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72319. Uluslararas ı Metalurj i ve Malzeme Kongresi | IMMC 2018

Fabrication and Characterization of a Silk Fibroin Based Cardiac Patch

Yiğithan Tufan¹, Hayriye Öztatlı², Bora Garipcan², Batur Ercan¹,³

¹Middle East Technical University, Faculty of Engineering, Department of Metallurgical and Materials Engineering, Ankara/Turkey²Institute of Biomedical Engineering, Boğaziçi University, Istanbul/Turkey

³Biomedical Engineering Program, Middle East Technical University, Ankara/Turkey

Abstract

Coronary heart disease accounted for nearly 40.5% of all deaths (~ 64,000 deaths) in Turkey in 2015 [1]. The progression of the disease is slow where gradual narrowing of the coronary artery due to plaque formation limits transportation of sufficient oxygenated blood and nutrients to the left ventricle of the heart, which in turn induces death of myocardium (heart muscle) and heart attack. Since the ability of cardiac tissue to regenerate itself is limited, different therapies were investigated to heal damaged hearth muscle. In the last decade, cardiac patches were offered as a potential remedy to heal injured heart muscle [2, 3, 4]. For cardiac patch applications, many scaffold chemistries were investigated, including poly(ethylene glycol), poly(lactic-co-glycolic acid), gelatin and collagen [4]. Among these materials, silk fibroin gained significant popularity in recent years due to its biocompatible nature. Briefly, silk fibroin is a natural material and its mechanical properties can be fine-tuned to mimic that of human myocardium. It is biodegradable and possesses some of the similar amino acids present in the extra cellular matrix of the heart tissues [5]. In this study, silk fibroin scaffolds having different pore sizes were fabricated in a 3D fashion to design a cardiac patch with optimum physical and chemical properties.

Introduction

The healing capacity of myocardium (heart muscle) after a heart attack is insufficient to regenerate itself, leading to scar tissue formation rather than functional heart cells, which occasionally leads to heart failure in the upcoming years [2]. Pharmacologics, ventricle assist devices, artificial hearts and total heart transplantation are among the existing solutions for heart failure [3]. However, none of these solutions has the ability to regenerate injured myocardium after a heart attack. Thus, alternate solutions are required to recover the functionality of the heart muscle, where cardiac patches can be a potential remedy [4]. The approach of using cardiac patches aims the creation of contractile heart muscle tissue to repair the infarcted region through biomimetic scaffolds seeded

with different types of cells [4]. There are multiple studies in the literature showing the beneficial influence of cardiac patches to promote cardiamyocyte (heart muscle cell) proliferation and induce contractile functionality of the heart tissue in vitro [6].

In recent years, silk based biomaterials have been investigated for various tissue engineering applications [7]. Silk is a protein based natural material having fibroin core encapsulated inside sericin outer lining [7]. Due to the biocompatible nature of silk fibroin, its use as a cardiac patch could be a potential remedy to heal infarcted myocardium [8]. For instance, Nazarov et. al. [5] modified mechanical and degradation properties of silk fibroin via controlling its crystallinity and porosity by altering scaffold fabrication route. Since it was important to match the mechanical properties of the cardiac scaffold with that of myocardium, silk fibroin could offer many advantages. In addition, the scaffold used for cardiac patch needed to have a proper degradation rate so that it could maintain its mechanical integrity upon interacting with a contractile myocardium while it could degrade within an adequate time interval allowing for the regeneration of healthy heart tissue. In another study, Lu et al. [9] claimed that porous silk fibroin scaffolds provided a suitable microenvironment for stem cells as the porous structures enabled them to have a direct connection with the surrounding tissue, and thus increased cell survival and biocompatibility of the scaffolds. Patra et. al. [8] showed that cardiomyocytes effectively attached and showed functionality on a 3D fibroin scaffold in vitro. As the above examples indicated, a 3D porous fibroin scaffold having tunable properties could be a favorable biomaterial in cardiac tissue applications. In this research, fibroin scaffolds having different pore sizes were produced and their morphological, chemical and structural analyses were performed to investigate optimum properties for cardiac patch applications.

