Bioactivity and Biocompatibility Studies on Silk-Based ... - UM Repository

Journal of Medical and Biological Engineering, 33(2): 207-214

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Bioactivity and Biocompatibility Studies on Silk-Based Scaffold for Bone Tissue Engineering Sahba Mobini1,2 Noor Azuan Abu Osman3

Mehran Solati-Hashjin2,3,*

Habibollah Peirovi4

Mazaher Gholipourmalekabadi5

Mahmoud Barati6

Ali Samadikuchaksaraei7 1

Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran 19615, Iran Nanobiomaterials Laboratory, Biomaterials Center of Excellence, Amirkabir University of Technology, Tehran 15914, Iran 3 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, Kuala Lumpur 50603, Malaysia 4 Nanomedicine and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran 4739, Iran 5 Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran 4739, Iran 6 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran 4739, Iran 7 Department of Biotechnology and Cellular and Molecular Research Center, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran 141761, Iran 2

Received 13 Nov 2011; Accepted 27 Jul 2012; doi: 10.5405/jmbe.1065

Abstract Novel materials with promising properties can be used to achieve scaffold-based tissue engineering goals. Natural silk (NS) polymer has remarkable biomedical and mechanical properties as a material for bone tissue engineering scaffolds. This study describes the fabrication of a silk-based composite, in which natural silk and regenerated silk (RS) are combined to achieve better mechanical properties in the three-dimensional (3D) porous form. The biocompatibility and bioactivity of these scaffolds are evaluated. RS was made using mulberry-silk cocoons. RS/NS composite scaffolds were fabricated using the freeze-drying technique. Silk protein extract was evaluated by Fourier transform infrared spectroscopy (FTIR), with sharp amide peaks appearing at 1655 cm-1 and 1530 cm-1 in the FTIR spectrum, confirming the existence of fibroin. The fabricated 3D scaffolds were morphologically analyzed by scanning electron microscopy (SEM). An inter-connective spongy structure was found. Mechanical characterizations were carried out using a universal testing machine. Results show that the mechanical properties of the RS/NS composites are better than those of scaffolds fabricated with RS alone. In addition, in vitro tests, including those for cell viability and adhesion, were carried out with osteoblast cells by the MTT assay with a new calculation approach, which confirmed biocompatibility. The bioactivity potential of the RS and composites fibers was tested by introducing scaffolds to normal simulated body fluid for 21 days. Energy-dispersive X-ray spectroscopy and SEM analyses proved the existence of CaP crystals for both configurations. Thus, reinforced silk composite is a bioactive and biocompatible alternative for bone tissue engineering applications. Keywords: Bone tissue engineering, Biocompatible materials, Scaffold, Silk

1. Introduction Bone is a complex and highly specialized connective tissue [1,2]. As the provision of structural support for the body is bone’s main role, any structural bone defect that leads to functional deficits can dramatically affect an individual’s wellbeing and quality of life [3]. Tissue engineering can provide an alternative to traditional treatment protocols by replacing living * Corresponding author: Mehran Solati-Hashjin Tel: +98-21-64542360; Fax: +98-21-66468186 E-mail: [email protected]

tissue with tissue grown in vitro to meet each patient’s individual needs [4,5]. Various materials and scaffold fabrication techniques have been investigated over the past two decades for bone tissue engineering with the aim to increase mechanical stability and improve scaffold-tissue interactions [6]. Requirements for mechanical properties, biocompatibility, and bioactivity must be met to ensure scaffold utility, particularly for bone tissue regeneration [7,8]. Silks are natural fibers produced by various insects, of which mulberry silkworms (Bombyx mori (B. mori)) are of high economic importance, as they can be reared in captivity [9,10]. Recently, it has been shown that silks can be employed for a variety of

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biomedical applications, including support of osteoblast cell growth and differentiation for bone tissue engineering purposes [11-13]. The present study combines natural silk (NS) and regenerated silk (RS) to achieve better mechanical properties in the three-dimensional (3D) porous form. The biomimetic capability of fibroin in both configurations is investigated. In addition, the biocompatibility of RS and RS/NS is investigated by the MTT method using a calculation method that overcomes the inaccuracy caused by the stain absorption of 3D scaffolds.

