were shown in Fig.6. One obvious diffraction

peak at 2θ=31.7° emerged, corresponding to the (211) reflections of hydroxide apatite. The other

apatite diffraction peaks indexed to (002), (300), (222) and (322), et al, also became evident. We also

can observe that the intensity of diffraction peaks of crystalline decrease with increasing SF

concentration. This also supports the results of the FT-IR spectra and FE-SEM observations.

3500 3000 2500 2000 1500 1000 500

Ab

sorb

ance

/a.u

.

Wavenumber (cm-1

)

560cm-1

603cm-1

MBG/3SF

MBG/6SF

MBG

MBG/8SF

10 20 30 40 50 60 70 80

2 Theta ( degree )

▼21

1

▼00

2

▼22

2

▼21

2-23

0▼

300

▼20

2

MBG

MBG/8SF

MBG/6SF

MBG/3SF

▼32

2

▼ HA

Inte

nsi

ty/a

.u.

Fig. 5 FT-IR spectra of MBG scaffold and MBG/SF Fig.6 WA-XRD patterns of NBG scaffold

composite scaffolds after soaking and NBG/SF composite scaffolds

in SBF for 3 days after soaking in SBF for 3 days

Conclusion

In this study, hierarchically 3D porous MBG scaffolds were successfully prepared by a

combination of DBM and P123 surfactant as co-templates and evaporation-induced self-assembly

process. And MBG/SF composite scaffolds were also successfully developed for bone tissue

engineering through a SF solution dip-coating method. After dip-coating process, a thin silk film was

formed on the surface of the pore walls. And these scaffolds all have the similar interconnected

macroporous network and the mesoporous wall. The in vitro bioactivity among these scaffolds

showed a few differences because of SF film affecting biomineralization process.

A1 A2 B1 B2

C1 D1 C2 D2

Advanced Materials Research Vol. 796 13

Acknowledgments

We gratefully thank the National Natural Science Foundation of China (grant nos. 51273181),

Commonweal Project of Science and Technology Department of Zhejiang Province (grant

No.2012C21027)and the Science Foundation of Zhejiang Sci-Tech University (grant nos.

1113808-Y).

References

[1] D.L. Batchelar, M.T.M. Davidson, W. Dabrowski, et al. Bone-composition imaging using

coherent-scatter computed tomography: Assessing bone health beyond bone mineral density [J].

Medical physics, 2006, 33: 904-910.

[2] W. Thein-Han, R. Misra. Biomimetic chitosan-nanohydroxyapatite composite scaffolds for bone

tissue engineering [J]. Acta Biomaterialia, 2009, 5(4): 1182-1197.

[3] X. Yan, C. Yu, X. Zhou, et al. Highly Ordered Mesoporous Bioactive Glasses with Superior In

Vitro Bone-Forming Bioactivities [J]. Angewandte Chemie International Edition, 2004, 43(44):

5980-5984.

[4] S. Sofia, M.B. McCarthy, G. Gronowicz, et al. Functionalized silk-based biomaterials for bone

formation [J]. Journal of biomedical materials research, 2001, 54(1): 139-148.

[5] B. Chen, H. Lin, Y. Zhao, et al. Activation of demineralized bone matrix by genetically engineered

human bone morphogenetic protein-2 with a collagen binding domain derived from von Willebrand

factor propolypeptide [J]. Journal of Biomedical Materials Research Part A, 2007, 80(2): 428-434.

[6] A. López-Noriega, D. Arcos, I. Izquierdo-Barba, et al. Ordered mesoporous bioactive glasses for

bone tissue regeneration [J]. Chemistry of materials, 2006, 18(13): 3137-3144.

[7] T. Kokubo, H. Kushitani, S. Sakka, et al. Solutions able to reproduce in vivo surface-structure

changes in bioactive glass-ceramic A-W3 [J]. Journal of biomedical materials research, 1990, 24(6):

721-734.

[8] M. C. von Doernberg, B. von Rechenberg, M. Bohner, et al. In vivo behavior of calcium

phosphate scaffolds with four different pore sizes [J]. Biomaterials, 2006, 27(30): 5186-5198.

[9] L.L. Hench. Bioceramics: from concept to clinic [J]. Journal of the American Ceramic Society,

1991, 74(7): 1487-1510.

14 Silk, Protective Clothing and Eco-Textiles

Silk, Protective Clothing and Eco-Textiles 10.4028/www.scientific.net/AMR.796 Preparation and Characterization of Biomimetic Mesoporous Bioactive Glass-Silk Fibroin Composite

Scaffold for Bone Tissue Engineering 10.4028/www.scientific.net/AMR.796.9

DOI References

[2] W. Thein-Han, R. Misra. Biomimetic chitosan-nanohydroxyapatite composite scaffolds for bone tissue

engineering [J]. Acta Biomaterialia, 2009, 5(4): 1182-1197.

http://dx.doi.org/10.1016/j.actbio.2008.11.025 [3] X. Yan, C. Yu, X. Zhou, et al. Highly Ordered Mesoporous Bioactive Glasses with Superior In Vitro

Bone-Forming Bioactivities [J]. Angewandte Chemie International Edition, 2004, 43(44): 5980-5984.

http://dx.doi.org/10.1002/anie.200460598 [4] S. Sofia, M.B. McCarthy, G. Gronowicz, et al. Functionalized silk-based biomaterials for bone formation

[J]. Journal of biomedical materials research, 2001, 54(1): 139-148.

http://dx.doi.org/10.1002/1097-4636(200101)54:1<139::AID-JBM17>3.0.CO;2-7 [5] B. Chen, H. Lin, Y. Zhao, et al. Activation of demineralized bone matrix by genetically engineered human

bone morphogenetic protein-2 with a collagen binding domain derived from von Willebrand factor

propolypeptide [J]. Journal of Biomedical Materials Research Part A, 2007, 80(2): 428-434.

http://dx.doi.org/10.1002/jbm.a.30900 [6] A. López-Noriega, D. Arcos, I. Izquierdo-Barba, et al. Ordered mesoporous bioactive glasses for bone

tissue regeneration [J]. Chemistry of materials, 2006, 18(13): 3137-3144.

http://dx.doi.org/10.1021/cm060488o [7] T. Kokubo, H. Kushitani, S. Sakka, et al. Solutions able to reproduce in vivo surface-structure changes in

bioactive glass-ceramic A-W3 [J]. Journal of biomedical materials research, 1990, 24(6): 721-734.

http://dx.doi.org/10.1002/jbm.820240607 [8] M. C. von Doernberg, B. von Rechenberg, M. Bohner, et al. In vivo behavior of calcium phosphate

scaffolds with four different pore sizes [J]. Biomaterials, 2006, 27(30): 5186-5198.

http://dx.doi.org/10.1016/j.biomaterials.2006.05.051 [9] L.L. Hench. Bioceramics: from concept to clinic [J]. Journal of the American Ceramic Society, 1991,

74(7): 1487-1510.

http://dx.doi.org/10.1111/j.1151-2916.1991.tb07132.x