Experimental Procedure

The production method of 3D porous fibroin scaffolds used in this study could be divided in three parts. For the

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initial step (fibroin extraction), fibroin was extracted from the Bombyx Mori (silkworm) cocoons by dissolving sericin outer protein layer using 0.02 M Na2CO3 solution by boiling cocoons for 30 min. Extracted fibroin was dried in a fume hood overnight. In the second step (lyophilization), fibroin was dissolved in 12 M LiBr solution and dialysed against distilled water for 2 days to remove Li+ and Br - ions. Dialyzed fibroin was centrifuged for 30 min (7830 RPM) at 4 oC to remove left-over impurities. Afterwards, the fibroin/water solution was frozen in -20 oC for one day and lyophilized in Christ Alpha 2-4 LDplus to remove water and store at room temperature. In the final stage (fabrication of porous scaffold), 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was used to dissolve fibroin and obtain 5 wt/vol % HFIP/fibroin solutions. Six different fibroin solutions were prepared in this study. NaCl particles were sieved to obtain 50-90 μm and 180-200 μm particle sizes. For each fibroin solution, 3 g. of sieved NaCl particles were used. NaCl particles were added into the solutions, the mixture was capped with the parafilm and waited for 24hrs. Afterwards, samples were placed in a fume hood for another 24 hrs to evaporate HFIP. At the end of evaporation, scaffolds were kept in methanol for 1, 6 and 24 hrs to induce crystallization. The scaffolds having 50-90 μm NaCl particles were labeled as A1, A6, and A24 accordingly to their immersion time in methanol; 1, 6 and 24 hours, respectively. Similarly, the samples in the second group having 180-200 μm NaCl particle size were labeled as B1, B6 and B24 indicating the duration of their methanol immersion. Schematic drawing of production route was given in Figure 1.

Morphology and chemistry of the samples were characterized using FEI Nova Nano SEM 430 microscope at 3 kV accelerating voltage. Prior to SEM characterization, samples were coated with a thin layer of gold using Quorum SC7640 high resolution sputter coater. Samples were investigated using FTIR Perkin Elmer 400 using attenuated total reflection in 400-2000 cm-1 range. Further structural analysis was completed using Rigaku D/Max-2200 X-ray diffractometer with monochromatic Cu K radiation (1.54056 Å).

Results and Discussions

In this research, altering the NaCl particle size used to form porosity led to the formation of different scaffold microstructures and morphologies. In Figure 2, cross sectional images of fibroin scaffolds was shown. Samples produced using NaCl having a particle size range of 50-90 μm had all similar microstructures (Figure 2, A1, A6 and A24) having a finer pore structure compared to sample group B (Figure 2, B1, B6, B24). Unlike sample group A, a coarser pore structure was obtained for B1, B6 and B24. In addition, NaCl particles were not visible

in SEM investigations, indicating their removal from the scaffolds. Since pore size is important for infiltration of cell inside the scaffold, both group A and B can potentially be a candidate for cardiac patch applications. However, the amount of porosity inside the scaffold would certainly influence mechanical properties of the scaffold, and thus selection of precise parameters for tissue engineering applications require mechanical and biological characterization of these scaffolds.

Figure 1. Schematic drawing of the three part fibroin scaffold production, namely extraction, lyophilization and the fabrication of porous scaffolds.

Table 1. Chemical compositions of the silk fibroin samples.

SamplesElements(wt %)

A1 A6 A24 B1 B6 B24

C 58 58.7 57 56.6 56.9 56.3N 13.4 12.3 14.2 13.4 12.8 14.7O 25.1 24.4 26.6 26.1 26.4 26.4Na 0.3 0.7 0.4 0.7 0.5 0.3Cl 0.3 0.9 0.3 0.5 0.5 0.4Au 3.1 3.1 1.6 1.9 2.8 2

TMMOB Metalurj i ve Malzeme Mühendisleri Odas ı Eğ i t im MerkeziBildir i ler Kitab ı

72519. Uluslararas ı Metalurj i ve Malzeme Kongresi | IMMC 2018

Results of EDS analyses revealed complete removal of Li+ and Br- ions from all scaffold types (Table 1), highlighting the efficacy of dialysis process in this study. This was a promising result since left-over Li+ and Br-

ions in the scaffold could potentially activate immune response and compromise biocompatibility.