2. Materials and methods 2.1 Materials Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) were obtained from Gibco. (United Kingdom) Penicillin-streptomycin (pen-strep), Trypsin/EDTA, and MTT substrate (3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrasodium bromide) were obtained from Sigma Chemical Co. (Germany) Cell culture plastics were purchased from Nunch (Denmark). The osteoblast cell line G 292 was purchased from National Cell Bank of Iran, Pasteur Institute. LiBr, Na2CO3, methanol, and all other salts for the simulated body fluid (SBF) were supplied by Merck (Germany). The cellulose dialysis tube (cut-off value of 12000 Da) was purchased from Sigma (Germany) and B. mori cocoons were generously provided by the Iranian Silkworm Research Center. 2.2 Scaffold preparation Fibroin extraction was performed using the protocol reported by Kaplan et al. [14]. Briefly, B. mori cocoons were boiled for 1 h in 0.02 M aqueous sodium carbonate solution and then rinsed thoroughly with cold and hot water to extract sericin proteins. The degummed silk was dissolved in 9 M LiBr at 55 °C for 4 h. Then, the fibroin solution was filtered and dialyzed against distilled water for 3 days to yield the fibroin water solution. The solution was allowed to lyophilize for 3 days to get dry storable fibroin. Fibroin solutions with 4% w/v and 8% w/v concentrations were prepared by resolving the above dried fibroin with stirring. These solutions were put either into Teflon molds or 24-well polystyrene plates and then frozen at -20 °C for 4 h and then at -80 °C for 1 h. The ice/silk composites were then lyophilized for 12 h, leaving a porous matrix. After drying, the porous matrices were immersed in methanol (99.9%) for about 1 h to induce crystallization, transforming into a β-sheet structure and becoming insoluble in water. Insoluble RS 3D scaffolds were then prepared by removing methanol and allowing further lyophilization. Continuous NS fibers were used as reinforcements for RS fibroin to fabricate composite 3D scaffolds. In order to determine the optimal amount of silk fibers, fiber concentrations of 30%, 50%, and 90% v/v were tested. NS and RS 4% w/v solution were mixed in proper molds and then frozen and lyophilized, as done with the RS samples. Dried scaffolds were treated with methanol to make them insoluble via a transformation into the β-sheet structure. Sample codes and compositions are shown in Table 1.

Table 1. Codes and compositions of samples. Sample code

F4

F8

4 wt% 8 wt% Composition Fibroin Fibroin

C1F4 90-10 v% RS/NS Composite

C2F4 C3F4 70-30 v% 50-50 v% RS/NS RS/NS Composite Composite

2.3 Characterization of 3D scaffolds Fibroin protein was analyzed by Fourier transform infrared spectroscopy (FTIR, EQUINOX 55FTIR spectrophotometer). Freeze-dried fibroin was mixed with KBr and pressed according to the ASTM E1252-07 standard. The pellets were analyzed in the range of 500 to 4000 cm-1 at a resolution of 4 cm-1 to determine the functional groups. The surface and cross-section morphologies and pore distributions, size and interconnectivity of scaffolds, morphology of fibers, and cell adhesion on scaffolds were observed using scanning electron microscopy (SEM, XL30 ESEM, Philips) with a field-emission gun. The specimens were sputter-coated with gold using a voltage of 15 or 20 keV. Additionally, the chemical composition of the crystals in the biomimetic experiments was semi-quantitatively measured via SEM equiped with an energy-dispersive X-ray spectroscopy (EDX) device (Rontec, Germany). Compression properties of 3D cylinder-shaped scaffolds were measured using a universal testing machine (HCT 25/400, servo hydraulic PID controller, Zwick/Roell, Germany). The setup employed in this study had a 0.01-N force and a 0.001-m axial resolution. Cylindrical samples, 7 × 28.26 mm2, were examined at a crosshead speed of 1 mm/min according to a modified method based on ASTM method F451-95 [15-17]. The compressive strength was determined from the stress-strain curve. Calculations were performed using RBCA Toolkit 1998 software. Hexane, a non-solvent agent for silk, was used as the displacement liquid. It permeates easily through the interconnected scaffold pores and causes no swelling or shrinkage. The silk scaffold was then immersed in a known volume (V1) of hexane in a graduated cylinder for 5 min. The total volume of hexane-impregnated scaffold and hexane was recorded (V2). The residual hexane volume in the cylinder after the removal of hexane-saturated scaffold was also recorded (V3). The porosity of the scaffold (ε) was calculated as [17]: ε (%) = [(V1-V3)/(V2-V3)] × 100