Figure 2. SEM images of scaffolds

In Figure 3, XRD analysis of the samples were given. There was a broad peak in all samples centered around 23o, which was attributed to -Sheet crystal structure [11]. In crystallographic analysis, no difference was present between different sample groups upon altering porosity or duration of methanol treatment. -sheetformation was evident for all samples investigated in this study.

In the FTIR analysis (Figure 4), amide I (1700-1600 cm-

1), amide II (1600-1500 cm-1) and amide III (~1200 cm-1)vibrations were investigated. Previous researchers determined that 80% of the amide I vibration came from C=O stretching and there was also a minor effect of N-H plane bending on this vibration [9]. On the other hand, chief contribution to amide II vibration was the C-N stretching and N-H in-plane bending [9]. In the FTIR characterization of fibroin scaffolds, transmission peaks obtained at 1623 cm-1 and 1516 cm-1 were attributed to -sheet crystalline structure of fibroin [9, 10, 12] and the peak at 1234 cm-1 belonged to amide III band. These

results confirmed the formation of -sheet crystalline structure in all samples.

Figure 3. X-ray diffraction patterns of samples having different porosities and crystallinities.

Although a significant difference could not be found in the XRD analyses between sample groups, it was evident in the FTIR spectra that samples having a coarser porous structure exhibited higher crystallinity. Comparing the FTIR spectra of group A and B, stronger signal with deeper peaks was observed for the sample group B compared to A. It can be speculated that coarser porous structure could allow better penetration of methanol with the silk fibroin, which in turn increased the crystallinity of the scaffolds.

Figure 4. FTIR spectra of samples having different porosities and crystallinities.

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This research showed the influence of process parameters on the microstructure and crystallinity of silk fibroin scaffolds. To further assess the suitability of fabricated scaffolds for cardiac patch applications, mechanical and biological characterizations will be completed.

Conclusion

Three dimensional porous silk fibroin scaffolds were fabricated using two different NaCl particle sizes. These samples were treated with methanol for three different time intervals to reveal the effect of methanol treatment duration on the crystallization of the fibroin. The formation of -sheet crystalline structures was confirmed by structural analyses for all samples. In addition, higher crystallinities were observed for the samples having coarser pore structures in FTIR analyses. Though the fabricated scaffolds show promise for cardiac-tissue engineering applications, further research is required to assess their mechanical properties and biocompatibility for their successful use as a cardiac patch material.

Acknowledgement

The authors would like to thank The Scientific and Technological Research Council of Turkey (Grant no: 117M754) for providing financial support.

References

[1] < http://ww.tuik.gov.tr>Dated: 16.05.2018 [2] Prabhakaran, M. P., Venugopal, J., Kai, D., & Ramakrishna, S, Materials Science and Engineering: C, 31(3) (2011) 503-513.[3] Jackman, C. P., Ganapathi, A. M., Asfour, H., Qian, Y., Allen, B. W., Li, Y., & Bursac, N., Biomaterials, 159 (2018) 48-58. [4] Amezcua, R., Shirolkar, A., Fraze, C., & Stout, D. Nanomaterials, 6(7) (2016) 133. [5] Nazarov, R., Jin, H., & Kaplan, D. L., Biomacromolecules, 5(3) (2004) 718-726. [6] Zhou, J., Chen, J., Sun, H., Qiu, X., Mou, Y., et. al., Scientific Reports, 4(1) (2014). [7] Vepari, C., & Kaplan, D. L.. Progress in Polymer Science, 32(8-9) (2007) 991-1007. [8] Patra, C., Talukdar, S., Novoyatleva, T., Velagala, S. R., Mühlfeld, et. al., Biomaterials, 33(9) (2012) 2673-2680.[9] Lu, G., Liu, S., Lin, S., Kaplan, D. L., & Lu, Q., Colloids and Surfaces B: Biointerfaces, 120 (2014) 28-37.[10] Ling, S., Qi, Z., Knight, D. P., Shao, Z., & Chen, X., Polymer Chemistry, 4(21) (2013) 5401. [11] Um, I. C., Kweon, H., Park, Y. H., & Hudson, S., International Journal of Biological Macromolecules, 29(2) (2001) 91-97.

[12] Chen, X., Knight, D. P., Shao, Z., & Vollrath, F., Polymer, 42(25) (2001) 09969-09974..