(1)

2.4 Cell culture experiments 2.4.1 Osteoblast seeding on scaffolds The human osteoblast cell line G 292 was expanded in DMEM (low glucose), containing 10 U/ml penicillin and 100 µg/ml streptomycin, and 10% FBS (normal medium) at 37 °C in a humidified 5% CO2/95% air incubator. Cultivation volume and duration depend on the number of cells. 5 × 105 cells were used for each sample. The disc-shaped scaffolds were rinsed with alcohol (70% v/v) in a biological laminar hood and washed several times with sterile phosphate-buffered saline (PBS) before cell seeding. Then, the scaffolds were pre-incubated in a normal culture medium for 2 h before

Silk scaffolds for bone tissue engineering

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seeding. The scaffolds were seeded with high-concentration cell suspension and incubated for 2 h. The volume of the medium for each sample of scaffolds was then increased to 1 ml to cover the scaffold completely. The polystyrene surface of the cell culture plates was used as the control.

mentioned above (as recommended by Bohner et al. [19]). SBF was changed every 3 days to introduce fresh ions to the scaffolds. Samples were dried in the air at room temperature and investigated by SEM and EDX to determine the probable crystal formation and chemical composition, respectively.

2.4.2 Cell response analysis Cell viability and biocompatibility assessments were carried out using the MTT assay after 48 h of osteoblasts culture. Due to their absorption of the dye, the 3D scaffolds affect the degree of color change in the MTT assay. Control scaffolds were thus placed into the polystyrene control wells for normalization. The scaffolds were soaked in the culture medium for 30 min before the tests were run. The growth medium of each scaffold sample as well as that of the control was replaced with 0.02 ml of MTT solution (5 mg/ml). Then, 0.1 ml serum-free medium was added to each well to cover the entire surface. The wells containing MTT solution were incubated for 4 h in a humidified atmosphere of 5% CO2 in air. The MTT was removed and 0.1 ml of dimethyl sulphoxide (DMSO) was added into each well in order to dissolve the formazan crystals. The stain from one sample of each type was transferred to its own control (scaffolds soaked in the culture medium). The plate was agitated for 20 min in the dark, and the viable cells in the colored solution were quantified using a scanning multi-well spectrophotometer (Anthos 2020, Austria) at 540 nm.

Table 2. Molarity of reagents used for preparing SBF [24].

2.4.3 Cell morphology One cell-seeded sample of each type after 4 days of the experiment was fixed by 2.5% glutaraldehide in PBS for 2 h and washed thoroughly with PBS. The samples were subsequently submerged in osmium tetroxide (0.1%) in 0.1 M PBS for 30 min and then washed thoroughly with PBS. Dehydration was accomplished by using a gradation series of acetone/distilled water solutions. Freeze drying was then performed at -40 to -75 °C for 12 h. Finally, samples were prepared for SEM imaging by being coated with a gold spattering machine. 2.4.4 Statistical analysis All experiments were performed with six replicates. The results are given as means ± standard deviation (SD). Statistical analysis was performed using the t-test; p < 0.05 was considered significant.

Reagent NaCl NaHCO3 Na2HCO3 KCL K2HPO4·3H2O MgCl2·6H2O 0.2 M NaOH HEPES CaCl2 Na2SO4

Amount 5.403 g 0.504 g 0.426 g 0.225 g 0.230 g 0.311 g 100 ml 17.892 g 0.293 g 0.072 g

3. Result and discussion 3.1 FTIR analysis of extracted fibroin There are three types of distinguishable vibration peak related to the amide groups in proteins. Amide has characteristic vibration bands between 1630cm-1 and 1650cm-1 for amide I (C = O stretching), between 1540cm-1 and 1520cm-1 for amide II (secondary NH bending), and between 1270cm-1 and 1230cm-1 for amide III (C–N and N–H functionalities) in its FTIR spectrum [20]. Miyazawa and Blout [21] studied the infrared spectra of polypeptides in various configurations. They demonstrated that except for some special proteins, polypeptides show amide I bands in α-helix form at 1655cm-1 and amide II bands in β-sheet form at 1540cm-1; in the extended configuration, they show these bands at 1630 cm-1 and 1520 cm-1, respectively. The conformation of extracted fibroin was determined from FTIR spectra, which revealed pure fibroin with a silk I structure [19,22-26]. Figure 1 shows the spectrum of the extracted fibroin, which is divided into four zones. Peaks at 1655, 1530, and 1239 cm-1 represent amide I, amide II, and amide III, respectively. The peak at 699 cm-1 confirms amide II.

2.5 Biomimetic experiment A bio-mineralization study was performed by placing the samples in SBF for 21 days. SBF was made using the protocol reported by Oyane et al. [18]. Briefly, HEPES, NaOH, and salts (weighs are given in Table 2) were mixed and the total volume was adjusted to 1000 ml by adding ultra-pure water. Pen-strep (in the proportion used for cell culture experiments) was added in a concentration of 1% to prevent contamination during the test. Finally, the pH was adjusted to 7.40 at 36.5 °C by adding an adequate amount of HCl. Two disc-shaped scaffold samples of each type as well as natural fibers were placed in the filtered SBF and incubated at 37 °C in 5% CO2 for the durations

Figure 1. FTIR spectrum of extracted fibroin.

3.2 Morphology of scaffolds The freeze-drying of fibroin solution with or without fiber, filled in appropriate molds, leads to porous 3D scaffolds, which

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can be produced in any size or shape. Samples were made with disc-shaped and cylindrical forms for mechanical and cell culture analyses, respectively, as shown in Fig. 2(a). SEM images of RS and NS/RS scaffolds with various concentrations are shown in Figs. 2(b)-(f). A higher concentration of fibroin solution led to a smaller pore size in the RS scaffold structure. The composite samples (RS/NS) had a completely different structure. Fibers dispensed homogenously and supported a porous structure in addition to the freeze-dried spongy structure. The porous structure of this blend, shown in Fig. 2(d), has fewer fibers, which gradually becomes more compact via accumulation, as shown in Figs. 2(e)-(f).

(a)

(b)

(c)

(d)

83%. In fact, there is an inverse relationship between the fibroin concentration and percentage of porosity. However, no prediction could be drawn from the introduction of fibers to the composite system. As it is summarized in Table 3, C3F4 scaffold has the most porous structure in a same time has the highest compressive module and strength. This can be due to grouping effect.

(a)

(b) (e)

(f)

Figure 3. (a) Compressive strength and (b) E module of scaffolds obtained from compressive tests.

Figure 2. (a) Porous scaffolds with various sizes and shapes. SEM images of scaffold samples (b) F8, (c) F4, (d) C1F4, (e) C2F4, and (f) C3F4.

3.3 Mechanical properties of scaffolds The mechanical properties obtained from static compressive tests of both RS and RS/NS composite samples are shown in Fig. 3. For RS samples, both mechanical strength and compressive modulus decreased with decreasing fibroin concentration. This trend is reasonable due to the ice/fibroin composite being enriched in the freeze-drying method by ice at the lower concentration of fibroin (4% w/v). Therefore, less fibroin and much larger pores, and thus a weaker structure, were obtained in F4 samples in comparison with F8 samples. In composite samples, the E module and compressive strength gradually increased with the accumulation of fiber. This indicates that the fibers reinforce the spongy composite. However, excess fiber in the system could limit the pore size and structure, as shown in Fig. 2(f). Fiber reinforcement makes a significant increase in compressive mechanical properties, although there is a slight drop in C1F4 samples. Figure 4 shows the results from the liquid displacement test for measurement of porosity. All of the scaffolds show porosity between 83 to 89% in which, C3F4 has the highest porosity with 89% and F8 has the less porosity with

Figure 4. Porosity of scaffolds (n = 3; mean ± SD). Table 3. Summary of porosity and mechanical properties of 3D scaffolds. Sample F4 F8 C1F4 C2F4 C3F4

Young's modulus (MPa) 1.114 1.87 0.795 2.75 3.42

Compressive strength Porosity (MPa) 1.027 87.49% 1.38 83.46% 0.75 88% 1.85 87% 1.87 89%

3.4 Cell seeding and cell response Osteoblast cells’ response to RS scaffolds and RS/NS composite scaffolds were investigated by MTT assay. As it has


also been noticed by other researchers [27] conventional MTT method does not yield consistent results when it is used for measurement of cellular proliferation on scaffolds, which tend to absorb Formozan stain. Therefore, we have taken into the account the stain abortion of the scaffolds for interpretation of MTT results. Figure 5 demonstrates the MTT result which reveals C1F4 has the least variance from its control. Furthermore, we have observed the highest stain absorption in C1F4. F4, as a simple fibroin scaffold shows less biocompatibility in comparison with the scaffolds containing fibers. It has been noted that addition of more fibers in the composite (C2F4) decreases the number of the cells on the scaffold. This could be explained by the fact that fibers decrease the porosity of the scaffold and thence, decrease the total surface area available for cell attachment and growth.

Figure 5. Viability of cells as measured using MTT assay (n = 3; mean ± SD). Figure 6 shows SEM image of seeded scaffolds. Osteoblasts covered the surface of both plane and composite scaffolds. The stretched and typical morphology of cells supports the results from MTT assay.

(a)

(b)

Figure 6. SEM images of osteoblast cells covering (a) composite RS/NS scaffold and (b) RS scaffold after 4 days of cell seeding.

3.5 Boimimetics Mineralization of RS scaffold, RS/NS composite scaffold as well as degummed fibers has been explored by SEM. To test which component plays the main role in mineralization, pure fibers also were immersed in SBF for 21 days. Figure 7 shows the crystal formation on fibers after 21 days in normal SBF and capacity of natural fibers in mineralization in SBF.

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(a)

(b)

(c)

(d)

(e)

(f)

Figure 7. (a) and (b) SEM images of natural silk fibers one day after immersion in SBF. (c) and (d) SEM images of natural silk fibers after 21 days of incubation in SBF. CaP needle-shaped crystals formed on the fibers. EDX chemical analysis results of fibers after (e) 1 and (f) 21 days in SBF. Crystals shows Ca and P peaks.

Element analysis using EDX, in 1 and 21 days after immersing showed CaP needle shape crystals were formed on fibers. NaCl peaks in EDX which is found in both are probably due to the supersaturation of solution. Figure 8 shows the SEM image of composite RS/NS scaffolds in SBF after 21 days. EDX chemical analysis reveals Cap formation on these scaffolds. To test if this formation is only supported by fibers or regenerated fibroin has also the same capacity, F4 scaffolds are also immersed in SBF for 21 days. Figure 9 demonstrates the formation of Cap crystals on RS scaffolds as well. However, the morphologies of the RS crystals are cubic, which is different from the needle-shaped crystals on the degummed silk fibers.

4. Conclusion The structural characteristics and properties of RS/NS composite 3D scaffolds were investigated. Cell growth and adhesion were evaluated on the composite scaffold. The RS/NS composite was synthesized using non-bioengineered silk fibroin protein from silkworms with an easy-to-follow protocol. The high compressive modulus, high compressive strength, highly porous structure, and simple fabrication technique of the scaffold make it a good choice for bone-tissue repair


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to thank Armin Springer, Max Bergman Center, Dresden, for running EDX analysis and taking SEM images.

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[6] (c)

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Figure 8. SEM images of C1F4 scaffold after (a) 1 and (b) 21 days of incubation in SBF. EDX chemical analysis of C1F4 scaffold after (c) 1 and (d) 21 days in SBF.

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Figure 9. SEM images of F4 scaffold after (a) 1 and (b) 21 days of incubation in SBF. EDX chemical analysis of F4 scaffold after (c) 1 and (d) 21 days in SBF.

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applications. Needle-shaped CaP crystals appeared in the RS/ NS scaffolds in SBF, showing the bioactive capability of the scaffolds. Adherence and growth of established osteoblast cells on the fabricated silk matrices are satisfactory. Since regenerated fibroin cannot form complete β-sheet structures, the utilization of NS for reinforcement is a promising strategy for future tissue engineering applications since RS loses some of its characteristics even after methanol treatment.

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Acknowledgments

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This work was partly funded by the Nanomedicine and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran. The authors would like

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